REGULATION OF ATP-SENSITIVE POTASSIUM CHANNELS IN THE HEART
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Pharmacy
in the Graduate School of The Ohio State University
By
Vivek Garg, M.Pharm
*********
The Ohio State University
2009
Dissertation Committee: Approved by
Professor Keli Hu, Advisor
Professor Terry S. Elton Professor Lane J. Wallace ------Advisor Professor Dale G. Hoyt Graduate Program in Parmacy
Copyright by
Vivek Garg
2009
ABSTRACT
ATP-sensitive potassium channels (KATP channels) link the cellular
energy levels to membrane potential and excitability in various cell types.
They control many important functions like insulin secretion in pancreatic β- cells; vascular tone in vascular smooth muscle cells; and action potential duration in cardiac myocytes and neurons under ischemic conditions. In cardiac myocytes, it has been shown that KATP channels on plasma
membrane are composed of Kir6.2 and SUR2A subunits in 4:4 stoichiometry.
Though many regulators of KATP channels and signal transduction
mechanisms regulating opening or closing of KATP channels have been identified, much less is known about the subcellular localization of KATP channels which can have a profound impact on the temporal and spatial regulation by its signaling modulators. Many studies have localized KATP channels to nucleus and mitochondria, besides cellular plasma membrane.
However, it not known if these are the same cell surface KATP channels, which
are also targeted to mitochondria, or some other isoform of KATP channel. The
plasma membrane itself is not homogenous through out. It is interspersed by
sub-domains rich in sterols and glycosphingolipids. In majority of cases, this sub-domain organization is orchestrated by special proteins called as
ii caveolins, which are the main structural components of caveolae. Caveolae
are small (50 to 100 nm), cholesterol and sphingolipid enriched “cave”-like
invaginations of the surface membrane. These specialized lipid microdomains have the ability to selectively compartmentalize many signaling molecules, including many modulators of KATP channels. Since little is known about the subcellular locations of KATP channel protein, we designed our studies to
characterize the localization of KATP channel protein in cardiac myocytes.
We first endeavored to look at KATP channel localization along the plasma membrane in rat cardiac myocytes. Using a variety of different
techniques on isolated murine cardiomyocytes, we found that majority of
cardiac KATP channels are localized to caveolin-enriched membrane
microdomains. Further, whole-cell voltage-clamp recording in both adult and
neonatal cardiac myocytes confirmed our hypothesis that caveolae integrity is essential for activation of KATP channel by its modulator adenosine (Chapter
1). Adenosine released from ischemic myocardium is a very important
modulator of KATP channels. These findings have significant implications for cardioprotective role of KATP channels during ischemic conditions.
A signaling function for caveolins either on their own (direct) or by
acting as scaffolding proteins (indirect) has also been described. To test its
relevance for KATP channels, we employed HEK293T cells transfected with
recombinant cardiac KATP channels (Kir6.2/SUR2A) with or without caveolin-3
(Cav-3, a muscle-specific caveolin isoform). We found that Cav-3 has
significant inhibitory effect on KATP channel current density which can be
iii reversed by a scaffolding domain peptide from caveolin-3 protein sequence
(CSD) (Chapter 2). This crucial experiment indicates a very interesting, dual
regulation of KATP channels by caveolins and caveolae. Though caveolae
structure ensures that KATP channel modulators are close to the channel proteins for efficient signal transduction, caveolin-3 protein through direct or
indirect interaction with channel proteins makes sure that they remain
inhibited until required.
Whether Kir6.2 containing KATP channel are present in mitochondria or
not is controversial; nevertheless no one has ever studied the trafficking
aspect of KATP channel to mitochondria. In our effort to elucidate the sub-
cellular localization of cardiac KATP channels, we hypothesized that
localization of Kir6.2-containing KATP in mitochondria can be increased by
activation of protein kinase C (PKC). Utilizing KATP-deficient COS-7 cells, we
reported a novel finding that a specific protein kinase C (PKC) isoform, PKCε,
promotes mitochondrial import of Kir6.2-containing KATP channels from cytosol. These findings were further corroborated with functional data using mitochondrial potential measurement studies.
Collectively, our data demonstrated that besides bulk plasma
membrane, Kir6.2 containing cardiac KATP channels are localized to two
distinct sub-cellular locations, namely caveolae and mitochondria. Their
localization can be modulated by specific regulatory pathways, and
furthermore
iv furthermore their regulation can be affected by their sub-cellular localization.
This provides valuable insight into the mechanisms regulating KATP channels,
which have been implicated for cardioprotection under ischemic conditions.
v
Dedicated to My parents & family
vi
Acknowledgements
I would like to pay a tribute to all my teachers (past and present) through this thesis. There are so many people to thank for who have contributed directly or indirectly to this work by, influencing my thinking, discussions, advice and of course blessings and wishes. My deepest gratitude is due to my advisor Dr. Keli Hu for her continued support, guidance and encouragement though out my graduate work; for allowing me to work independently on research areas of my interest. Her attention to detail, focus and fruitful suggestions made my work worthy of presentation. I wish to thank Dr. Jundong Jiao, who started me on patch clamp studies. I also thank Arun Sridhar, Veronique Lacombe for helpful discussions on perforated-patch and voltage-clamp studies. My thanks are due to Dr. Douglas Pfeiffer for discussions regarding mitochondrial studies and allowing me to work in his lab for some of the experiments. I would like to thank my committee members Dr. Lane Wallace, Dr. Terry Elton, Dr. Dale G. Hoyt for their time, effort and sharing their valuable opinions regarding this work. I also thank the faculty members of the pharmacology division especially Dr. Lakhu Keshvara, Dr. Popat N. Patil, Dr. Kari Hoyt, Dr. Cynthia Carnes for their encouragement, conversations, and time to time intellectual stimulation. My training as a research scientist is greatly enriched with your interactions. I thank all of my friends and wonderful colleagues in the division Vaibhav, Zhaogang, Tongzheng, Brandon, Ryan, Rachel, Raeann, Sarmistha who helped me in different ways, be it reagents or discussions regarding individual experiments. I will never forget my friends at OSU. You guys made
vii my stay here truly enjoyable. Thanks for sharing times of happiness and celebration; and emotional support in times of stress. I am just humbled by all kinds of contributions and sacrifices made by my parents Dr. Pawan Kumar Garg, Mrs. Kamla Garg and family members Priya, Rishubh, Upma, Anupma, Sanjeev, Tarun, Rohan, Honey, and Aryan. I would have never reached at the stage where I am now without them. Though far, you are always in my thoughts as a perpetual source of inspiration. I wish to thank you all for your unconditional love, blessings and happiness. Last but not the least, I am highly grateful to my wife Priyanka for being supportive and infinitely patient in tolerating my business. Her support and encouragement was in the end what made this dissertation possible in time. She was always there cheering me up in all my endeavors.
viii
VITA
July 18, 1977……………………...Born – Punjab, India
1995 – 1999.………………………Bachelor of Pharmacy
Punjab University, India
2000 – 2002………………………..Master of Pharmcy
Punjab University, India
2004 – Present……………………Graduate Teaching Associate
The Ohio State University, Columbus, Ohio
PUBLICATIONS
1. Garg V, Jiao JD, Hu K. (2009) ATP-sensitive K+ channels are
regulated by caveolin-enriched microdomains in cardiac myocytes.
Cardiovasc Res 82: 51-58.
2. Jiao J, Garg V, Yang B, Elton TS, Hu K. (2008) PKC-epsilon induces
caveolin-dependent internalization of vascular ATP-sensitive K+
Channels. Hypertension 52: 499-506.
ix 3. Jiao JD*, Garg V*, Yang B, Hu K (2008) Novel functional role of heat
shock protein 90 in ATP-sensitive K+ channel-mediated hypoxic
preconditioning. Cardiovasc Res 77: 126-33 (*equal contribution).
4. Garg V, Hu K (2007) Protein kinase C isoform-dependent modulation
of ATP-sensitive K+ channels in mitochondrial inner membrane. Am J
Physiol Heart Circ Physiol 293: H322-332.
5. Singh A, Garg V, Gupta S, Kulkarni SK (2002) Role of antioxidants in
chronic fatigue syndrome in mice. Indian J Exp Biol 40: 1240-1244.
FIELDS OF STUDY
Major Field: Pharmacy
x
TABLE OF CONTENTS
PAGE
Abstract……………………………………………………………….…………..ii
Dedication…………………………………………………………….………….vi
Acknowledgements………………………………………………….………….vii
Vitae…………………………………………………………………….………..ix
List of Figures………………………………………………………….………..xiv
Abbreviations………………………………………………………….………...xvi
CHAPTER 1. INTRODUCTION………………………………..………..1
1. ATP-sensitive potassium channels……………………………………...1
1.1. Molecular basis……………………………………………...………...... 3
1.2. Biophysical properties………………………………………...…………6
1.3. Regulation……………………………………………………………...... 7
1.3.1. Nucleotide regulation………………………………...... ….……7
1.3.2. Pharmacological regulation………………………...……………9
1.3.3. Regulation by signaling molecules……………………………..11
1.3.3.1. Phosphoinsotides (PIP2)…………………………:...... 13
1.3.3.2. Protein kinase C ……………………..…………...……14
1.3.3.3. Subsarcolemmal ATP………..………………....…...... 17
xi 1.3.3.4. Nitric oxide ……………………………………………….21
1.3.3.5. G-protein coupled receptors (GPCRs)……………...... 22
1.4. Functional role………………………………………………...... …..…....30
2. Caveolae……………………………………….…………………………...... 38
2.1. Caveolae and caveolins……………………………….………...………38
2.2. Functional role of caveolae/caveolins……………….……………..…..41
2.3. Compartmentalization of ion channels…………..………..…………...44
2.3.1. Cardiac ion channels……………………………………….…….45
2.3.2. KATP channels……………………………………………………..48
3. Objectives……………………………………………………………...……..49
CHAPTER 2. REGULATION OF ATP-SENSITIVE POTASSIUM
CHANNELS BY CAVEOLIN-ENRICHED
MICRODOMAINS IN CARDIAC MYOCYTES………..51
1. Introduction……………………………………………...…….…...... 53
2. Materials and Methods……………………………………………....54
3. Results……………………………………………...…….…….…...... 59
4. Discussion…………………………………………….....…..………..81
5. Acknowledgements…………………………………….…….……....85
CHAPTER 3. CAVEOLIN-3 NEGATIVELY REGULATES
RECOMBINANT CARDIAC ATP-SENSITIVE
POTASSIUM CHANNELS IN HEK293T CELLS….....86
xii 1. Introduction………………………………….……………...….…...... 88
2. Materials and Methods…………………….………………………...89
3. Results……………………………………..……….…….…………...93
4. Discussion……………………………………………..……….……..105
5. Acknowledgements………………………...…………….….……....108
CHAPTER 4. PROTEIN KINASE C ISOFORM-DEPENDENT
MODULATION OF ATP-SENSITIVE POTASSIUM
CHANNELS IN MITOCHONDRIAL INNER
MEMBRANE………………………………...…………...109
1. Introduction…………………………………………...... ….……...111
2. Materials and Methods……………………………...………….…...113
3. Results……………………………………………...... ….…………...119
4. Discussion……………………………………………....….………...141
5. Acknowledgements…………………………………...…….…..…...147
CHAPTER 5. CONCLUSIONS AND FUTURE DIRECTIONS……....148
BIBLIOGRAPHY…………………………………………….…………………...154
xiii
LIST OF FIGURES
Figure Page
1.1. Topology and assembly of KATP channels……………………..……..3
1.2. Comparison of biophysical and pharmacological properties
of different isoforms of KATP channels………………………….……..4
1.3. Topology and predicted membrane insertion of Cav-1………….….39
2.1. Kir6.2 is enriched in caveolar plasma membrane in
adult rat cardiomyocytes…………………………………………...…..62
2.2. KATP channels are associated with caveolin-3 (Cav-3) in
adult rat ventricular myocytes……………………………………..…..65
2.3. Colocalization of Kir6.2 and caveolin-3 (Cav-3)……………….…….67
2.4. Caveolar disruption with MβCD eliminates adenosine
receptor-mediated stimulation of KATP channels…………….………71
2.5. Knockdown of caveolin-3 expression with siRNA prevents
adenosine receptor-mediated activation of KATP channels…………75
2.6. Immunoblot analysis of samples from neonatal rat hearts
immunoprecipitated with anti-caveolin-3 (Cav-3) or
anti-Kir6.2 antibodies……………………………………………...……78
xiv 2.7. Effect of MβCD on Kir6.2, adenosine A1 receptors (Ade A1)
and caveolin-3 (Cav-3) coprecipitation………………………………..80
3.1. Modulation of spontaneous KATP currents by caveolin-3………….…95
3.2. Modulation of pinacidil-activated KATP currents by caveolin-3………98
3.3. Effect of caveolin-3 scaffolding domain peptide (SDP) on
KATP currents……………………………………………………………..102
3.4. Co-immunoprecipitation and colocalization of Kir6.2/SUR2A
channels with caveolin-3………………………………………………..104
4.1. Immunofluorescence microscopy of mitochondrial localization
of Kir6.2 in mitoplasts isolated from COS-7 cells…………………….123
4.2. Proximity of Kir6.2-cyan fluorescent protein (CFP) to
Mito-yellow fluorescent protein (YFP) in mitochondrial matrix
yielded fluorescence resonance energy transfer (FRET) signals…..127
4.3. Immunoblot analysis of Kir6.2 protein in mitochondria……………....131
4.4. PMA-induced KATP-dependent changes in mitochondrial
membrane potential (∆ψm)…………………………………………..….136
4.5. Immunofluorescence microscopy of mitochondrial localization of
Kir6.2 in mitochondria isolated from rat adult cardiomyocytes….…..140
5.1 Hypothetical model for the targeting of KATP channels to plasma
membrane, caveolar microdomains and mitochondria……….……...153
xv
ABBREVIATIONS
5-HD 5-Hydroxy decanoate
ABC ATP-binding cassette
AC Adenylate cyclase
Ach Acetylcholine
APD Action potential duration
[ATP]I Intracellular ATP
BKca Calcium-activated potassium channel
Cav-1 Caveolin-1
Cav-2 Caveolin-2
Cav-3 Caveolin-3
CBD Caveolin binding domain
CFTR Cystic fibrosis transmembrane conductance
regulator channel
CK Creatine kinase
Cr Creatine
CRP Caveolae resident protein
CSD Caveolin scaffolding domain
xvi DAG Diacylglycerol
DEAE Diethylaminoethyl cellulose
E-C Excitation-contraction eNOS Endothelial nitric oxide synthase
Erev Reversal potential
FRET Fluorescence resonance energy transfer
GAPDH Glyceraldehyde phosphate dehydrogenase
GIRKs G-protein activated inwardly rectifying K+ channels
GPCR G-protein coupled receptor
HCN4 Hyperpolarization-activated cyclic nucleotide gated
HEK Human embryonic kidney
ICa,L L-type calcium channel current
If ‘funny’ current (mediated by HCN channels)
IKACh Muscarinic-gated potassium channel
+ IKr Rapidly activating delayed rectifier K channel
current
IMM Inner membrane of mitochondria
INa Voltage-activated sodium channel current
IP3 Inositol triphosphate
IPC Ischemic preconditioning
Ito Transient outward potassium current
ITP Inosine triphosphate
KATP ATP-sensitive potassium channel
xvii KCOs Potassium channel openers
Kir Inwardly rectifier potassium channel
KO Knock out
Kv Voltage-activated potassium channel
M-LDH Muscle isoform of Lactate dehydrogenase
MβCD Methyl-β-cyclodextrin
Na/K-ATPase Sodium-potassium ATPase
NBD Nucleotide binding domain
NBF1 Nucleotide-binding fold-1
NBF2 Nucleotide-binding fold-2
NCX Sodium-calcium exchanger
NDP Nucleoside diphosphate
PC preconditioning
PFK Phosphofructokinase
PI Phosphoinositide
PIP2 Phosphatidylinositol-4,5-bisphosphate
PKA Protein kinase A
PKC Protein kinase C
PLC Phospholipase C
PMA Phorbol-12-myristate-13-acetate
PTX Pertussis toxin
RACK Receptor for activated C kinase
ROS Reactive oxygen species
xviii SarcKATP Sarcolemmal KATP channel siRNA Small interfering RNA
SUR Sulphonylurea receptor
TIM Translocases of the inner membrane of
mitochondria
TOM Translocases of the outer membrane of
mitochondria
TRP Transient receptor potential channel
UDP Uridine diphosphate
UTP Uridine-5'-triphosphate
xix
CHAPTER 1 INTRODUCTION
1. ATP-sensitive potassium channels
Ion channels are specialized membrane proteins which provide
hydrophilic conduction pathways for various ions across the hydrophobic
interior of lipid bilayers. They exhibit ion-selectivity based on size, charge and water of hydration. Based on ion-selectivity, they are classified broadly as sodium channels, potassium channels, calcium channels and chloride channels. Typically, they are composed of multi-subunits which are arranged in a circular fashion around a water-filled tunnel/pore which has openings on both extracellular and intracellular side. The pore-forming subunits are called as α-
subunits while the accessory subunits are called as β-, γ-subunits and so on. In
many ion channels, the opening or closing of the pore is governed by a ‘gate’
which is controlled by various signals like chemical, electrical or mechanical
signals. In the heart most of the ion channels are voltage gated while some are
ligand (chemical) gated. For the purposes of a review relevant to the content of
this work, I will mainly focus on a ligand gated potassium channel called as
ATP-sensitive potassium channels.
1 Originally discovered in the heart, ATP-sensitive potassium channels
(KATP channels) are widely distributed in various other tissues and cell types
like pancreas, smooth muscles and neurons (197). KATP channels activation/inhibition and cellular energy metabolism are coupled in most of the
excitable tissues where they are expressed. Inhibition of KATP channels by ATP
and stimulation by nucleotide diphosphates regulate insulin release in
pancreatic β-cells; vascular tone in smooth muscle cells; and action potential
duration (APD) shortening in cardiomyocytes and neurons (192). Pancreatic β-
cells offer the classical and simplistic paradigm to understand KATP channel
function. These channels are open under the basal conditions in the pancreas,
leading to significant K+ outflux and hyperpolarized state of the membrane.
However, when plasma glucose levels increase; an increase in pancreatic β-
cell [ATP/ADP]i ratio causes inhibition of these channels leading to opening of
calcium channels, consequent membrane depolarization, and insulin release.
This is considered to be the fundamental mechanism responsible for insulin
release in response to glucose. Interestingly, in the heart, these channels are
closed under the basal conditions, leading many people to question their
physiological relevance. Nevertheless, these channels shorten APD in
response to metabolic or ischemic stress and mediate a robust cardioprotective
response. As evidenced by recent reports and will be discussed later, these
channels do play an important role in normal physiological functions of the
heart as well (section 1.4).
2 1.1. Molecular basis
KATP channels are essential hetero-octamers, consisting of two subunits:
four pore forming subunits (Kir6.x), and four regulatory subunits (SURx) (2)
(Fig. 1.1). While Kir6 is a member of inwardly rectifier potassium channel
family; the other subunit SUR (for sulphonylurea receptor), is a member of
ATP-binding cassette (ABC) protein superfamily, which includes CFTR (Cystic
fibrosis transmembrane conductance regulator channel, a chloride channel)
and P-glycoprotein etc. Two isoforms of Kir6 have been identified: Kir6.1,
Kir6.2; and three major isoforms have been described for SUR namely, SUR1,
SUR2A and SUR2B. Further, many small isoforms and splice variants have
also been described in the various tissues in mice (260). The exact
physiological role of these isoforms is unclear. Different subunit composition in
different tissues gives rise to KATP channels with different biophysical and
pharmacological properties: pancreatic β-cells (SUR1/Kir6.2), cardiac myocytes
(SUR2A/Kir6.2), brain (SUR1/Kir6.2), and vascular smooth muscle cells
(SUR2B/Kir6.1) (summarized in Figure 1.2).
It is now widely accepted that sarcolemmal KATP channels in cardiomyocytes are composed of Kir6.2 and SUR2A subunits (77). Both
-/- -/- Kir6.2 and SUR2 mice have no KATP channel current in cardiomyocytes (44,
159, 269). Nevertheless SUR1, SUR2B and Kir6.1 immunoreactivity and
transcripts have also been detected in the heart in several studies (104, 187,
221, 265).
3
Figure 1.1. Topology and assembly of KATP channels. The topologies of
SUR (Upper panel, left), and Kir6.2 (Upper panel, right) are illustrated. (Lower panel) The channel is assembled from SUR1/2 and KIR6.2 subunits as an octameric complex in 4:4 stoichiometry, with four Kir6.2 subunits forming the inner pore and each Kir6.2 subunit attached to one SUR subunit from outside.
Abbreviations: N = N-terminus, C = C-terminus, TMD = transmembrane domain, M = membrane domain, P = pore region, A and B = walker domains A and B, NBF = nucleotide binding fold. Adapted from Aguilar-Bryan and Bryan,
1999 (2).
4
Type Subunit Unitary Ki for IC50 by KCO’s Composition Conductance ATP Glibenclamide sensitivity (pS) inhibition (nM) (μM)
Cardiac & SUR2A/ 80 100-170 6-9 P=C Skeletal Kir6.2 muscle Pancreatic SUR1/ Kir6.2 76 10 5 D>P>C Vascular SUR2B/ 33 >1000 20-100 P=C>D Smooth Kir6.1 Muscle
Figure 1.2. Comparison of biophysical and pharmacological properties of
different isoforms of KATP channels. Abbreviations: KCO = Potassium
channel opener, P = Pinacidil, C = Cromakalim, D = Diazoxide.
A recent study by Flagg et el. 2008 (77) has shown that SUR1 is an essential component of sarcolemmal KATP channels in atrial myocytes but not in
ventricular myocytes in mice. This might be responsible for different
pharmacological profile (different sensitivities to KCOs) and different functional
responses of atrial and ventricular myocytes during ischemic conditions. Using
transgenic approaches, it was further deduced that SUR1 containing channels
open much more readily as compared to SUR2A containing KATP channels in
the heart. Further, mice overexpressing SUR1 in the heart were more prone to
arrhythmic events as compared to SUR2A overexpressing hearts. Why SUR2A
containing KATP channels are silent in the heart as compared to SUR1 is not
clear.
5 Based on different sensitivities to ATP depletion and differential APD
shortening in epicardial versus endocardial myocytes under ischemic
conditions, there are reports proposing that there may be transmural
heterogeneity of KATP channel within the heart (81, 182). Adding to the diversity
and complexity of KATP channels are the studies showing that there are KATP channels in the subcellular organelles also, like mitochondria (119, 227, 265) and nucleus (227). Though the molecular identity of surface KATP channels is
much clearer, the subunit structure of KATP channels at intracellular locations is
still enigmatic.
1.2. Biophysical properties
Like other potassium channels, KATP channels are selectively permeable
to potassium and negligibly permeable to sodium. They exhibit time- and
voltage-independent kinetics and are ligand gated (ATP). The single-channel
conductance in symmetrical K+ solution is ~70-90 pS which is much higher than
any other potassium channel except calcium-activated potassium channels
(BKCa, ~210 pS). The kinetics of KATP channel openings are characterized by a
unique ‘flickering behavior’ consisting of ‘burst’ of channel openings followed by
relatively long closed periods or silent ‘gaps’. The main effect of ATP is by
reducing the burst duration (affects both mean open time and number of
openings per burst) and prolonging the gap period.
These channels are weak inwardly rectifiers at very positive potentials
as conduction of K+ ions is better in inward direction as compared to outward 6 direction at those potential. Rectification describes the relationship between the
voltage and the conductance of an ion. In non-rectifying current, conductance
is voltage independent and follows Ohm’s law as current is directly dependent
on voltage (V=IR). A channel is an inward rectifier when it passes current
(positive charged ion) more readily in inward direction (into the cell) as
compared to outward direction at potentials positive to reversal potential (Erev).
The main cause of rectification is the high-affinity voltage-dependent block by
intraceullar cations, principally Mg2+ and Na+, and polyamines, namely
spermine, spermidine and putrescine that block the channel pore at positive potentials (2).
1.3. Regulation
1.3.1. Nucleotide regulation
The most characteristic property of KATP channels is its inhibition by
intracellular free ATP which does not require Mg2+. The intracellular free ATP
appears to bind to the pore-forming subunit Kir6.2, while most pharmacological
agents (K+ channel openers and sulphonylureas) and MgADP target SUR2A
subunit. The ATP inhibition on Kir6.2 can be modulated by the respective
binding of ATP and ADP to NBF1 (Nucleotide binding fold 1) and NBF2 of
SUR2A subunit. Since Kir6.2 and SUR2A coassemble in 4:4 stoichiometry, as
per the widely accepted model, there are four ATP binding sites on KATP
channel and two MgADP binding site on each SUR2A subunit. Though only
one ATP site needs to be occupied to inhibit the channel, ATP binding at other 7 sites stabilizes the channel inhibition indicating a cooperative interaction. Thus,
ATP sensitivity of KATP channel is not constant throughout (183). Further,
considerable patch to patch variation besides transmural heterogeneity in ATP
sensitivity of KATP channels has also been noted (182). The ATP sensitivity also
decreases during metabolic stress and ischemic conditions (284). The
adenosine ring is important for high affinity inhibition, as other nucleoside
triphosphates like GTP (Guanosine triphosphate), ITP (Inosine triphosphate)
and UTP (Uridine triphosphate) are much less effective as compared to ATP.
Most of the nucleoside diphosphates (NDP) like ADP and UDP (uridine
diphsophate) antagonize ATP dependent inhibition in Mg2+-dependent manner.
MgADP interaction at SUR2A allosterically affects the interaction of ATP at
Kir6.2 subunit. The effect of MgADP is paradoxical; it activates the channel at
lower concentrations while concentrations higher than 500 μM have inhibitory
effect (183).
An interesting aspect of many ion channels, including KATP channels is
‘run-down’, which is time-dependent decrease in open probability (events of
channel opening) which is often observed in cell-free excised patches (a patch
membrane excised from the cell by recording pipette). This indicates the
requirement of a cytoplasmic factor to maintain channel activity. Divalent
cations like Ca2+ and Mg2+ promote run-down when applied on the intracellular side, while ATP, ADP and UDP decrease run-down in Mg2+-dependent manner.
Even though there is no direct evidence indicating that ATP hydrolysis at NBFs
of SUR is required for channel opening, non-hydrolysable analogs of ATP like 8 ATP[γS] or βγ-methylene ATP are ineffective in preventing run-down, suggesting the requirement for possible ATP hydrolysis at NBFs of SUR subunit (203). Channel phosphorylation by protein kinases was ruled out using specific inhibitors. However, wortmannin was found to inhibit MgATP dependent recovery of KATP channels, indicating that Phosphoinositide-kinases are likely involved. Since PIP2 (Phosphatidylinositol-4,5-bisphosphate) can
activate and recover run-down KATP channel, it was proposed that lipid
phosphorylation may be involved in MgATP-dependent recovery of run-down
channels (306).
1.3.2. Pharmacological regulation
KATP channels are the primary target for classical antidiabetic drugs,
sulphonylureas (glibenclamide, tolbutamide etc) and selective potassium
channel openers (KCOs) (pinacidil and cromakalim etc). The binding of
sulphonylurea follows two-site model with a very high-affinity binding site on
SUR (nanomolar range) and a low-affinity site on Kir6.x (millimolar range,
clinically irrelevant site). The binding affinity and potency of inhibition by
sulphonylureas varies depending on the specific isoform of the KATP channel
(pancreatic > cardiac > vascular) and type of sulphonylurea drug (tolbutamide,
gliclazide, and mitiglinide bind and block pancreatic isoform, Kir6.2/SUR1 with
much higher affinity). Sulphonylureas bind to the cytoplasmic domains of SUR
subunit and close KATP channel, however, the exact transduction mechanism is
not known. Further, this interaction is significantly modulated by intracellular 9 nucleotides as increased ATP increases sulphonylurea sensitivity. There is a
competitive antagonistic interaction between MgADP (at least at lower
concentrations) and sulphonylurea, which might be one of the reasons for
decreased sulphonylurea sensitivity during metabolic stress in cardiac
myocytes (224).
Potassium channel openers (KCOs) are a group of structurally diverse
compounds that open KATP channels (176). These drugs bind to and act on
SUR subunit and require ATP hydrolysis (Mg2+ dependent) at NBFs as non-
hydrolysable analogs of ATP are ineffective. The C-terminus of SUR was also found to significantly affect the binding affinity with relative affinity among different isoforms following the order: SUR2B ≈ SUR1 > SUR2A. Mg- nucleotides are essential for the action of KCOs (255). Pinacidil and cromakalim are more effective on vascular and cardiac KATP channels (vascular
> cardiac) as compared to pancreatic-isoform; while diazoxide is ineffective on cardiac sarcolemmal KATP channels. The effect of KCOs is increased by
intracellular decrease in pH, indicating that protons also modulate ATP-
sensitivity. Furthermore, lactate accumulation during ischemic conditions has been shown to activate KATP channels and shorten action potential duration;
however the effect was later found to be mainly due to acidosis caused by
lactate instead of lactate itself (24).
10 1.3.3. Regulation by signaling molecules
Under normal circumstances, KATP channels do not play any discernible
role in the heart as they are inhibited by high ATP concentrations inside the
cells. However, under the conditions of metabolic stress or ischemia, KATP
channel mediated outward potassium current increases rapidly, leading to APD
shortening (with decrease in calcium influx) and cardiac protection (93, 194).
This APD shortening and decrease in calcium influx helps in preserving ATP during energy deficient conditions. Moreover, using Kir6.2 knockout mice, it has been demonstrated that KATP channels play an important role of homeostatic
control in adaptation to stress (168, 320).
The normal physiological levels of ATP in a cell fluctuate between 6mM to 10mM (7, 193). Even under severe metabolic stress (when both oxidative phosphorylation and glycolysis are inhibited), intracellular ATP levels ([ATP]i)
fell down only by ~80% of control over the time (~5 mins) when significant APD
shortening occurs. At the same time, developed pressure is significantly
reduced and KATP channels are activated (7, 69, 194). On the other hand,
studies have consistently shown that for KATP channels, Ki(ATP) ([ATP]i causing
half maximal inhibition of channel activity) in cardiac myocytes is 100 µM in
excised inside-out patches (197) and ~500 µM in open-cell attached patch and whole cell configurations (76, 136). This wide discrepancy between the
observations obtained in intact cells and in any of the patch configurations
studied, points out that the regulation of KATP channels is probably much more
complex than can be explained by the simple hypothesis of opening by bulk 11 decrease in [ATP]i (192). An open question in the field is what exactly makes
these KATP channels open up at millimolar concentration of ATP inside an intact cell (or in-vivo conditions) when even micromolar amounts are enough to inhibit the channel?
Recent studies have shown that besides nucleotides KATP channels are
also regulated by a large number of other modulators. These modulators
include adenosine (114, 274), bradykinin (196), acetylcholine (274), and
adrenergic agents (116, 251) which act through G-protein coupled receptors;
protein kinase C (112, 163); protein kinase A (164); nitric oxide (NO) (36); and
membrane phospholipids, especially PIP2 (21, 263). These modulators apparently regulate ATP-dependent gating of the KATP channels by decreasing
ATP-sensitivity and thus increasing open probability. Since many of these
mediators/ receptors are released or activated during metabolic stress
conditions, they might affect the channel microenvironment and thus alter the
ATP sensitivity (Grover and Garlid, 2000; Zhuo et al., 2005). However, there
might be “fuzzy space” like barriers to diffusion (below the sarcolemma) which
would slow the access of modulators to KATP channels (156). Therefore, the
spatial and temporal localization of these modulators should be very close to
KATP channels to realistically explain the rapid activation of KATP channels
during metabolic stress. In other words, it may be possible that this signaling is compartmentalized. There are no reports of KATP channel compartmentalization
in the literature, even though the KATP channel microenvironment is considered to be different from “bulk” cytosol. One of the reasons for this different local 12 environment is that these channels exist in mutlimolecular complexes with
many metabolic enzymes: glyceraldehyde-3-phosphate dehydrogenase, triose-
phosphate isomerase, pyruvate kinase (64) and adenylate kinase (39). In the
following sections, I will discuss some of the important regulators of KATP
channels and evidence implicating their compartmentalization either alone or in
combination with KATP channels on membrane microdomains.
1.3.3.1. Phosphoinositides (PIP2)
Phosphoinositides (in particular phosphatidylinositol-4,5-bisphosphate,
PIP2) regulation of ion channels was discovered about a decade ago for the
+ 2+ first time in KATP channels and Na /Ca exchanger in cardiac myocytes (110).
Similar results were later found in skeletal muscle, pancreatic β-cells and
recombinant KATP channels (72). Later on, many other ion channel targets of
2+ PIP2 were discovered like other Kir channels, Kv channels, L-type Ca
channels, TRP channels and CFTR etc (111). PIP2 is produced by the
phosphorylation of phosphatidylinositol (PI) and phosphatidylinositol-4-
monophosphate (PI-4P) on the plasma membrane by the sequential action of
PIP-4-kinase (PIP4K) and PIP-5-kinase (PIP5K). This PIP2 is further
hydrolyzed by receptor activated Phospholipase C (PLC) or dephosphorylated by inositolpolyphosphate phosphatase.
PIP2 activates KATP channel current by increasing the open probability to
almost double of the original value in the absence of ATP. Moreover, using
recombinant channels, it was shown that 10 min exposure of 5 μg/ml of PIP2 to 13 the cytosolic side decreased the ATP sensitivity by 340 times, made the
channel activity stable and increased the run-down time constant. Similar
values (100-700 fold) for shift in ATP sensitivity were obtained in native cardiac
and pancreatic cells. This may in part explains the opening of KATP channels at
physiological ATP levels. It was also shown that the effect is mediated by direct
binding of PIP2 to the Kir6.2 subunit, which is influenced by the SUR subunit. A
negatively charged head group and a lipid tail were found to be essental for effect as inositol trisphosphate (no lipid tail) or phosphatidylcholine (no
negatively charged head group) were ineffective; while PIP2, with three
negative charges, is more effective than PIP (which has two), and PI (which has only one) is ineffective (12, 21, 263). However, the exact kind of interaction between PIP2 and ATP on Kir6.2 subunit is ambiguous, as it has been shown
that the phosphate groups of PIP2 and ATP may compete for the same binding
site (262), but others have shown there are also non-overlapping positive
residues which mediate binding (235, 253). However, using homology
modeling and ligand docking, a recent study proposed that a direct competition
for same binding site is highly unlikely. Instead an allosteric model was
proposed, where PIP2 binding site is just above the ATP binding site, which can explain the experimental observations at large (236).
1.3.3.2. Protein kinase C
Phosphorylation of ion channels (including KATP channels) by kinases
plays an important role in regulating channel function by G-protein coupled 14 receptors (GPCRs). Though a variety of protein kinases have been shown to
affect cardiac KATP channels, the best characterized is the phosphorylation by
PKC (163). PKC is a family of serine-threonine protein kinases that depend on
lipids for activity and consist of atleast 12 isozymes. They are divided into three
subfamilies, based on their structure and second messenger requirements:
conventional or cPKC (α, βI, βII and γ) require Ca2+ and diacylglycerol (DAG)
for their activation, novel or nPKCs (δ, ε, η and θ) require DAG but not Ca2+
whereas atypical or aPKCs (λ, ι, ζ and μ) require neither Ca2+ nor DAG for
activation. GPCR which couple with Gq, activate enzyme PLC-β which causes hydrolysis of phosphatidylinositol 4,5-biphosphate into IP3 (Inositol 1,4,5-
phosphate) and DAG (Diacylglycerol). IP3 provides the second messenger of
this pathway by releasing Ca2+ from intracellular stores while DAG remains
embedded in the membrane and either activates the already membrane-bound
PKC isoform or provides the docking site for activated PKC from cytosol (33).
There are many potential PKC phosphorylation sites in both Kir6.2 and
SUR2A subunits (257). Of these sites, phosphorylation at a specific conserved
site, Thr-180 of Kir6.2 is the major site of PKC phosphorylation and is mainly
responsible for stimulatory effect of PKC on KATP channel (163). However,
prolonged stimulation (more than 15 minutes) of PKC can cause internalization
of KATP channels (113). Though, which PKC isoform is responsible for cardiac
KATP channel phosphorylation is not clear, majority of evidence points toward a
crucial role of PKCε. Various studies using KO or transgenic mice using either
deletion or overexpression of PKCε respectively, have clearly established its 15 role in cardioprotective (33). PKCε is the major isoform present in the adut
heart (27), and a membrane-bound, phorbol ester-sensitive,
compartmentalized pool of PKC (distinct from membrane-translocated PKC)
which includes PKCε and PKCδ has been detected in cardiomyocytes even
under non-stimulated conditions (27, 28). On activation, PKCε has been shown
to translocate to Z-line/T-tubule region of myocytes (region rich in ion channels,
anchoring structural proteins and signaling molecules) and bind to it much
more effectively as compared to PKCδ (66, 117, 118, 238, 239). Moreover, a
subset of PKC isoforms including PKCε are present in cardiomyocyte caveolae
at very low levels under basal, and much higher levels under PMA stimulated
conditions (185, 242). In cardiomyocyte, both caveolin-3 and RACK (receptor
for activated C-kinase) proteins can anchor PKC in caveolae. However, the
identity of PKC substrates in caveolar domains is unknown at present. Two
recent studies have shown that for vascular KATP channels, PKCε is responsible for inhibition of KATP current via caveolae dependent mechanism
(128, 244).
Likewise PKCε has also been linked to cardiac KATP channels in signal
transduction pathways mediating ischemic preconditioning (4, 33). Using whole cell patch clamp studies, it was found that PKCε primes the sarcKATP channel in
guinea pig myocytes to open by isoflurane, which is preconditioning-mimetic,
while PKCδ was not that effective (4). A majority of preconditioning studies
implicate mitoKATP channel as the primary candidate for the cardioprotective
phenomenon, and PKCε has been shown to be present endogenously in 16 mitochondria (15, 50) and also translocate to mitochondria (4, 201) on PC
stimulus. Accordingly, it was proposed that mitoKATP might be the primary target
of PKCε. Because of the complexity and redundancy of ischemic
preconditioning pathways, it is not clear whether PKCε is upstream (49, 125),
downstream (201, 293) or in positive feedback loop with mitoKATP. Some
studies have even proposed mitoKATP channel to act as a RACK for activated
or endogenous PKCε in mitochondria (133). Regardless, delineation of the
exact nature of relationship between PKCε and mitoKATP, has to await the
determination of molecular identity of mitoKATP channel (33).
1.3.3.3. Subsarcolemmal ATP
The concept of subsarcolemmal “fuzzy space” was first proposed to
conceptualize the observation that excitation–contraction (E–C) is possible
even in the absence of the L-type Ca2+ current, provided there was influx of Na+
via the Na+ channel. It was speculated that there is a diffusion-restricted
+ + subsarcolemmal space (“fuzzy space”), where [Na ]i is elevated (by Na influx via the Na+ channel) to such a level that NCX is activated in reverse mode
causing Ca2+ influx (19, 155, 156). These observations were later supported by
many studies which have been reviewed recently (19, 285). Similarly, the subsarcolemmal space in cardiac myocytes is an area which is envisaged to be quite different from bulk cytosol in terms of ATP levels depending on rate of production, rate of diffusion and rate of consumption (129, 135). In myocytes, there are many plasma membrane ATPases (e.g. ion channels, ion exchangers 17 and membrane pumps) which maintain the local ATP/ADP ratio quite distinct
from bulk cytosolic levels. Besides, molecular crowding in this space also restricts the free exchange of nucleotides.
In a highly compartmentalized cell like myocyte, elaborate mechanisms
exists which ensure high free energy of hydrolysis of ATP in all compartments
whether center or periphery of the cell. In cardiomyocytes, mitochondria
constitute ~30-35% of the cell volume and are regularly arranged in a longitudinal lattice between myofibrils along the length of the sarcomere (208,
283). Two populations of mitochondria have been described namely, subsarcolemmal (located beneath plasma membrane) and interfibrillar (located between myofibrils). Except for some minor biochemical differences, no major functional or morphological differences were found between these two
populations. It is speculated that interfibrillar mitochondria provide much of the
ATP required for contractile machinery, while subsarcolemmal mitochondria
cater to the membrane pumps and channels (209). To ensure smooth ATP
delivery at all times, muscle cells have also evolved an ATP-shuttling
mechanism via creatine kinase (CK) (ATP+Cr<=>Cr-P+ADP) and adenylate
kinase (2ADP<=>ATP+AMP) which rapidly equilibrate and maintain high ATP
levels in all parts of the myocyte (1, 140).
However, evidence is accumulating indicating that membrane ATPases
including KATP channels preferentially use glycolytic-ATP over mitochondrial-
ATP (65, 298). Studies using CK knockout mice (either mitochondrial or
cytosolic isoform, or both) have shown that these mice have normal cardiac 18 functions under at least moderate workload. This indicates that probably the
dependence on this ATP shuttling system is not absolute, especially for
membrane pumps which are far from mitochondria (249, 250). Secondly, when
cells were perfused with respective substrates for glycolysis, oxidative
phosphorylation or creatine kinase, they supported membrane and contractile
functions with different competencies (23, 174, 297). Finally, many studies
have shown that KATP channels (which act as metabolic sensors) are mainly
regulated by glycolytic ATP and metabolites. It has been shown that in
permeabilized myocytes; glycolytic substrates (with mitochondrial inhibitor) in
the presence of external ATP consuming system were far more competent in
inhibiting KATP channel activation as compared to substrates for mitochondrial
oxidative phosphorylation or creatine kinase system (296). Many glycolytic
enzymes like GAPDH (glyceraldehydes phosphate dehydrogenase),
triosephosphate isomerase, and pyruvate kinase have been shown to be
associated with KATP channel subunits using co-immunoprecipitation, yeast
two-hybrid screen or patch clamp studies (53, 64). Similarly, muscle isoform of
lactate dehydrogenase (M-LDH) (52), adenylate kinase (39) and creatine kinase (53) also associate with KATP channels. These proteins would either bind
directly to KATP channel subunits or indirectly via some scaffolding protein. In
this regard, it is interesting that some of the glycolytic enzymes, namely
phosphofructokinase-M (PFK) and aldolase, have been shown to be present in
caveolae in vascular smooth muscle. Also, in skeletal muscle, this
compartmentalization of PFK-M was shown to involve caveolin-3 interaction 19 and is glucose dependent (229, 252, 267, 282). Since the role of caveolae in
integrating signal transduction pathways is well-known, it is quite possible that
caveolae could be one of the hot spots for metabolic integration of diverse
signals as well (37, 73, 200).
There is a strong evidence implicating physical and functional
compartmentalization of glycolytic enzymes at sarcolemma and sarcoplasmic
reticulum in cardiac muscle and other tissues (65, 91, 145, 222, 307). This
would place the energy generating mechanism closer to the site of utilization,
especially membrane ATPases and metabolic sensors like KATP channels. It is worthwhile to note here that KATP channel microenvironment is considered to
be quite different from bulk cytosol and is secluded from brief oscillations in
cytosolic nucleotides levels (during normal APD). However, if there is any
major change in the cytosolic ATP, the ATP shutting mechanism complements
the metabolic signaling circuit by amplifying and conveying cellular energetic
state to KATP channels (1, 258). An interesting functional interaction between
KATP channel and Na/K-ATPase has been shown, where both compete for the same subsarcolemmal ATP (during metabolic stress) and regulates each other’s activity in the process (106, 135, 280). In fact, literature is replete with similar reports proposing that subsarcolemmal ATP is an important contributor to KATP channel regulation by various receptors like β–adrenergic (251), P2-
purinergic (14), endothelin-1 (295) and angiotensin-II receptor (281).
Interestingly, both pertussis toxin sensitive (Gi, increase ATP by inhibiting adenylate cyclase) and cholera toxin sensitive (Gs, decrease ATP by activating 20 adenylate cyclase) G-proteins were shown to be involved. It is important to realize here that such a regulation by subsarcolemmal ATP may not be tight enough during normal physiological functions but might have implications during metabolic impairment or ischemic conditions when mitochondrial supply of ATP is restricted.
1.3.3.4. Nitric oxide
The role of nitric oxide (NO) in cardioprotection during ischemia and in
ischemic preconditioning is well-known (59, 230). The main source of NO in cardiomyocytes is endothelial nitric oxide synthase (eNOS), and NO released can further upregulate iNOS (inducible). NO activation of KATP channels in cardiomyocytes is thought to involve cGMP-protein kinase G signaling pathway. NO acting via this pathway has been shown to activate sarcolemmal
KATP channels in gold-fish cardiomyocytes using patch clamp recordings (36)
and in intact hearts in various preconditioning studies (16, 186).
Pharmacological evidence also points towards a substantial role of mitoKATP
channels in NO-mediated cardioprotection (48, 247). Whether sarcKATP and mitoKATP are regulated differentially or in parallel by NO is not clear. The source
of NO might play a crucial role in determining which subtype plays an important
role. Although nitric oxide released from endothelium can diffuse to larger
distances and may act in a paracrine fashion, it has been proposed that a small
and local release of NO by eNOS present in cardiomyocyte caveolae (in
21 association with caveolin-3) might act in an autocrine fashion on nearby proteins (40).
1.3.3.5. G-protein coupled receptors (GPCRs)
KATP channels in cardiomyocytes are regulated by a variety of neurotransmitters and hormones via GPCRs namely adenosine (114, 274), bradykinin (196), acetylcholine (274), and adrenergic agents (116, 251). These
GPCRs are coupled to heterotrimeric G-proteins (Gαβγ). Upon activation by ligand, GPCRs promote exchange of Gα-bound GDP for GTP, which in turn favors the dissociation of this trimeric complex into Gα-GTP and Gβγ subunits.
Both of these active signaling molecules modulate various target effector systems and can modulate a majority of ion channels indirectly (via second messenger system pathways) or by direct interaction of ion channel subunits with G-protein subunits without the involvement of diffusible cytosolic factors
(membrane delimited activation by Gα or Gβγ). The Gα subunit has intrinsic
GTPase activity which terminates activation by hydrolyzing GTP to GDP and promoting the reassociation of Gα with βγ subunits.
Indirect G-protein regulation
The G-protein regulation of ion channels via second messengers is the most common and widely investigated pathway. GPCRs regulate multiple second messenger pathways namely: adenylyl cyclase (cAMP-PKA), guanylate
cyclase (cGMP-PKG), phospholipase C (PIP2→DAG/IP3/PKC), phospholipase
22 A (arachidonic acid), receptor tyrosine kinase etc. The relevant pathways pertaining to regulation of KATP channels have been discussed before.
Membrane delimited direct G-protein regulation
The membrane delimited G-protein activaton of ion channels has been firmly established only for some members of voltage-activated calcium
channels (Cav) and G-protein activated inwardly rectifying potassium channels
(GIRKs). Interestingly, Gβγ inhibits Cav while it activates GIRKs (Kir3). Since
there are membrane-delimited G-protein dependent pathways like membrane
bound PKC activation, PLC activation and PIP2 generation, a set of criterion
has been proposed to distinguish direct G-protein regulation of an ion channel
(57). These criteria need to be satisfied for an ion channel to be characterized
as a direct G-protein regulated ion channel.
Criterion 1. Purified or over-expressed G-protein subunit replicates the effects
of GPCR-ligand; while deletion or withdrawal of the subunit using binding-
peptide or antibody abrogates the effect of the ligand.
Criterion 2. G-protein subunit act in a membrane-delimited fashion (using
‘inside-out’ or ‘cell-attached’ patch configuration).
Criterion 3. G-protein subunit binds directly to channels protein either in-vitro or preferably in-vivo (using co-precipitation or fluorescence resonance energy transfer (FRET)).
Criterion 4. Identification of a G-protein binding site on ion channel protein
(using mutational or competition assays).
23 Extensive evidence points out a role of pertussis toxin sensitive G-
proteins, Gαi (αi1, αi2, αi3) in the activation of KATP channels via GPCRs in native
cells and recombinant channels in transfected cells (246, 274). Studies using
excised inside-out patches in guinea pig myocytes have shown that GTP (in
the presence of external acetylcholine and adenosine), GTPγS (non-
- hydrolysable analog of GTP), AlF4 (mimics the action of terminal γ-phosphate
of GTP on the GDP-bound α-subunit) and purified G-protein subunits (αi1, αi2,
αo), all antagonize the ATP-dependent inhibition of KATP channels (274). Similar
results were obtained in other studies for Gαi (143, 246), while conflicting evidence was presented for Gβγ showing either inhibition (122) or no effect
(246).
A recent study (287), however, made a strong case proposing a role of
Gβγ for the direct activation of KATP channels. These authors expressed
recombinant channels (Kir6.2/SUR) in COS cells and showed that Gβγ directly
activates KATP channels by reducing ATP inhibition. Using in-vitro binding assay, these authors provided conclusive evidence by mapping the Gβγ
interaction site to both nucleotide binding loops (NBD1 and 2) of SUR subunit.
They also found that a single arginine residue in one of the binding segment
(656 in SUR2A and 665 in SUR1) is essential for the functional effect.
However, it does not mean that Gα-subunit does not play any role.
Since the affinity for G-protein subunits in the excised patches was
found to be very high, 5-10 pM for Gβγ and 30-50 pM for Gαi (much higher than
GIRK channel or adenylyl cyclase, respectively), it has been speculated that 24 “KATP channels might be already saturated by G-proteins at their ‘basal’ levels”
(57). Or it may be possible that KATP channels are compartmentalized with the respective GPCRs within the membrane microdomains ensuring efficient coupling with G-protein subunits. Nonetheless, compartmentalization of many
GPCRs with their heterotrimeric G-proteins and effectors in caveolar microdomains has been shown recently and is firmly established (17, 120,
153).
Adenosine
Adenosine is the main mediator released during ischemia due to alteration in degradation and regeneration of AMP (by adenosine kinase). The main effect is possibly due to decreased regeneration. Adenosine mediated cardioprotection is very well established, though it is debatable which mediator and end-effectors are involved. In addition to the heart, adenosine mediated protection against ischemia has been shown in liver, kidneys and brain also
(60, 188).
Three main subtypes of adenosine receptors (AR) have been identified so far: A1, A2 (A2a and A2b) and A3, and all have been shown to be present in cardiomyocytes (79). Among these subtypes, cardioprotective effect and KATP channel activation is thought to be mainly mediated by either A1 or A3 or both and is a subject of intense debate. Both A1 and A3-AR are coupled to G- proteins (Gαi, Gαo or Gαq) and mediate either inhibition of adenylate cyclase
(AC) or activation of phospholipases and PKC (via IP3/DAG pathway).
25 Most of the evidence for the involvement of adenosine in KATP channel
activation is derived from preconditioning studies where glibenclamide inhibited
the adenosine mediated cardioprotective effect. These studies implicate either
Gαi or PKC as the second messenger in KATP channel activation. However,
PKC hypothesis is the focus of a lot of interest and probably a major contributor
to the cardioprotection and activation of KATP channels, and thus gained much
more prevalence in literature (47, 114, 163, 170, 188). Using KO mice, it was
shown that deletion of A1-AR increases ischemic damage and abrogates the
protective effects of both pre- and post-conditioning while transgenic
overexpression of A1-AR gives additional cardioprotection compared to
respective wild-type strains (151, 179, 303). There is abundant in-vivo and in-
vitro pharmacological data which implicate the involvement of A1-AR in
cardioprotection and KATP channel activation (98, 162, 188, 240, 312). On the
other hand, A3-AR KO mice were more tolerant to ischemic damage and have
intact intrinsic preconditioning response (100, 105). However, pharmacological
data using a recently available selective agonist for A3-AR proved otherwise
(88, 105, 289). There are only few studies which directly investigated the subtype of AR involved in activation of KATP channels using patch clamp
technique. A majority of these studies using selective antagonist showed that
A1 receptor antagonism inhibit the activation of KATP channels by adenosine
(114, 123, 124, 141, 143, 274). One recent study using selective A3-agonist
and A3-AR knockout mice implicates the involvement of A3-AR (289), though it
does not rule out any contribution from A1-AR. Possibly, both of these receptors 26 play a role in the activation of KATP channels, with A1-AR being the major contributor.
However, recent data paint a complex picture of adenosine mediated regulation of KATP channels. Though acute stimulation (~5 minutes) of AR activates KATP channels (114, 162), prolonged stimulation (15-30 minutes) causes internalization of KATP channels (113). Interestingly, A1-AR including the downstream signaling components (Gαi and PKC) has been shown to localize to caveolae in cardiomyocytes. Since, these A1-AR translocate out of caveolae upon 15 minutes of agonist stimulation (17, 153, 185, 242), it can be easily inferred that adenosine mediated regulation of KATP channels is probably more complex.
Noradrenaline
Noradrenaline released through sympathetic nerve stimulation in the heart causes strong ionotropic, chronotropic and dromotropic effects; increasing heart rate, strength of contraction and simultaneous reduction in action potential duration. The adrenergic regulation of many ion channel currents is well established like ICaL, INa, Ito and Ikr. However, there are very few studies investigating the adrenergic regulation of KATP channels, and the exact mechanism is unclear. Using isoproterenol as agonist and propranolol as antagonist, it was shown that β-AR (β-adrenergic recptor) can activate KATP channels in feline and guinea pig ventricular myocytes. Since this modulation was not mimicked by cAMP analog and was independent of PKA inhibition, it was proposed that isoproterenol activates KATP current via Gs, by consuming 27 subsarcolemmal ATP through the activation of adenylate cyclase activity (251,
279, 295). As mentioned before, this regulation might have more importance
during ischemic conditions as compared to normal physiological situations
(when ATP supply is abundant). However, a recent study has challenged this
view. Using isoproterenol and exercise challenge as physiological stressors,
these authors showed that though wild-type mice respond with reduced APD and more efficient calcium handling, this adaptive response was absent in
Kir6.2 KO mice. Under the same conditions, Kir6.2 KO mice were vulnerable to
electrical instability, dysregulated calcium handling and ultimately lethal
ventricular arrhythmias. Most importantly, no major shift in bulk adenine
nucleotide content was noted between normal or stressed conditions, indicating
a direct or indirect β-adrenergic mediated activation of KATP channels during
physiological conditions. Though detailed mechanism of KATP activation was
not investigated, a compartmentalized regulation of KATP channels by β-AR was
emphasized (320). Nevertheless, β-AR and Gs (including adenylate cyclase) have been shown to be present in caveolae (107, 108, 305). The presence of
2+ KATP channels in the same microdomains with β-AR and L-type Ca channels
would allow more efficient signaling and direct control of calcium influx (17).
Acetylcholine
All the five subtypes of muscarinic receptors (M1, M2, M3, M4, M5) have
been localized to heart, with M2 being the predominant one which mediates the
negative ionotropic and chronotropic effects. Though acetylcholine (ACh)
mediated Gβγ-dependent activation of IKACh and inhibition of ICaL is well 28 established, the exact role and mechanism of muscarinic receptors in KATP channel regulation is unclear. Previous studies have implicated both PTX- sensitive Gα subunit and PKC-dependent pathways in KATP channels activation
(226, 274). On the other hand, there are reports purporting inhibition of KATP channels by muscarinic receptor stimulation (295) involving similar mechanisms as described before including PKC activation, PIP2 depletion and augmentation of subsarcolemmal ATP (123, 294). As such, the cholinergic regulation of KATP channels is still ambiguous.
Bradykinin
Bradykinin (BK), primarily released from endothelial cells during ischemia, mediates cardioprotection via BK2 receptors on cardiomyocytes (22).
The signaling mechanism of BK in ischemic preconditioning has been linked to activation of KATP channels in mitochondria (218) and sarcolemma (67).
Though direct electrophysiological evidence for KATP current activation by BK is lacking, putative mechanisms include generation of prostanoids and nitric oxide or activation of PKC (22). BK2 receptors have been shown to be present in caveolae in endothelial, smooth muscle and cardiomyocytes (61, 132, 228).
Endothelin
It is believed that during acute myocardial ischemia, the majority of endothelin-1 (ET-1) comes from the heart, as isolated-perfused hearts also release significant amounts of ET-1. The effect of ET-1 on cardioprotection is complex, as it mediates protection at lower concentrations while higher doses have no effect (109). Using patch clamp analysis, previous studies have 29 consistently shown that ET-1 inhibits KATP channels via ETA receptors coupled
to PTX sensitive G-proteins by inhibiting adenylate cyclase activity and thus
increasing subsarcolemmal ATP (248, 295). However, later studies showed
that ET-1 is also a preconditioning mimetic and may act as trigger for ischemic
preconditioning by opening KATP channels subsequent to PKC activation. Direct
involvement of Gαi was also speculated, though the receptor subtype was not
investigated (34, 109). Inline with the lingering controversy for ET-1 role in
cardioprotection, the role of ET-1 in KATP channel regulation also needs
clarification and dissection in terms of specific subtype of receptor involved.
Further, it would be interesting to know if this signaling is compartmentalized or
not, as ET receptors and associated signaling partners have been shown to be
present in caveolae (29, 43).
1.4. Functional role of KATP channels
It’s been known for a while that KATP channels in the pancreas mediate
insulin secretion. Pancreatic KATP channels remain open under the basal
conditions and close when plasma glucose levels increase leading to insulin
secretion. Surprisingly, cardiac KATP channels are closed under the basal conditions. However, they open-up during metabolic stressed conditions (like ischemia) and play significant role in outward potassium influx and resultant action potential duration (APD) shortening. This is expected to lead to decreased calcium influx and decreased calcium overload and resulting energy sparing will have cardioprotective effect (93, 194). The APD shortening effect of 30 KATP channels during metabolic stress has been confirmed in Kir6.2 knockout
mice also (270). Since KATP channel are sensitive to micromolar amount of
ATP, the exact mechanism leading to KATP channel opening under ischemic conditions (when intracellular levels of ATP are still in millimolar range) is not very clear. The putative mechanisms and associated signaling are discussed in
detail in section 1.3.3.
The protective effect of KATP channel opening during
ischemia/reperfusion has been demonstrated using KCOs and selective KATP
channel blockers (glibenclamide) also. Interestingly, ‘ischemic preconditioning’,
a cardioprotective phenomenon can be mimicked by KCOs and inhibited by
sulphonylureas (47). Ischemic preconditioning (IPC) is the protection of
myocardium from prolonged ischemic damage by a prior, brief and reversible
ischemic insult. The phenomenon originally discovered by Murry in canine
model in 1986 (189) has since been shown to occur in almost all other species,
including humans, and can be demonstrated in single cells and in isolated
perfused hearts in-vitro. Earlier, much emphasis was on two main types,
namely, early or classical preconditioning (effective within 2-3 hours) and
delayed or late preconditioning (effective between 24 to 96 hours). However,
recent intense effort is being directed towards a new-type called ‘post- conditioning’, which is thought to be more clinically relevant (207). Originally
shown as a reduction in infarct size in the heart, other end-points of IPC
protection include decrease in apoptosis and necrosis, decrease in post-
ischemic arrhythmic events, improvement in post-ischemic dysfunction and 31 decrease in stunning (a reversible and temporary loss of contractile ability that remains persistently evident despite restoration of coronary blood flow). There is wide variability and conflicting results in literature in terms of many correlates of IPC like duration and timing of preconditioning stimulus; duration of protection window; signaling pathways mediating IPC and; end-points of protection afforded by IPC. This variability could be due to interspecies variability and different model systems used in various studies (317). Because of redundancy and involvement of large number of signaling pathways in IPC
(207, 317), a comprehensive discussion is not possible here. So, I will mainly focus on relevance of KATP channels signaling in IPC. Since the discovery of the IPC, KATP channels have emerged as one of the prime candidates mediating the protective effects of IPC. It has been shown in many studies that
KCOs can mimic while glibenclamide can abolish the protective effects of preconditioning (94, 149, 150). Many mediators released during ischemia, like adenosine, bradykinin, prostacyclin and nitric oxide, have been shown to activate or facilitate opening of KATP channel (36, 114, 116, 196, 251, 274). The activation of PKC which is considered to be the key player for many signaling pathways (47) has also been linked to activation of KATP channel (discussed in section 1.3.3.2). Recently, Kir6.2 knockout mice have been shown to be resistant to the protective effects of preconditioning, indicating their importance in IPC. However, these knockout mice had more intense and rapidly developed contractures and decreased functional recovery in the absence of IPC stimulus than the wild-type controls. Though this indicates their prime role in protection 32 during ischemia-reperfusion, it is possible that ischemic injury is artefactually
enhanced and IPC effect is nullified (270). Nevertheless, there are studies
showing negligible role of KATP channels in IPC (169). As will be discussed
later, evidence also implicates the presence of another isoform of KATP
channels in mitochondria (mitoKATP) (119) which might mediate the protective effects of IPC either alone or alongwith sarcolemmal KATP (sarcKATP) channel .
Since most of the studies were done in healthy and young animals, it is further
debatable whether IPC can occur or is preserved in senescent or diseased
heart (207).
The role of KATP channels in arrhythmias is probably the most
controversial aspect currently. Since KATP channels shorten APD, they should be antiarrhythmic as they hyperpolarize the membrane, decrease calcium overload and decrease ischemic injury. Paradoxically, there is always a theoretical possibility for reentrant arrhythmias on the background of ischemic substrate, though experimental evidence is limited (41). It can be argued that the propensity of KATP channel to act as antiarrhythmic or arrhythmogenic
depend on the type of arrhythmias whether long QT arrhythmias or reentrant
arrhythmias respectively (190, 234). As mentioned earlier, SUR1 is recently
shown to be an essential component of sarcolemmal KATP channels in atrial
myocytes but not in ventricular myocytes in mice. Same group also showed that transgenic mice overexpressing SUR1/Kir6.2 containing KATP channels in
the heart were prone to arrhythmias including atrial fibrillation. Based on the
findings, it was speculated that SUR2 is cardioprotective while SUR1 is 33 arrhythmogenic (77). Nevertheless, several studies have demonstrated the protective effects of KCOs in prevention of post-ischemic arrhythmias (46, 134,
173).
Even though the role of cardiac KATP channel in ischemic conditions is well-characterized, their role under normal physiological conditions is just beginning to unravel. Recent studies (320) have shown that KATP channels in the heart may not be as silent as assumed previously. Though Kir6.2 knockout mice seem to develop normally, these mice have significantly less exertional capacity; less cardiac performance, disruptive calcium handling and increased vulnerability to lethal ventricular arrhythmias under sympathetic challenge. The metabolic demands of the sympathetic stimulation put a significant demand on the energy resources of the heart (increased cellular Ca2+ overload/ handling and associated energy depletion). To maintain proper cellular homeostasis, this requires some degree of fine tuning between the adequate escalation of Ca2+ influx and increase in compensatory outward hyperpolarizing current (via KATP current). This indicates that KATP channels play an important role of homeostatic control in adaptation to acute stress (168, 320).
Sarcolemmal vs. mitochondrial KATP channels
A detailed dissection of KCOs action in preconditioning (PC) led many to believe that channels on the sarcolemmal membrane (sarcKATP) may not be the only site of action of these drugs. Using a low-dose KCO bimakalim, it was established that cardioprotection is independent of APD shortening in dogs
(311). Further, protection can be shown in isolated non-beating cardiomyocytes 34 in culture (10). Much of the attention shifted to mitochondria, once it was discovered that there are KATP channels (mitoKATP) in inner membrane of mitochondria (IMM) (119). Using proteoliposomes containing reconstituted KATP channels from bovine heart mitochondria, it was found that KCO diazoxide is
2000 times more selective for mitoKATP channel as compared to sarcKATP channels (87). A selective mitoKATP inhibitor, 5-hydroxy decanoate (5-HD), was also identified. HMR1098, which is selective for cardiac sarcKATP channels, was found to be minimally effective on mitoKATP. Based on these findings, two groups independently showed that mitoKATP may be the prime target of KCOs induced PC (86, 171).
Most of the evidence available for the existence of mitoKATP is based on: mitoKATP selective pharmacological agents; use of antibodies; and three main techniques to study its properties namely, patch clamp, flavoprotein fluorescence and spectrophotometeric measurement of mitochondrial swelling.
Many properties of the mitoKATP resemble those of sarcKATP channels, although some significant differences have been noted-especially the selectivity of respective pharmacological agents (198). MitoKATP channels exhibit similar inhibition by ATP (Ki ≈ 0.8 mM) when applied from the matrix side and exhibit sensitivity to 4-amino pyridine (a K+ channel inhibitor) and glibenclamide.
MitoKATP channel conductance was found to be well below the sarcKATP channel conductance (10 pS in 100 mM cytosolic and 33 mM matrix K+) (119).
However, a recent report using reconstituted human inner mitochondrial membrane (IMM) in lipid bilayer found that there could be multiple conductance 35 states depending on the concentration of ATP and other nucleotides (127).
Over the years, many investigations have been carried out to elucidate the
molecular composition of mitoKATP channels without any concrete results. Since
the subunit composition of sarcKATP channels is known for a while, it was
hypothesized that probably mitoKATP might share the same composition. Using
antibody approach, it has been shown by many that varied amounts of both
Kir6.2 and Kir6.1 are present in cardiac mitochondria, but of course, with
conflicting results (54, 147, 256, 265, 268). A recent study investigated specificity of the currently available commercial Kir6.1 antibodies using mass spectrometer proteomics approach. They concluded that most of them bound
only to non-specific mitochondrial proteins at best and could be one of the
reasons for discrepancy in literature (78). However, using DEAE-affinity
column, a 54 KDa protein was isolated from mitochondrial fraction and was
tentatively identified as mitoKATP subunit (216). Further, low affinity binding sites for sulphonylureas have also been identified in purified mitochondrial fractions.
It is not clear whether these binding sites represent the already identified SUR
clone or other minor splice variant (97, 272).
The physiological role of mitoKATP (198) is thought to be mitochondrial
matrix volume control via counterbalancing mitochondrial uniporter activity with
K+/H+ exchange. This volume control is thought to be important for controlling the redox-state of the mitochondria (K+ influx is supposed to decrease
mitochondrial potential and decrease Ca2+ overload) and also prevent bursting
of the mitochondrial membranes during pathological swelling as happens 36 during ischemic-reperfusion injury. Another leading hypothesis is the
generation of reactive oxygen species (ROS) after mitoKATP opening which can
further activate many other protective signaling pathways including activation of
PKCε which can also be redox-activated (discussed in section 1.3.3.2). It is
hypothesized that mitoKATP opening causes increased protective ROS during
PC and inhibits pathological post-ischemic ROS generation. A recent study
investigating the mechanism of PC has given credence to the hypothesis that
mitoKATP channel opening and consequent mitochondrial swelling and ROS
generation are important components of the memory of PC (133).
However, it does not mean that sarcKATP channels do not play any role.
Concerns have been raised on the selectivity of so called ‘mitoKATP selective’
pharmacological agents (198, 317). These mitoKATP-selective agents have
been shown to act on many other mitochondrial targets significantly affecting
the function of mitochondrial respiratory enzymes (68, 103). Possibly, both sarcoplasmic and mitochondrial KATP channels play complementary roles in the protection of intact beating heart (96). Nevertheless, 17 years after its discovery (119), a uniform consensus for the molecular identity of mitoKATP is
not established and has to await cloning of the channel subunits.
37 2. Caveolae
2.1. Caveolae and caveolins
Discovered in 1950s (309) as the 50-100 nm flask-shaped invaginations
of the plasma membrane, caveolae, are now firmly established as a subcellular
organelle. Caveolae are a special type of lipid rafts (sterol- and sphingolipid-
enriched domains of the cellular membrane) tagged on the inner-leaflet of the membrane by caveolin proteins. Caveolae are found in almost all cell types
(except neurons and lymphocytes) and are most abundant in terminally- differentiated epithelial cells, endothelial cells, adipocytes and muscle cells
(232).
Though the techniques to specifically label raft structures for imaging are
controversial (121), caveolae can be labeled by their marker proteins,
caveolins. Caveolae are considered to be insoluble in non-ionic detergents at
low temperature because of the tight packing of cholesterol and spingolipids.
Since detergents can promote non-physiological interactions by raft-fusion, detergent-free extraction procedures are currently employed to isolate caveolae from cellular fractions (261, 266). They can be isolated by their buoyant density on sucrose-gradient.
There are four isoforms of caveolins: Cav-1 (α and β), 2 and 3, all of which have been shown to be present in cardiac myocytes. They are all small proteins of molecular weight ranging between 20-25 KDa. Cav-1 and 3 are most identical (65%) while Cav-2 is most divergent (38% identical to human
Cav-1). Cav-2 is dependent on Cav-1 for full expression and can not form 38 caveolae on its own. Cav-3 is the predominant isoform present exclusively in muscle tissue only (107, 273). Deletion of Cav-1 in most cell-types and specifically Cav-3 in muscle cells leads to loss of caveolae in respective cell- types. The molecular structure of caveolins is unusual with N and C terminus in the cytosol and a hairpin domain which traverses only the inner-leaflet of membrane (Fig. 1.3). They are synthesized as membrane proteins in endoplasmic reticulum (ER) and once in the golgi-complex go through first stage of oligomerization (~14-15 monomers). At some point during the exit from golgi, they associate with cholesterol and arrange themselves into higher order detergent-resistant complexes (complexes of ~10-11 oligomers) of ~350-400
KDa (210). Besides plasma membrane, studies have found Cav-1 in some intracellular structures like secretory vesicles of some exocrine and endocrine cells and also in mitochondria from airway epithelial cells (161). A detailed investigation for subcellular location of Cav-3 is lacking but it is thought to play an important role in developing T-tubules. Nevertheless, in adult cardiac myocytes, caveolin-3 is present in T-tubules and sarcolemma (107, 211, 266).
39
Figure 1.3. Topology and predicted membrane insertion of Cav-1. For simplicity a dimer of Cav-1 is shown. Both N- and C-terminal have membrane attachment domains and are exposed to cytosol. Most of the C-terminus is associated with membrane and is extensively palmitoylated as shown. The yellow color hairpin loop can be seen traversing the lipid membrane, but is not exposed to extracellular side. The position of the caveolin scaffolding domain
(CSD) is also shown. Adapted from Williams and Lisanti, 2004 (300).
40 2.2. Functional role of caveolae/caveolins
Vesicular trafficking
Caveolae play an important role in endocytosis and transcytosis of some
proteins. They are relatively immobile and stable structures on the plasma membrane. However, this immobility is significantly altered by cholesterol
depletion and various agents. Some viruses like SV40 and polyoma virus as
well as sterols and glycosphingolipids can initiate caveolar budding and are
endocytosed by caveolae. Interestingly, many components of endocytosis are
shared with other endocytic pathways like requirement of dynamin, actin
cytoskeleton and Rab5 etc (217, 259). After endocytosis, these lysosome-
resistant caveosomes (equivalent to endosome) can be directed towards a
variety of destinations, including recycling back to the plasma membrane.
Though a preferred route of endocytosis for the above mentioned agents or
some of the caveolar-resident proteins (CRPs), CRPs are not entirely
dependent on caveolae and can follow other endocytic pathways in their
absence (142).
Lipid homeostasis
Caveolins have high affinity for, and bind to cholesterol and fatty acids.
Cholesterol depletion/binding agents like methyl-β-cyclodextrin (MβCD), nystatin or filipin can flatten caveolae (175, 241). Cav-1 has been found to be associated with lipid droplets, and Cav-1 overexpression has been shown to increase fatty acid uptake and increase free cholesterol levels and export (80,
181). Since, caveolae are extremely abundant in adipocytes, the main storage 41 sites for fats, Cav-1-/- mice show decreased cholesterol in lipid droplets in
adipocytes. These mice show an interesting lean phenotype, have defect in
insulin-stimulated lipogenesis, and are resistant to atherosclerotic lesions or
diet-induced obesity (37, 231). These mice have highly impaired liver
regenerative response, with reduced lipid droplets in hepatocytes and low
survival rate, indicating the potential importance of Cav-1 in lipid homeostasis
(74). Further, cholesterol levels have been shown to control Cav-1 expression
(102). Thus, a unique relationship exists whereby both regulate each other’s
level of expression.
Tumor suppression
The role of caveolin in tumor suppression dates back to 1989 when Cav-
1 was first discovered as a tyrosine phosphorylated protein from v-Src-
transformed fibroblasts (89). It was later established that transformation of NIH
3T3 cells leads to decreased Cav-1 expression and loss of caveolae.
Overexpression of Cav-1 in H-Ras G12V-transformed cells can reverse transformation and prevent anchorage-independent growth (144). Accordingly,
Cav-1 is thought to recruit and organize proteins at focal adhesions for substrate attachment (62). The phenotype of Cav-1-/- mice support their role in
tumor suppression. Though these mice do not form spontaneous tumors, they
are highly vulnerable to oncogenic stimulus (38). This illustrates their role in
cell-cycle regulation and transformation.
42 Compartmentalized signal transduction: caveolae as signalosome
The ‘caveolae/raft signaling hypothesis’ was first proposed (165) based on the observation that a large number of signaling molecules are present in caveolae at much higher concentrations as compared to bulk plasma membrane. It states that caveolae serve as structural platform to specifically segregate and concentrate selective signaling molecules. It was hypothesized that this will lead to more efficient, selective and regulated signal transduction events instead of random chance interaction of signaling molecules. Over the years, most of the findings have corroborated this hypothesis to a large extent as an array of proteins like many G-protein coupled receptors (including their downstream components), kinases, adaptor proteins, structural proteins and ion channels have been preferentially localized to these membrane microdomains (120, 232, 316). Some of those molecules worth mentioning here are adrenergic receptors (β2>>β1 and α1), bradykinin receptor (B2),
adenosine receptor (A1), muscarinic receptor (M2), PKC (α, β and ε), PKA,
eNOS and Phosphoinositides and PI-kinases. Caveolin proteins act as
scaffolding proteins for many of these and regulate their functions. After it was
shown that they inhibit G-proteins in GTP hydrolytic assays (160), many other
proteins like eNOS, PKC isozymes, Src family tyrosine kinses and H-Ras were shown to be inhibited by interaction with caveolins. Though there are few
exceptions, the general inhibitor role goes well with the tumor suppressor role
of Cav-1 (214, 232).
43 An important question would be what mediates the localization of these
proteins to caveolae or lipid rafts. Though there is no absolute criterion, some
generalities have been found in many of these proteins. Many of these
caveolar-resident proteins (CRPs) have lipid modifications (lipid-protein
interactions); protein-protein interaction with caveolar resident scaffolding
protein; direct interaction with caveolins (120, 177). Till now experimental
evidence is limited to and has favored the third hypothesis. A scaffolding
domain in caveolin-1 (CSD) has been identified (160) which is capable of
binding with a broad array of signaling molecules. Analogous to Cav-1, a highly homologous CSD is present in Cav-3. Using CSD of Cav-1 as bait, consensus motifs were identified in some of the caveolar resident proteins termed as caveolin binding domains (CBD). These motifs are: ΦXΦXXXXΦ,
ΦXXXXΦXXΦ, ΦXΦXXXXΦXXΦ, where Φ means an aromatic residue (Phe,
Tyr, or Trp), and X means any amino acid. Most of the CRPs have at least one
copy of any of these motifs, indicating that this may be the site of direct
interaction (51). Nevertheless, presence of CBD does not mean that the protein
resides in caveolae, and absence does not negate caveolar localization.
Probably the most interesting insight in the caveolar field has come from
the investigations of dynamic interaction of GPCRs with caveolae before and
after ligand stimulation. It was shown that β2-adrenergic receptor and A1-
adenosine receptor (preferential CRPs) exit caveolae after agonist stimulation
(153, 205). On the other hand, the agonist stimulation of B1-, B2-bradykinin
receptors and M2-muscarinic receptor translocate them into caveolae (75, 243). 44 This was further shown to be important for their respective fates after
stimulation like internalization and termination or activation of signaling. This
kind of dynamic interaction was later found to have general applicability to
other signaling molecules too and further established the role of caveolae as
dynamic signalosomes (232).
2.3. Compartmentalization of ion channels
2.3.1. Cardiac ion channels
Though Cav-3 is the predominant isoform expressed in cardiac, skeletal
and vascular myocytes, current evidence has now established that Cav-1 and -
2 are also present in significant amounts in all three cell types (108, 237).
Though it is not clear whether Cav-1 and Cav-3 have over-lapping roles or not,
Cav-3-/- mice lack identifiable caveolae from muscle cells. These mice show severe skeletal muscle abnormalities with muscle degeneration and T-tubule abnormalities (83, 101). These mice also develop severe cardiomyopathy by 3-
4 months of age, with hypertrophy, dilation and reduced fractional shortening.
Analogous to the inhibitory role of Cav-1 in other cells, Cav-3-/- mice show
hyperactivation of the Ras-p42/44 MAPK cascade in cardiomyocytes (301).
In cardiac myocytes, compartmentalization of many ion channels or
exchangers (like voltage dependent Na channel (hNav1.5), voltage dependent
+ 2+ potassium channel (Kv1.5), the Na /Ca exchanger, the pacemaker channel
(HCN4), Na+/K+-ATPase, L-type Ca2+ channel) and signaling molecules (like G-
protein coupled receptors; G-proteins: Gαs, Gαi, Go, Gq; various types of 45 receptor and non-receptor kinases, GTPases, cellular proteins/adaptors,
structural proteins and other enzymes) have been demonstrated in lipid rafts
and caveolae (17, 18, 30, 177, 313). Cardiac muscle caveolae are very rich in
many of the already mentioned signaling molecules: adrenergic receptors (β2-
AR and α1-AR), bradykinin receptor (B2), adenosine receptor (A1), muscarinic
receptor (M2), PKC (α, β and ε), PKA, eNOS, nNOS, PIP and PIP2 (30, 35, 167,
175, 232). Caveolin-3 is present in T-tubules and sarcolemma of cardiac
myocytes (107, 211, 266) and modulates the function of many ion channels by
direct interaction.
The earlier work utilizing immunogold electron microscopy suggested the
lipid raft localization of sodium channels in electric eel and 1,4-dihydropyridine
receptor in skeletal muscle caveolae in rabbit (70, 130). Later on, a variety of
other ion channels from different ion channel families including potassium and
chloride were also found to localize to caveolae (175, 177). This raft/caveolae
localization of several ion channels was found to profoundly affect their
function. Since methyl-β-cyclodextrin (MβCD) depletes membrane cholesterol
and disrupts caveolae, it was extensively employed (along with other
cholesterol binding agents like nystatin, filipin) to study the functional effect
caveolar localization of ion channels. It was shown that MβCD treatment
decreased the frequency, amplitude and spatial size of Ca2+ sparks in
myocytes, indicating that caveolae might be the site for their formation (172).
MβCD treatment also decreased the amount of Na+/Ca2+ exchanger protein
(which has five potential CBDs) that can be co-immunoprecipitated with Cav-3 46 antibody (30). An interesting interaction between caveolae and cardiac sodium
channel (INa; Nav1.5) has been shown. Using Cav-3 antibodies it was shown
that Cav-3 inhibited the direct G-protein activation of sodium channels by
isoproterenol. It was concluded that caveolae increase the surface presentation
of sodium channels in response to β-AR receptor activation (313). Further, in
addition to MβCD treatment, siRNA against caveolin-3 also disrupted the β2-
2+ adrenergic activation of L-type Ca channel current while β1-mediated
activation remained intact (17).
Recent investigations have found that caveolins do play a direct role in
regulation of some ion channels, and this regulation is independent of caveolar
structure. Using Cav-1 scaffolding domain (CSD) in pipette solution, it was
found that Cav-1 exerts a strong inhibitory effect on Ca2+-activated K+ channels
(BKCa) in vascular endothelial cells (290). Following these observations,
another group showed that in human myometrial smooth muscle cells, actin
cytoskeleton is part of caveolar-complex regulating BKCa channels (31).
Overall, available data indicate the importance of caveolae and caveolin proteins in normal physiological functioning of ion channels and compartmentalization with respective modulators. Since dysregulated cell surface expression and functioning of ion channels is increasingly linked to ion channelopathies, understanding the lipid regulation of membrane excitability might provide new therapeutic avenues for those diseases.
47 2.3.2. KATP channels
Intriguingly, there are reports indicating that KATP channels in other tissues
do localize to caveolae. In vascular smooth muscle cells, KATP channels consist of Kir6.1 and SUR2B. These channels are tonically activated by cAMP- dependent PKA because of the constitutive activity of adenylyl cyclase. This
KATP current is essential in giving vasodilating drive to resting muscle tone
(245). It was found that caveolae integrity is essential for cAMP-dependent,
protein kinase A (PKA)-activated component of KATP current (Kir6.1/SUR2B).
Since diffusion of cAMP to distant sites is restricted by phosphodiesterases, it
was postulated that caveolae help in compartmentalizing the regulatory
pathways including adenylyl cyclase with vascular KATP channels. Further, it
was noticed that MβCD was unable to disrupt the interaction between Kir6.1
and Cav-3 indicating involvement of direct protein-protein interaction (245).
For pancreatic KATP channels (Kir6.2/SUR1), the evidence is less clear. In hamster pancreatic β-cells where Cav-1 is localized to both plasma membrane and insulin granules, it has been shown that both lipid rafts and caveolae are involved in insulin secretion (191). In a pancreatic β-cell line, it was shown earlier that both Kir6.2 and SUR1 are present in bulk-membrane fraction after detergent-free extraction and sucrose-gradient ultracentrifugation (304).
However, a recent study (225) demonstrated a direct protein-protein association between Cav-1 and Kir6.2 in a mouse pancreatic β-cell line.
Further, using siRNA mediated Cav-1 knockdown, same authors also showed that Cav-1 is essential for sulphonylurea or glucose induced insulin secretion 48 (225). Whether, the difference in two studies is related to species difference or
method of analysis is not clear. Other possibilities could be that KATP channels may associate with Cav-1 outside of caveolae; or the association and localization may depend on specific stimulation.
3. Objectives
ATP-sensitive potassium channels (KATP channels) play an important role in
cardioprotection under stress conditions. They link the cellular energetic state
to membrane excitability, and thus control Ca2+ influx. These channels are
evolutionary conserved across animal species and show protection under
various kinds of stressful conditions, especially under ischemic conditions.
Identification and elucidation of their subcellular localization and mechanism of
regulation in cardiac myocytes will be potentially useful in understanding the
endogenous cardioprotective mechanisms of the heart and their alteration in
some pathological conditions. Therapeutic strategies can be further designed
to enhance their cardioprotective regulatory mechanisms.
Accordingly, the objectives of the present investigation were three-fold,
each of which served as a basis for a submitted or published manuscript:
Hypothesis 1: Cardiac KATP channels (Kir6.2/SUR2A) reside in caveolin
enriched membrane microdomains and are regulated by caveolae-dependent adenosine receptor signaling.
49 Hypothesis 2: Caveolin-3 can functionally modulate the activity of KATP channels (Kir6.2/SUR2A).
Hypothesis 3: Kir6.2 containing KATP channels reside in mitochondrial
inner membrane and can be regulated by PKC activation.
50
CHAPTER 2
REGULATION OF ATP-SENSITIVE POTASSIUM CHANNELS BY
CAVEOLIN-ENRICHED MICRODOMAINS IN CARDIAC MYOCYTES
ABSTRACT
ATP-sensitive potassium (KATP) channels in the heart are critical regulators of
cellular excitability and action potentials during ischemia. However, little is
known about subcellular localization of these channels and their regulation. The
present study was designed to explore the potential role of caveolae in the
regulation of KATP channels in cardiac ventricular myocytes. Both adult and
neonatal rat cardiomyocytes were used. Subcellular fractionation by density
gradient centrifugation, Western blotting, co-immunoprecipitation and
immunofluorescence confocal microscopy were employed in combination with
whole-cell voltage clamp recordings and siRNA gene silencing. We detected
that the majority of KATP channels on the plasma membrane of cardiac myocytes were localized in caveolin-3-enriched microdomains by cell fractionation and ultracentrifugation followed by Western blotting.
Immunofluorescence confocal microscopy revealed extensive colocalization of 51 KATP channel pore-forming subunit Kir6.2 and caveolin-3 along the plasma
membrane. Co-immunoprecipitation of cardiac myocytes showed significant
association of Kir6.2, adenosine A1 receptors and caveolin-3. Furthermore,
whole-cell voltage clamp studies suggested that adenosine A1 receptor- mediated activation of KATP channels was largely eliminated by disrupting
caveolae with methyl-β-cyclodextrin or by small interfering RNA, whereas
pinacidil-induced KATP activation was not altered. We demonstrate that KATP
channels are localized to caveolin-enriched microdomains. This microdomain
association is essential for adenosine receptor-mediated regulation of KATP
channels in cardiac myocytes.
52 1. Introduction
The ATP-sensitive potassium (KATP) channel is a highly abundant
plasma membrane protein responsible for linking the cellular energy levels to
membrane potentials (2, 197). In the heart, KATP channels on the plasma
membrane are critical regulators of cellular excitability and action potentials
during ischemia. They are closed under the basal conditions due to inhibition by
intracellular ATP, but open under metabolic stress such as ischemia or hypoxia.
Cardiac KATP channels have been shown to protect against the metabolic insult of ischemia and contribute to adaptive responses to metabolic stress (139, 193,
212).
It’s been known that cardiac KATP channels open at millimolar range of intracellular ATP during ischemia while ATP required to inhibit this channel is in micromolar range (69, 197). This discrepancy is probably due to the fact that some signaling molecules, which are released during ischemia, regulate ATP- dependent gating of the KATP channels by decreasing ATP-sensitivity and thus
increasing open probability (36, 112, 263, 274). Since many signaling
molecules and their downstream mediators/effectors concentrate in the
microdomains of the plasma membrane, the localization of KATP channels to
these sites may have important implications for channel function and regulation.
For rapid and efficient activation of KATP channels during metabolic stress, the
spatial localization of KATP channels should be of immense importance. It is
likely that the KATP channel signaling is compartmentalized.
53 Caveolae are specialized plasma membrane microdomains involved in
numerous signaling transduction events. Caveolins are the main structural
components of caveolae that can bind cholesterol and coat the cytoplasmic
surface of these organelles. They comprise a family of three distinct 21- to 24-
kDa isoforms; caveolin-1 and -2 are almost ubiquitously expressed, whereas
caveolin-3 is a muscle-specific isoform (8, 232). In the present study, we tested
the hypothesis that subcellular localization of KATP channels to caveolin-
enriched microdomains on the plasma membrane of cardiac myocytes is
essential for regulation of KATP channels by adenosine receptors.
2. Materials and Methods
Cell Isolation
The investigation conforms to the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH Publication N0.
85-23, revised 1996). Adult rat ventricular myocytes were isolated from
Sprague Dawley (SD) rats (250 to 300 g) by enzymatic dissociation (112). In
brief, hearts were excised and retrogradely perfused via aorta with Tyrode's
solution containing (in mM) NaCl 136, KCl 5.4, CaCl2 1.0, MgCl2 1.0, NaH2PO4
0.33, HEPES 10, and glucose 10 at 37°C. The perfusate was then changed to a
Tyrode's solution that was nominally Ca2+ free but otherwise having the same
composition. The hearts were perfused with the same solution containing collagenase for 20 min. Softened ventricular tissues were removed, cut into small pieces, and mechanically dissociated by trituration. The digested cell 54 suspension was gently centrifuged, after which the supernatant was removed
and the remaining pellet was resuspended in a storage solution containing
(mM) KCl 20, KH2PO4 10, glucose 10, potassium glutamate 70, ß- hydroxybutyric acid 10, taurine 10, mannitol 5, and EGTA 5, along with 1%
albumin.
Neonatal myocytes were isolated from 1- or 2-day-old SD rats by
collagenase digestion as described previously (113). Hearts were removed and
collected in ice-cold Hank’s balanced salt solution (HBSS) and rinsed free of
excess blood. The tissue was minced and collected in 1 ml of fresh HBSS to
which 1 ml of 2 mg/ml collagenase type 2 was added. A total of seven rounds of
5-min digestions were performed at 30°C on a rocking platform. After each digestion, the tissue was gently aspirated up and down three times through a
10-ml pipette and allowed to settle for 1 min. The supernatants of the first three digestions were discarded. The supernatants collected from digestions 4-7 were collected, adjusted to 20% FCS, and centrifuged at 400 x g for 5 min. The cell pellets were immediately resuspended in 1 ml DMEM containing 10% FCS.
After preplating for 1 hr in a tissue culture grade petri dish at 37°C and 5% CO2
to allow for the attachment of fibroblasts, cell suspension was plated onto
collagen-coated coverslips. On second day, cytosine-β-D-arabinofuranoside (10
µg/ml) was added to the medium to inhibit fibroblast growth. Adherent viable
myocytes started to contract spontaneously 24 hr after plating. Nonmyocyte
cells were less than 10% of total cells.
55 Small Interfering RNA (siRNA) and transfection
The siRNA oligonucleotide targeting caveolin-3 was purchased from
Ambion Inc (Austin, TX, USA). A negative control siRNA (scrambled) was
included to monitor non-specific effects. Neonatal ventricular myocytes were transfected with siRNA and pEGFP using Amaxa kit (Amaxa, Gaithersburg,
MD) immediately after preplating step. Forty-eight to 72 hr after transfection,
Western blot was carried out to examine the knockdown of targeted proteins.
Fractionation of Caveolin-enriched Membrane
Caveolin-rich fractions from cardiomyocytes were prepared by using a
previously described detergent-free method with some modification (266).
Briefly, cell pellet was resuspended in 0.5 M sodium carbonate (pH 11.0; 2 ml) and homogenized sequentially by using a loose-fitting Dounce homogenizer, a
Polytron tissue grinder and a sonicator. The homogenate was adjusted to 45%
sucrose by addition of an equal volume of 90% sucrose in MBS (25 mM Mes,
pH 6.5/0.15 M NaCl) and placed at the bottom of an ultracentrifuge tube. A 5–
35% discontinuous sucrose gradient (in MBS containing 250 mM sodium
carbonate) was formed above, by overlaying with 4 ml of 35% sucrose
(prepared in MBS with 250 mM sodium carbonate) and then 4 ml of 5% sucrose
(again prepared in MBS with 250 mM sodium carbonate). Tubes were
centrifuged at 39,000 rpm for 18-20 hr in a SW41 rotor. Twelve 1-ml fractions
were collected from the top to the bottom of the gradient for subsequent
analysis by Western blot. 56 Western Blotting
Immunoblot analysis was carried out as described previously (84). The
density gradient fractions or cell lysates were denatured in a sample buffer,
electrophoresed on 10% SDS-polyacrylamide gels and transferred onto
nitrocellulose membranes. The transferred blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS, 150 mM NaCl, 20 mM Tris-HCl, pH 7.4) and
incubated for 1 hr at room temperature with primary antibodies in TBS, 0.1%
Tween 20. After washing, the blots were reacted with peroxidase-conjugated secondary antibodies for 45 min and developed using the ECL detection system.
Co-immunoprecipitation
Immunoprecipitation experiments were performed as reported previously
(128). Adult rat ventricular myocytes or neonatal rat hearts were homogenized
in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2 mM
EGTA, pH 7.6) containing 1% Triton X-100 and 1 mM sodium orthovanadate
with protease inhibitor tablets (Roche). Insoluble materials were pelleted by
centrifugation at 10,000 x g for 15 min and the lysates were precleared by
incubation with r-protein-G agarose (Invitrogen; 1 hr, 4°C). 500 µl of cleared
lysate (1 µg/µl) was incubated with 5 µg of primary antibodies or 5 µg of
appropriate control IgG (or without primary in some cases) overnight at 4°C.
Antigen-antibody complexes were captured with r-protein-G agarose (4°C, 2
57 hr). Agarose beads were washed 4-times with solubilization buffer before
removal of bound proteins by boiling in SDS sample buffer. In some experiments, co-immunoprecipitation was performed in caveolin-rich fractions.
The fraction 4, 5 and 6 from sucrose-gradients centrifugation were combined,
diluted by adding MBS and centrifuged at 40,000g for 2 hr to pellet caveoale,
which was then suspended in lysis buffer and sonicated prior to immunoprecipitation. Samples were resolved by SDS-PAGE, transferred onto
nitrocellulose membrane, and analyzed by probing with various antibodies.
Immunofluorescence
As described previously (84), the cells were fixed with 4% formaldehyde
in PBS for 30 min, blocked, permeablized in 5% goat serum in PBS with 0.1%
Triton X-100 (30 min), and labeled with primary antibody for 2 hr. Cells were
then washed three times and labeled with fluorescence -conjugated secondary
antibody for 1 hr. Immunofluorescence was visualized with confocal laser
scanning microscopy. All images were analyzed using a background
subtraction method offline.
Electrophysiological Recordings
Membrane currents were recorded from adult and neonatal rat myocytes
using whole-cell perforated patch and conventional whole-cell configurations,
respectively. In the siRNA transfected cells, only GFP-positive neonatal
myocytes were used for recording. Cells were perfused with the bath solution
58 containing (mM) NaCl 135, KCl 5.4, MgCl2 1.0, CaCl2 1.0, NaH2PO4 0.33,
HEPES 10, and glucose 10 at pH 7.4. The pipette solution contained (mM) KCl
140, MgCl2 1.0, HEPES 10, EGTA 5, and GTP 0.1 at pH 7.3. For conventional
whole-cell recordings, 0.25 mM ATP was added to pipette solutions for
recordings. For perforated whole-cell configuration, amphotericin B stock
solution (30 mg/ml in DMSO) was diluted with the pipette solution to reach the
final concentration (300 µg/ml) with the aid of sonication. All experiments were
conducted at room temperature (22°C to 25°C).
Borosilicate glass electrodes (outer diameter, 1.5 mm) were used with
resistances in range of 3 to 5 MΩ when filled and connected to a patch-clamp
amplifier (Axopatch 200B, Axon Instruments, Union City, CA, USA). For the
time course of KATP currents, the cell membrane was held at -40 mV. KATP
currents were calculated as glibenclamide-sensitive currents.
Data analysis
Group data were presented as means ± SE. Multiple group means were
compared by ANOVA followed by LSD post hoc test. Differences with a two-
tailed P<0.05 were considered statistically significant.
3. Results
Localization of KATP channels in caveolin-enriched microdomains
To determine whether KATP channels are present in caveolin-rich
membrane fractions in cardiac myocytes, we used a detergent-free sucrose 59 gradient extraction-procedure for adult rat ventricular myocytes. Figure 2.1A
shows Western blot analysis of ten 1-ml fractions collected from top to bottom
of the sucrose density gradient. The marker proteins for caveolae, caveolin-1 and caveolin-3, were found predominantly in the lower-density fractions 4 and 5
(Fig. 2.1A, upper panel). In the same caveolar-enriched fractions, we detected
immunoreactivity for a significant amount of KATP channel pore-forming subunit
Kir6.2 (~ 80%). As reported previously (107), caveolin-3 was also detected in heavier samples (fractions 9-12) though enrichment for caveolin-3 occurred only in samples 4 and 5. Clathrin heavy-chain was detected with antibody against clathrin heavy chain across a broad range of the gradient fractions
similar to that reported by Sampson et al (245). Although there was some
overlap between caveolins and clathrin in fraction 5, it should be noted that the
distribution of Kir6.2 did not follow that of clathrin but only in caveolin-enriched
fraction. Coomassie blue staining of the gel showed that little protein was found
in the earlier fractions (fractions 4 and 5) that contain substantial amounts of
caveolin-3 and caveolin-1 but excludes most of the cellular protein (Fig. 2.1A, lower panel). Measurement of cholesterol levels within each fraction showed cholesterol to be enriched in fraction 4 and 5 (Fig. 2.1B), consistent with the observation that these two fractions represent caveolae-containing membrane fraction of the density gradient. Therefore, KATP channel pore-forming subunit
Kir6.2 is present in the caveolin-rich membrane fractions of cardiac ventricular
myocytes.
60
Figure 2.1. Kir6.2 is enriched in caveolar plasma membrane in adult rat
cardiomyocytes. A, 1 ml fractions were collected from the top of the gradient and analyzed with antibody against Kir6.2, caveolin-1 (Cav-1), caveolin-3 (Cav-
3), clathrin heavy-chain (Clathrin-HC). Lower panel shows coomassie blue staining pattern of same sucrose gradient fractions. B, Relative cholesterol levels of each fraction. Three independent experiments were performed.
61
Figure 2.1. Kir6.2 is enriched in caveolar plasma membrane in adult rat cardiomyocytes.
62 Co-immunoprecipitation of KATP channels and caveolin-3 from adult rat cardiac myocytes
To determine whether muscle-specific caveolin isoform caveolin-3 and
Kir6.2 are associated with each other, we performed immunoprecipitation experiments from the cell lysates of adult rat cardiac myocytes. The cell homogenate was incubated with rabbit anti-Kir6.2 antibody to precipitate caveolin-3. The immune complexes were collected with r-protein G beads and analyzed by immunoblotting against mouse anti-caveolin-3 and anti-Kir6.2 antibodies. Figure 2.2 shows that anti-Kir6.2 antibody immunoprecipitated caveolin-3. Conversely, anti-caveolin-3 antibody immunoprecipitated Kir6.2.
Neither protein was immunoprecipitated with control IgGs (mouse IgG and rabbit IgG). These results suggest that the Kir6.2 associates with caveolin-3 in adult rat ventricular myocytes.
63
Figure 2.2. KATP channels are associated with caveolin-3 (Cav-3) in adult rat ventricular myocytes. Cav-3 was detected in the immunoprecipitates with anti-Kir6.2. Similarly, Kir6.2 was detected in the Cav-3 immunoprecipitates.
Control IgGs (mouse and rabbit IgG) did not immunoprecipitate the proteins.
64
Figure 2.2. KATP channels are associated with caveolin-3 (Cav-3) in adult
rat ventricular myocytes.
65 Colocalization of KATP channel pore-forming subunits and caveolin-3
To investigate whether KATP channel pore-forming subunit Kir6.2
colocalizes with caveolin-3, we employed immunofluorescence confocal
microscopy. Figure 2.3 shows fluorescence images from adult rat ventricular
myocytes. The antibody against Kir6.2 (red) or caveolin-3 (green) demonstrated a prominent surface sarcolemmal punctate staining area. Merged images showed significant punctuate areas of colocalization for Kir6.2 and caveolin-3 along the plasma membrane. In agreement with previous observations (187),
Kir6.2 was clearly localized in peripheral sarcolemma and T tubules. Caveolae
vesicles have been noted before by electron microscopic examination of the myocardium on both peripheral plasma membrane and T tubules. Our immunostaining also showed the presence of caveolin-3 in peripheral sarcolemma and T tubules (82, 157). These data suggests that cardiac KATP
channels are largely localized in caveolin-3-associated membrane domains.
67
Figure 2.3. Colocalization of Kir6.2 and caveolin-3 (Cav-3). Double labeling confocal images of adult rat ventricular myocytes with anti-Kir6.2 and anti-Cav-
3 antibodies. Punctate areas of colocalization (represented by yellow) are apparent along the plasma membrane. The white boxes represent enlarged areas shown in the lower panels.
68 Effect of disrupting lipid rafts with methy-β-cyclodextrin (MβCD) on adenosine
A1 receptor-mediated activation of KATP channels
KATP channels are activated by adenosine via adenosine A1-receptors
and an inhibitory G-protein (Gi) in the heart (113, 170). This adenosine-
mediated KATP activation has been linked to cardioprotection against ischemia.
Both A1-receptors and Gi are localized in caveolar-enriched fractions. To
determine whether cholesterol-enriched microdomains are essential for
adenosine receptor-mediated activation of KATP channels, we tested the effect
of disrupting caveolae with a cholesterol depleting agent MβCD on KATP channel
activity in adult rat ventricular myocytes. Non-selective adenosine receptor
antagonist 8-phenytheophlline (8-PT) was used to ensure that adenosine effect
involves activation of adenosine receptors. Due to strong inhibition of KATP
channels by intracellular ATP, most of modulators including adenosine
receptors are shown to activate KATP channels only at reduced intracellular ATP level or in the presence of a potassium channel opener. Since we employed the whole-cell perforated patch-clamp technique where physiological ATP level was preserved, we studied the effect of adenosine on KATP channels in the
presence of pinacidil (50 μM). The effect of MβCD on KATP channels evoked by
pinacidil alone (100 μM) was used as a negative control since pinacidil
activates KATP channel by interacting with SUR subunit and its effect should be
independent of lipid rafts/caveolae microdomains. As previously reported,
adenosine alone did not activate KATP channels due to stronger inhibition by
intracellular ATP (data not shown). Pinacidil alone at 50 μM elicited a small K+ 69 current that was completely blocked by selective KATP channel blocker
glibenclamide (10 μM, Fig. 2.4A). However, when pinacidil (50 μM) and
adenosine (100 μM) were superfused together in the bath solution, they
activated a large K+ conductance channel that was completely inhibited by
glibenclamide (Fig. 2.4B). When cells were superfused with 10 mM MβCD for
10 min, which was previously shown to disrupt the function of caveolar associated proteins (17), prior to application of adenosine plus pinacidil, we found that MβCD pre-treatment almost completely eliminated the response of
KATP channels to adenosine receptors (Fig. 2.4C, 13.19±2.28 vs. 2.37±1.21
pA/pF, adenosine + pinacidil vs. adenosine + pinacidil + MβCD, p< 0.05, n=3-
4). In contrast, MβCD did not reduce KATP currents activated by 100 μM
pinacidil (15.37±3.41 vs. 19.67±4.15 pA/pF, pinacidil vs. pinacidil + MβCD,
p=NS), indicating that KATP channels evoked by pinacidil is independent of
cholesterol-rich plasma membrane microdomains. These observations indicate
that disrupting cholesterol-rich microdomains by MβCD significantly reduced
effect of adenosine receptors on KATP channels.
70
Figure 2.4. Caveolar disruption with MβCD eliminates adenosine
receptor-mediated stimulation of KATP channels. Time course of KATP
currents in perforated whole-cell configuration was recorded in the presence of
pinacidil 50 μM alone (Pina, A), pinacidil plus Adenosine 100 μM (Pina+Ade, B)
at a holding potential of -40 mV in control cells or cells pretreated with MβCD for 10 min (C). Glibenclamide (Glib) was added subsequently to ensure KATP
channel activation. D, Averaged current density in the presence of various agents as indicated, which was calculated by dividing glibenclamide-sensitive currents by cell capacitance. *p<0.05 vs. Pina. n=3-4.
71
Figure 2.4. Caveolar disruption with MβCD eliminates adenosine receptor-mediated stimulation of KATP channels.
72 Effect of disrupting caveolae with siRNA targeting caveolin-3 on adenosine A1
receptor-mediated activation of KATP channels
Although acute MβCD treatment of adult rat myocytes caused caveolar
disruption and resulted in the loss of regulation of KATP channels by adenosine
receptors, interpretation of results could be complicated by MβCD-induced
cholesterol depletion that may influence the regulation of KATP channels outside
of caveolae. To determine whether specifically destroying caveolae by siRNA knockdown of caveolin-3 expression alters the regulation of KATP channels by adenosine A1 receptors, we investigated the effect of caveolin-3 siRNA on KATP
channel regulation. We first examined whether a predesigned siRNA
oligonucletide for rat caveolin-3 can reduce endogenous caveolin-3 protein
level in neonatal rat cardiomyocytes. We found that caveolin-3 siRNA
significantly suppressed caveolin-3 but not caveolin-1 protein expression 48 hr
after transfection with siRNA. In contrast, control siRNA had no effect (Fig.
2.5B). Cells exhibiting GFP that was co-transfected with siRNA were used for
whole-cell voltage clamp studies. As shown in Fig. 2.5A, adenosine alone can
activate KATP channels after dialysis of pipette solution with a low ATP (0.25 mM). Transfection of neonatal cardiomyocytes with caveolin-3 siRNA largely
eliminated the stimulatory effect of adenosine A1 receptors on KATP channels
whereas control siRNA showed no significant changes (1.5±0.6 vs. 24.1±4.0
pA/pF, caveolin-3 siRNA vs. control siRNA, p< 0.01, n=4-5). In contrast, KATP
channels activated by pinacidil (100 μM) were not altered by siRNA targeting caveolin-3 (38.21±13.02 vs. 45.85±10.40 pA/pF, control vs. siRNA, p=NS). 73 Pinacidil followed by glibenclamide was applied to confirm there were functional
KATP channels in the cells transfected with caveolin-3 siRNA. Further, the effect of adenosine on KATP channels was almost completely blocked by a selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX,
1 μM, Fig. 2.5C). Taken together, these findings suggest that adenosine receptor-mediated activation of KATP channels in the cardiac myocytes requires intact caveolae where KATP channels and adenosine receptors associate.
74
Figure 2.5. Knockdown of caveolin-3 expression with siRNA prevents adenosine receptor-mediated activation of KATP channels. A, Conventional whole-cell voltage clamp recordings of KATP currents were performed at a holding potential of -40 mV in neonatal rat cardiomyocytes transfected with nothing (Control), control siRNA or siRNA targeting caveolin-3 (Cav-3 siRNA).
Adenosine (Ade), glibenclamide (Glib), or pinacidil (Pina, 100 μM) were applied to the bath solutions at various times as indicated by horizontal lines. B,
Western blot shows significant reduction of endogenous caveolin-3 (Cav-3) by siRNA against caveolin-3 (Cav-3 siRNA) and no change in caveolin-1 (Cav-1).
C, Averaged current density induced by adenosine (100 μM) was shown in cells transfected with nothing, control siRNA or Cav-3 siRNA. DPCPX was used to ensure that KATP channel activation by adenosine was through the stimulation of adenosine A1 receptors. **p<0.01 vs. control for DPCPX group, vs. control siRNA for Cav-3 siRNA group. n=4-5.
75
Figure 2.5. Knockdown of caveolin-3 expression with siRNA prevents adenosine receptor-mediated activation of KATP channels.
76 Co-immunoprecipitation of KATP channels and caveolin-3 from neonatal rat hearts
To determine that caveolin-3 and KATP channels reside in the same membrane compartment in neonatal rat hearts as those in adult rat hearts, we subjected the caveolin-rich fractions (fractions 4-6) to immunoprecipitation with anti-caveolin-3 or anti-Kir6.2 antibodies. The immunoprecipitates were analyzed by immunoblotting with antibodies against Kir6.2 or caveolin-3. As shown in Fig.
2.6, Kir6.2 co-precipitated caveolin-3 and caveolin-3 co-precipitated Kir6.2. The control samples without primary antibody did not immunoprecipitate the proteins. We also examined the presence of adenosine A1 receptors in the samples obtained by immunoprecipitation with anti-caveolin-3 antibodies.
Adenosine A1 receptors were detected in these samples. These data suggest that KATP channels and adenosine A1 receptors associate with caveolin-3-rich membrane compartments in the neonatal rat heart.
77
Figure 2.6. Immunoblot analysis of samples from neonatal rat hearts immunoprecipitated with anti-caveolin-3 (Cav-3) or anti-Kir6.2 antibodies.
Caveolin-rich fractions were immunoprecipitated with specified primary antibodies or no primary antibody (No ab). Kir6.2 and adenosine A1 receptors were detected in anti-caveolin-3 or anti-Kir6.2 immunoprecipitates, but not detected in the samples without primary antibody.
78
Figure 2.6. Immunoblot analysis of samples from neonatal rat hearts immunoprecipitated with anti-caveolin-3 (Cav-3) or anti-Kir6.2 antibodies.
79 Association of adenosine A1 receptors and KATP channels in caveolin-3-rich fractions of cardiomyocytes
We have shown that Kir6.2 associates with caveolin-3 in the total cell lysates of adult rat cardiomyocytes. In order to determine whether Kir6.2 and adenosine A1 receptors reside in the same caveolin-enrich microdomains and further elucidate the mechanism involved in adenosine-mediated KATP activation, we performed co-immunoprecipitation experiments in the caveolin-3- enriched fractions of adult rat ventricular myocytes. Cells were pretreated with
MβCD (10 mM) for 30 min prior to sucrose gradient fractionation. Caveolin-rich fractions (fractions 4-6) were immunoprecipitated with antibodies against Kir6.2, adenosine A1 receptors and caveolin-3. As shown in Fig. 2.7, Kir6.2, adenosine
A1 receptors and caveolin-3 coimmunoprecipitated with anti-Kir6.2, anti- adenosine A1 receptors or anti-caveolin-3 immunoprecipitates. The 50 kDa IgG heavy chain band was detected in the blot when probing with the antibody against adenosine A1 receptors. There was no detection of Kir6.2, adenosine A1 receptors and caveolin-3 in the homogenates without the treatment of primary antibodies. MβCD diminished caveolin-3 that co-precipitated with Kir6.2 and adenosine A1 receptors, while the levels of Kir6.2 and adenosine A1 receptors were also reduced. These data indicate that KATP channels and adenosine A1 receptors may associate with each other in caveolin-rich membrane microdomains and this association depends on the level of cholesterol.
80
Figure 2.7. Effect of MβCD on Kir6.2, adenosine A1 receptors (Ade A1) and caveolin-3 (Cav-3) coprecipitation. Adult rat cardiomyocytes were pretreated with or without MβCD (10 mM) for 30 min prior to homogenation and sucrose gradient centrifugation. The light fractions (fractions 4-6) were immunoprecipitated with antibodies against Kir6.2, Ade A1 or Cav-3, or without
primary antibody (No ab). Immunoblots were probed for the protein indicated.
81 4. Discussion
In the present study, we demonstrate that localization of KATP channels to
caveolin-enriched microdomains in the plasma membrane of cardiac myocytes
is essential for adenosine receptor-mediated activation of KATP channels.
Specifically, we show that KATP channel pore-forming subunit Kir6.2 is localized
in caveolin-enriched membrane fractions, and is associated and colocalized with caveolin-3. Patch-clamp studies reveal that this microdomain association is necessary for KATP channel regulation by activation of adenosine receptors.
KATP channels are regulated by a number of endogenous activators,
most of which are released or increased during ischemia such as noradrenaline
(116, 251), adenosine (114, 274), protein kinase C (112, 163), nitric oxide (36)
and membrane lipids especially phosphatidylinositol-4,5-bisphosphate (263). All
of these regulators activate KATP channels by decreasing ATP-sensitivity and
thus increasing open probability. It has been proposed that activation of KATP
channels by these endogenous regulators may be one of reasons why KATP
channels can be activated at millimolar range of intracellular ATP during
ischemia while KATP channels are inhibited by micromolar range of ATP in
patch-clamp studies (192). It is likely that those molecules decrease the ATP- sensitivity of KATP channels within caveolae and thus activates the channels
even though intracellular ATP level is relatively high. A recent study shows that
KATP channel subunit Kir6.2 is mandatory for optimal adaptation capacity under
stress, further indicating that cardiac KATP channels may not be as silent as
originally thought and the mechanism may involve regulation of the channel 82 microenvironment (320). The present study revealed a novel mechanism by
which KATP channels in the plasma membrane of cardiac myocytes are
regulated by caveolin-rich microdomains. This finding indicates that the
regulation of KATP channels is more complex than that can be explained by the simple hypothesis of opening by bulk decrease in intracellular ATP.
In the heart, several ion channels and exchangers are localized in
caveolae and have shown functional modulation by caveolae-associated mechanism including the voltage-dependent Na+ channel, voltage-dependent
Ca2+ channel, a voltage-dependent K+ channel (Kv1.5), the Na+/Ca2+
exchangers and Na+/K+-ATPase (17, 18, 30, 167, 178, 313). In vascular smooth
muscles KATP channels (Kir6.1 and SUR2B) are also present in caveolae (244,
245). It is not known that whether cardiac KATP channels, which are composed
of Kir6.2 and SUR2A, are localized in caveolin-rich microdomains. While the
adenosine A1 receptor is well known to couple to cardiac KATP channels via
inhibitory G proteins, and inhibitory G proteins often couple to their effectors by
protein kinase C (114, 141, 143), it is also not clear whether cholesterol-rich microdomains on the plasma membrane of cardiac myocytes impacts the
regulation of KATP channels by adenosine A1-receptor signaling. The present
studies provide the first evidence that intact caveolin-rich microdomains are
essential for adenosine A1-receptor-mediated KATP channel signaling. This is
consistent with the previous finding that adenosine A1-receptors and their
downstream signaling molecules including Gi protein and protein kinase C are
localized in caveolae (17, 153, 206, 305). 83 In the present study, disruption of caveolae may affect other modulators
of KATP channels within caveolae. If adenosine-induced activation of KATP
channels is obscured by disrupting other caveolar-associated signaling
molecules that are active under basal conditions, pinacidil-evoked KATP currents
should also be reduced. Our data showed that disrupting caveolae by either
MβCD or caveolin-3 siRNA did not alter the amplitude of pinacidil-evoked KATP
currents, indicating that basal KATP activity may not be significantly affected by
the net effect of disrupting caveolae.
It is known that caveolin-3 is essential for the formation of caveolae in
cardiac myocytes, whereas caveolin-1 is expressed and important in cardiac
myocytes. We noticed that we detected significant amount of caveolin-1 in
addition to caveolin-3 in our cardiomyocyte preparations. This could raise a
question whether caveolin-1 is involved in adenosine-mediated regulation of
KATP channels. Our data from neonatal rat cardiomyocytes show that suppression of caveolin-3 by specific siRNA did not alter the expression of caveolin-1 but significantly affected the regulation of KATP channels by
adenosine receptors. This observation indicates that caveolin-3 not caveolin-1
play an important role in KATP channel regulation by adenosine receptors in
cardiomyocytes.
We understand that there are significant developmental changes in ion
channel expression and/or distribution. The subsarcolemmal localization of KATP
channels in adult rat heart may not be the same as that in neonatal rat heart.
However, our immunoprecipitation experiments from caveolin-3-enriched 84 fractions of neonatal rat heart demonstrate that Kir6.2 indeed associates with
caveolin-3, which is consistent with the observation that KATP channels are
functionally regulated by intact caveolae or caveolin-3.
We have previously shown that acute stimulation (<5 min) of adenosine
receptors activates KATP channels (114, 141) and prolonged stimulation (15-30
min) causes internalization of KATP channels in cardiac myocytes (113). In the
present study, the KATP currents started to increase significantly around 5 min
after superfusion with adenosine, and reached the steady state within 5-10 min.
The time course for adenosine to induce a detectable increase in KATP currents
and then reach a steady state depends on the rate of superfusion, the level of
intracellular ATP, metabolic state of the cell and the concentration of pinacidil.
Although adenosine A1 receptor has been shown to translocate out of caveolae
after agonist stimulation, this translocation occurs after prolonged stimulation
which lasts for 15 min (153). It is thus likely that the acute effect of adenosine
A1 receptors on KATP channels in cardiac myocytes is caveolae-dependent.
In recent years, it has become apparent that the regulation of ion
channel function is not the only means of controlling cell excitability. The
trafficking and localization of ion channels with signaling molecules also play an
important role. The precise subcellular localization of ion channels is often
necessary to ensure efficient integration of both intracellular and extracellular events. The present study has identified a novel mechanism for regulation of cardiac KATP channel. Giving that caveolae are emerging as important surface- associated organelles, our elucidation of caveolar microdomain-dependent 85 regulation of KATP channels in cardiac myocytes will significantly enhance our knowledge of the complexity of regulation of this macromolecular channel and provide mechanistic insight into how KATP channels control cellular functions.
Acknowldgement
This work was supported by the American Heart Association National
Center [0835117N to K.H.].
86
CHAPTER 3
CAVEOLIN-3 NEGATIVELY REGULATES RECOMBINANT CARDIAC ATP-
SENSITIVE POTASSIUM CHANNELS IN HEK293T CELLS
ABSTRACT
We have recently shown that ATP-sensitive potassium (KATP) channels in the heart are localized in the caveolae of cardiac myocytes and regulated by caveolae-related signaling. However, little is known about the role of caveolins, signature proteins of caveolae, in cardiac KATP channel function. The present study was designed to explore the potential functional interaction between caveolin-3 and KATP channels. The cardiac KATP channel subunits Kir6.2 and
SUR2A were transiently transfected in HEK293T cells with or without co- transfection of caveolin-3. Our data demonstrated that the recombinant KATP channel activity in HEK293T cells was inhibited by expression of caveolin-3 but not caveolin-1. Application of caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, blocked the inhibitory effect of caveolin-3 on KATP channels. However, the same peptide did not have any significant effect on KATP channels in HEK293T cells without 87 caveolin-3 expression. We further confirmed that expressed caveolin-3 co- localized and co-immunoprecipitated with the KATP channel pore-forming subunit Kir6.2. Thus, our results indicate that caveolin-3 negatively regulates
Kir6.2/SUR2A channel function.
88 1. INTRODUCTION
A variety of proteins are located within caveolae. Caveolae bring
signaling molecules together into a confined microdomain, whereas caveolins,
structural proteins of caveolae, interact with a range of key proteins localized
within caveolae. Many signaling molecules are shown to interact with caveolins
such as G proteins, G protein–coupled receptors, Src family protein kinases,
Ha-Ras, and nitric oxide synthases (51). Caveolin is a scaffolding protein that
can bind and assemble multiple signaling molecules in a large complex and are
known to modulate the activity of most of its interacting partners. Direct
interaction between caveolins and interacting proteins may serve to either
anchor proteins to caveolae or modulate protein function.
Regulation of ion channel function is not the only means of controlling
cell excitability. The localization and trafficking of ion channels play an
important role in cellular function. The precise subcellular localization of ion
channels is important for rapid and efficient integration of cellular signaling events. Lipid raft association has been identified as a novel mechanism for the subcellular sorting of specific ion channels to the plasma membrane microdomains rich in signaling molecules (175, 210).
The ATP-sensitive potassium channel (KATP) is a highly abundant
plasma membrane protein responsible for linking the cellular energy levels to
membrane potential and cellular excitability (2, 197). KATP channels are
heterooctamers consisting of two structurally unrelated proteins: four pore-lining
subunits that belong to the Kir6 subfamily of inwardly rectifying potassium 89 channels, and four regulatory subunits of the ATP-binding cassette superfamily.
Cardiac KATP channels have been shown to protect against the metabolic insult of ischemia and contribute to adaptive responses to metabolic stress (139,
212). We have recently shown that KATP channels in the heart are primarily localized in the caveolae of cardiac myocytes and regulated by caveolae-
related adenosine receptor signaling (85). Giving that caveolin has been shown
to regulate the function of certain molecules accumulated in caveolae; we
sought to examine the potential functional role of caveolins on cardiac KATP
channels. In the present study, HEK293T cells were transfected with cardiac
type KATP channel subunits Kir6.2/SUR2A with or without caveolins. The effect
of caveolin-3 or caveolin-1 on recombinant KATP channel function was
determined by expression of caveolin-3, caveolin-1 or use of caveolin-3
scaffolding domain peptide (CSD). We demonstrate that caveolin-3 not
caveolin-1 negatively regulates recombinant cardiac KATP channel function.
2. Materials and methods
Chemicals and reagents
Caveolin-3 scaffolding domain peptide (CSD) containing the putative
scaffolding domain of caveolin-3 (amino acids 55–74,
DGVWRVSYTTFTVSKYWCYR) was purchased from AnaSpec Inc. (San Jose,
CA, USA). Pincidil and glibenclamide were purchased from Sigma-Aldrich (St.
Louis, MO, USA).
90
Cell culture and transfections
Human embryonic kidney (HEK)293T were cultured in DMEM,
supplemented with 10% fetal bovine serum, L-glutamine (2 mM), penicillin (100
IU/ml), and streptomycin (100 µg/ml). HEK293T cells were plated onto 35-mm culture dishes and transfected with cardiac KATP channel subunits, HA-tagged
Kir6.2 and SUR2A, with or without cavoelin-3-ECFP (180) or caveolin-1-EGFP
(204) by using FuGENE6 (Roche). Cells were also transfected with GFP to
ensure there is transfection. All mammalian expression constructs are either in
pcDNA3, pCMV, pECFP or pEGFP. Experiments were conducted 2-3 days after
transfection.
Electrophysiology Studies
The whole-cell KATP currents in HEK293T cells were recorded using
patch-clamp technique (112, 128). Only the cells exhibiting GFP were used for
recordings. Cells were superfused with the bath solution containing (mM) NaCl
135, KCl 5.4, MgCl2 1.0, CaCl2 1.0, NaH2PO4 0.33, HEPES 10, and glucose 10 at pH 7.4 (pH adjusted with NaOH). The pipette solution contained (mM) KCl
140, MgCl2 1.0, HEPES 10, EGTA 5, GTP 0.1, ADP 0.1 at pH 7.3 (pH adjusted
with KOH). 3 mM ATP was added to the pipette solution for spontaneous KATP
currents and 5 mM ATP was for pinacidil-evoked KATP currents. Data were
sampled at 13.3 kHz with an A/D converter (Digidata 1322A, Axon Instruments)
and stored on the hard disk of a computer for subsequent analysis. The 91 recordings were filtered with a low-pass corner frequency of 2 kHz. Borosilicate glass electrodes (outer diameter, 1.5 mm) were used with resistances in range of 3 to 5 MΩ when filled and connected to a patch-clamp amplifier (Axopatch
200B, Axon Instruments). The time course of KATP currents were recorded at a holding potential of 0 mV. The activation of KATP currents was verified by application of KATP channel selective blocker glibenclamide. All the Recordings were carried out at room temperature (22-25oC).
Co-immunoprecipitation and Western blotting
HEK293T cells were rinsed with ice-cold PBS (pH 7.4), homogenized in ice-cold lysis buffer (25 mM Tris-HCl; 250 mM NaCl; 10 mM EDTA; pH 7.6) containing 1% Triton X-100 and 2 mM phenylmethylsulfonyl fluoride with protease inhibitor tablets (Roche) (85). Insoluble material was pelleted by centrifugation at 10,000Xg for 15 min and the lysates were precleared by incubation with r-protein-G agarose (Invitrogen; 1 hour, 4°C). Approx. 800 µL of cleared lysate (1µg/µl) was incubated with anti-HA (1:100), anti-caveolin-3 antibody (1:100) for 2 hours at 4°C. Control sample was incubated without antibody. Antigen-antibody complexes were captured with r-protein-G agarose
(4°C, 2 hours). Agarose beads were washed 4-times with lysis buffer before removal of bound proteins by boiling in SDS sample buffer. Samples were resolved by SDS-PAGE (10–12% acrylamide gel) and transferred onto nitrocellulose membrane. The transferred blots were blocked with 5% nonfat- milk in Tris-buffered saline (TBST: 150 mM NaCl, 20 mM Tris-HCl, 0.05% 92 tween-20, pH 7.4) and incubated for 1 hr at room temperature with antibodies
against HA-tag or caveolin-3 in TBST. After washing, the blots were incubated
with peroxidase-conjugated secondary antibodies for 45 min and developed
using the ECL detection system (Amersham).
Immunofluorescence microscopy
Immunolabeling was performed in HEK293T cells transfected with
Kir6.2-HA/SUR2A/Cav-3-CFP (85, 113). Briefly, cells were fixed with 4% paraformaldehyde in PBS for 30 min, blocked, permeablized in 5% goat serum in PBS with 0.1% Triton X-100 (30 min), and labeled with a rat anti-HA antibody
for 2 hours (Roche, Indianapolis, IN, USA ). Cells were washed three times and
labeled with fluorescent secondary antibody for 45 minutes.
Immunofluorescence was visualized with a Nikon epifluorescence. Cells were
randomly selected for imaging and analysis.
Statistics
Group data were presented as means ± SE. Unpaired t-test was used to
compare between groups. Multiple group means were compared by ANOVA
followed by LSD post hoc test. Differences with a two-tailed P<0.05 were
considered statistically significant.
93 3. RESULTS
Effects of caveolin-3 on spontaneous Kir6.2/SUR2A channels
To determine whether caveolin-3 directly modulate KATP channel
function, we recorded the time course of spontaneous whole-cell KATP currents
at a holding potential of 0 mV in HEK293T cells transfected with Kir6.2/SUR2A or Kir6.2/SUR2A/caveolin-3 using a conventional whole-cell patch-clamp technique. Only cells show green fluorescence were used for recordings. The whole-cell spontaneous KATP currents were developed relative slowly when cells
were dialyzed with the pipette solution containing 3 mM ATP and 100 μM ADP.
We found that all 8 cells without transfection of caveolin-3 showed spontaneous
KATP currents whereas 6 out of 8 cells co-transfected with caveolin-3 did not
generate significant spontaneous KATP currents (Fig. 3.1A and 3.1B). As shown
in the figure 3.1, spontaneous KATP currents were blocked by application of the
KATP channel inhibitor glibenclamide (10 μM). The cells that did not show
significant spontaneous KATP currents were further tested by subsequent
application of the KATP channel opener pinacidil (100 μM) to ensure sufficient
expression of KATP channels (data not shown). Figure 3.1C summarizes the
current density from cells transfected with Kir6.2/SUR2A or
Kir6.2/SUR2A/caveolin-3 (66.79 ± 18.66 pA/pF vs. 8.58 ± 5.88 pA/pF, control
vs. caveolin-3, p<0.05, n = 8).
94
Figure 3.1. Modulation of spontaneous KATP currents by caveolin-3. (A)
The spontaneous Whole-cell KATP current was detected at 0 mV in HEK293T cells transfected with Kir6.2/SUR2A and blocked by 10 μM glibenclamide (Glib).
(B) The spontaneous Whole-cell KATP currents were detected at 0 mV in
HEK293T cells transfected with Kir6.2/SUR2A/caveolin-3. (C) Mean current
density (pA/pF) in HEK293T cells transfected with Kir6.2/SUR2A (Control) or
Kir6.2/SUR2A/caveolin-3 (Cav-3). Drugs were applied as indicated by the
horizontal lines. The gaps between the traces were interruptions for recording
step currents with various voltages. p<0.05, vs. control. n=8 cells.
95
Figure 3.1. Modulation of spontaneous KATP currents by caveolin-3.
96 Effect of caveolin-3 on pinacidil-induced Kir6.2/SUR2A channels
Since expression of caveolin-3 suppressed spontaneous KATP currents,
we decided to study further the effect of caveolin-3 on KATP currents evoked by
pinacidil. With pipette solution containing 5 mM ATP and 100 μM ADP,
spontaneous KATP currents in HEK293T cells transfected with Kir6.2/SUR2A
were usually not detected even after dialysis for 20-30 minutes. The whole-cell
KATP currents evoked by pinacidil were recorded 10 minutes after formation of whole-cell configuration. As shown in Figure 3.2, an outward current at a holding potential of 0 mV was activated in the presence of 50 μM pinacidil and completely blocked by 10 μM glibenclamide. Expression of caveolin-3 in
HEK293T cells significantly reduced pinacidl-induced currents (134.71 ± 30.19 pA/pF vs. 53.78 ± 15.15 pA/pF, control vs. caveolin-3, p<0.05, n=6-7).
However, when caveolin-1 was transfected into HEK293T cells, the amplitude of pinacidil-induced KATP currents was not different from that in control cells
(134.71 ± 30.19 pA/pF vs. 115.84 ± 36.85 pA/pF, control vs. caveolin-1, p=NS,
n = 6-7). Expression of caveolin-1 and caveolin-3 were verified by their tagged
fluorescent proteins. These observations indicate that the function of
recombinant cardiac KATP channels is specifically regulated by caveolin-3 not
caveolin-1.
97
Figure 3.2. Modulation of pinacidil-activated KATP currents by caveolin-3.
The time course of Whole-cell KATP currents were recorded at 0 mV in the presence of pinacidil (50 μM) or pinacidil plus glibenclamide (10 μM) in
HEK293T cells transfected with Kir6.2/SUR2A (A, Control),
Kir6.2/SUR2A/caveolin-3 (B, Cav-3) or Kir6.2/SUR2A/caveolin-1 (C, Cav-1). (D)
Mean current density (pA/pF) in HEK293T cells transfected with Kir6.2/SUR2A
(Control), Kir6.2/SUR2A/caveolin-3 (Cav-3) or Kir6.2/SUR2A/caveolin-1 (Cav-
1). The pipette solution contained 3 mM ATP and 100 μM ADP. Drugs were
applied as indicated by the horizontal lines. The gaps between the traces were
interruptions for recording step currents with various voltages. P< 0.05, vs.
control. n=6-7 cells.
98
Figure 3.2. Modulation of pinacidil-activated KATP currents by caveolin-3.
99 Effect of caveolin-3 scaffolding domain peptide on pinacidil-induced
Kir6.2/SUR2A channels
Next, we sought to examine whether functional disruption of caveolin-3 affects KATP channel activity in HEK293T cells transfected with
Kir6.2/SUR2A/caveolin-3. Caveolin-3 scaffolding domain peptide (CSD, 10 μM)
corresponding to putative scaffolding domain of caveolin-3 (amino acids 55–74,
DGVWRVSYTTFTVSKYWCYR) was introduced into the cell interior from the
patch pipette. Pinacidil (50 μM) was applied to induce KATP currents after 10-
min dialysis. This caveolin-3 CSD has been previously shown to block caveolin-
3 function (138). Figure 3.3 shows one representative experiment. The
amplitude of KATP current evoked by pinacidil in the cells transfected with
Kir6.2/SUR2A/caveolin-3 was significantly larger in the presence of CSD than
that in the absence of CSD.
Caveolin-3 CSD has been shown to directly modulate the function of
some proteins. To determine whether there is a direct functional interaction
between caveolin-3 CSD and KATP channels, we take advantage of HEK293T
cells that lack endogenous caveolin-3 to record pinacidil-induced KATP currents
in HEK293T cells without caveolin-3 transfection. Figure 3.3C summarizes the current density for control, caveolin-3, control plus caveolin-3 CSD and
caveolin-3 plus caveolin-3 CSD. While the expression of caveolin-3 inhibited
KATP channel currents, Caveolin-3 CSD significantly blocked the effects of caveolin-3 on KATP channels (53.78 ± 15.15 pA/pF vs. 131.53 ± 26.13 pA/pF,
caveolin-3 vs. caveolin-3 + caveolin-3 CSD, p<0.05, n=6-7). However, caveolin- 100 3 SDP did not have the direct functional effect on KATP channels (109.16 ±
12.05 pA/pF vs. 134.71 ± 30.19 pA/pF, control + caveolin-3 SDP vs. control, p=NS, n=5-7). The above data suggest that caveolin-3 negatively regulates the function of recombinant cardiac KATP channels.
101
Figure 3.3. Effect of caveolin-3 scaffolding domain peptide (CSD) on KATP
currents. The time course of pinacidil-activated whole-cell KATP currents in the
presence of caveolin-3 CSD (Cav-3 CSD, 10 μM ) was recorded at 0 mV in
HEK293T cells transfected with Kir6.2/SUR2A (A) or Kir6.2/SUR2A/caveolin-3
(B). Cav-3 CSD was applied in the pipette solution. The KATP currents evoked
by 50 μM pinacidl were verified by 10 μM glibenclamide (Glib). (C) Mean current density (pA/pF) in HEK293T cells transfected with or without caveolin-3
in the presence or absence caveolin-3 CSD. The pipette solution contained 5
mM ATP and 100 μM ADP. Drugs were applied as indicated by the horizontal
lines. The gaps between the traces were interruptions for recording step
currents with various voltages. p<0.05, vs. Cav-3 + Cav-3 CSD. n=5-7 cells.
102
Figure 3.3. Effect of caveolin-3 scaffolding domain peptide (CSD) on KATP
currents.
103 Colocalization and co-immunoprecipitation of KATP channel pore-forming
subunit Kir6.2 and caveolin-3
To determine whether caveolin-3 physically associates with KATP
subunits, co-immunoprecipitation experiments were performed from the cell
lysates of HEK293T cells transfected with Kir6.2/SUR2A and caveolin-3. The
cell homogenate was incubated with anti-HA antibody or caveolin-3 to
precipitate caveolin-3 or Kir6.2, respectively. The immune complex were
collected with protein G beads and analyzed by immunoblotting against anti-HA
and anti-caveolin-3 antibodies. Fig. 3.4A shows that anti-HA antibody
immunoprecipitated caveolin-3 and anti-caveolin-3 antibody immunoprecipitated
Kir6.2. Neither protein was immunoprecipitated in the samples without antibody
treatment.
To investigate whether KATP channels colocalizes with caveolin-3, we
employed immunofluorescence confocal microscopy. Fig. 3.4B shows the representative fluorescence images of cells transfected with
Kir6.2/SUR2A/caveolin-3. The plasma membrane of cells was strongly labeled with both Kir6.2 and caveolin-3. Substantial colocalization of Kir6.2 (red) with caveolin-3-ECFP (green) was observed in these cells, as indicated by yellow functate staining alone the plasma membrane in the merged image. These data suggests that recombinant cardiac KATP channels specifically associates with
expressed caveolin-3.
104
Figure 3.4. Co-immunoprecipitation and colocalization of Kir6.2/SUR2A channels with caveolin-3. HEK293T cells were transfected with Kir6.2-
HA/SUR2A/caveolin-3-ECFP (Cav-3-ECFP). (A) Immunoblots from immunoprecipitates with anti-caveolin-3 (Cav-3) or anti-HA antibodies (anti-HA).
Control sample was incubated without antibody (No ab). (B) Representative fluorescent images from transfected cells labeled with anti-HA antibody for
Kir6.2 (red) and ECFP for Cav-3-ECFP (green). The merged images showed significant colocalization between Cav-3-ECFP and Kir6.2.
105 4. Discussion
In the present study, we provide the first evidence that cardiac type KATP
channels expressed in HEK293T cells were negatively regulated by caveolin-3.
Specifically, we show that KATP channel activity was significantly inhibited by expression of caveolin-3 but not caveolin-1. Further, we demonstrate that
caveolin-3 CSD blocked caveolin-3-mediated suppression of KATP channels but
did not exhibit any direct functional effect on KATP channels. The physical
association of expressed KATP channels with caveolin-3 in HEK293T cells was confirmed by immunofluorecent co-localization and co-immunoprecipitation.
Caveolin is a major protein component of caveolae, flask-shaped plasma membrane invagination. Caveolae has been shown to participate in transmembrane signaling, such as G protein-coupled signaling (232). In addition, caveolin may directly interact with molecules that are accumulated in caveolae and regulate their function (202). KATP channels are regulated by a
number of endogenous activators (36, 112, 114, 116, 163, 251, 263, 274).
Interestingly, most of these modulators and receptors reside primarily in
caveolae (152, 153, 242, 305), suggesting a possible role for KATP channels in
caveolin-dependent cellular function. We have recently shown that KATP
channels are mostly localized in the caveolae of cardiac myocytes and
adenosine-induced KATP channel function is regulated by intact caveolae (85).
In the present study, we extend this finding by elucidating the functional role of
caveolin-3 on KATP channels. We expressed caveolin-3 or caveolin-1 in
HEK293T cells transfected with Kir6.2/SUR2A and studied the effect of 106 expressed caveolin on the basal KATP channel activity. Our data indicate that
caveolin-3 not caveolin-1 is a major regulator of cardiac KATP channel function.
It appears that caveolae is essential for regulation of cardiac KATP
channels by adenosine receptors while caveolin-3 negatively modulates KATP
channel function. Our previous data show that in cardiac myocytes disruption of
caveolae by suppressing caveolin-3 with siRNA affects adenosine-mediated
activation of KATP channels but does not alter the basal KATP channel function
(pinacidil-evoked KATP) (85). The observation that knockdown of caveolin-3 expression by siRNA did not significantly alter pinacidil-induced KATP currents
seems different from what we observed in the present study where caveolin-3
inhibits pinacidil-induced recombinant KATP channel activity. This discrepancy
could be attributed to the incomplete knockdown of endogenous caveolin-3 by
siRNA in native cardiac myocytes. This insufficient suppression of caveolin-3 by
siRNA appeared to be efficient to destroy caveolae structure and thus affect
adenosine-mediated KATP channel regulation (85) but did not provide enough
evidence for the role of caveolin-3 on KATP channel function.
Caveolin-3 CSD encode membrane proximal regions of caveolin-3 and
are known to functionally interact with some caveolin-associated proteins (138,
290). To assess the functional role of caveolin-3 on KATP channels, we tested
the effect of caveolin-3 CSD on pinacidil-evoked KATP channels in HEK293T
cells. Our data show that the caveolin-3 CSD blocked the inhibitory effect of
expressed caveolin-3 on KATP channels, indicating that the caveolin-3 has an
important functional role on KATP channel regulation. It is likely that the caveolin- 107 3 scaffolding domain peptide breaks the binding of caveolin-3 to other protein(s)
that regulates KATP channels. This notion was further confirmed by our observation that caveolin SDP did not have significant functional effect on KATP
channels in HEK293T cells without expression of caveolin-3. Although the exact
mechanism by which caveolin-3 suppresses KATP channel function remains
elusive, it is likely that caveolin-3 scaffolding domain does not exert a direct
functional effect on cardiac KATP channels expressed in HEK293T cells but
interacts with other binding partners to regulate KATP channel function indirectly
(138). The advantage of using HEK293T cells when compared with other cells
is that they lack endogenous caveolin-3, contain minimum amount of caveolin-1
and do not express detectable KATP channels. Thus we can dissect not only the
specific role of individual caveolin isoforms but also the functional effect of
putative scaffolding domain peptide on KATP channels without interference of
endogenous proteins.
While activation of KATP channels during metabolic stress is protective
against injury, excessive KATP channel opening may have deleterious
consequences. In the heart, active KATP channels cause the action potential
duration to shorten and contribute to cellular K+ loss, both are risk factors for
arrhythmia (126, 299). KATP channel blockers have been shown to prevent
ventricular arrhythmia in the canine model of sudden cardiac death (25). A tight
regulation of Caveolin-3 expression may play an important role in controlling
KATP channel function under metabolic stress to confer protection without
incurring serious damage. 108
Acknowledgments
We would like to thank Dr. Jeffery R. Martens (University of Michigan,
MI) for kindly providing caveolin-3-ECFP and Dr. Mark A. McNiven (Mayo
Clinic, MN) for caveolin-1-EGFP DNA clones. This work was supported by the
American Heart Association National Center [0835117N to K.H.].
109
CHAPTER 4
PROTEIN KINASE C ISOFORM-DEPENDENT MODULATION OF ATP-
SENSITIVE POTASSIUM CHANNELS IN MITOCHONDRIAL INNER
MEMBRANE
ABSTRACT
+ The ATP-sensitive K (KATP) channels in both sarcolemmal (sarcKATP) and mitochondrial inner membrane (mitoKATP) are the critical mediators in
cellular protection of ischemic preconditioning (IPC). Whereas cardiac sarcKATP
contains Kir6.2 and sulfonylurea receptor SUR2A, the molecular identity of
mitoKATP remains elusive. In the present study, we tested the hypothesis that protein kinase C (PKC) may promote import of Kir6.2-containing KATP into
mitochondria. Fluorescence imaging of isolated mitochondria from both rat adult
cardiomyocytes and COS-7 cells expressing recombinant Kir6.2/SUR2A
showed that Kir6.2-containing KATP channels were localized in mitochondria and
this mitochondrial localization was significantly increased by PKC activation
with phorbol 12-myristate 13-acetate (PMA). Fluorescence resonance energy
transfer microscopy further revealed that a significant number of Kir6.2- 110 containing KATP channels were localized in mitochondrial inner membrane after
PKC activation. These results were supported by Western blotting showing that
the Kir6.2 protein level in mitochondria from COS-7 cells transfected with
Kir6.2/SUR2A was enhanced after PMA treatment and this increase was
inhibited by the selective PKC inhibitor chelerythrine. Furthermore, functional
analysis indicated that the number of functional KATP channels in mitochondria
was significantly increased by PMA, as shown by KATP-dependent decrease in
mitochondrial membrane potential in COS-7 cells transfected with
Kir6.2/SUR2A but not empty vector. Importantly, PKC-mediated increase in
mitochondrial Kir6.2-containing KATP channels was blocked by a selective PKCε
inhibitor peptide in both COS-7 cells and cardiomyocytes. We conclude that the
KATP channel pore-forming subunit Kir6.2 is indeed localized in mitochondria
and that the Kir6.2 content in mitochondria is increased by activation of PKCε.
PKC isoform-regulated mitochondrial import of KATP channels may have significant implication in cardioprotection of IPC.
111 1. INTRODUCTION
Activation of ATP-sensitive potassium (KATP) channels could protect the
heart under metabolic stress (96, 189, 193, 195). Importantly, the modulation of
KATP channels accounts for the ability of brief ischemia and reperfusion to
protect the heart against infarction induced by subsequent prolonged ischemia,
a phenomenon known as ischemic preconditioning (IPC) (95, 189, 199). Not
only do molecularly defined cell surface sarcKATP channels in the heart activate
on metabolic stress and cause action potential shortening and less energy
consumption (113, 195, 275), KATP channels in the mitochondrial inner
membrane (mitoKATP channels) are also implicated in cardioprotection during ischemia (86, 95, 199). However, despite immense interest and serious
attempts to clone mitochondrial KATP channels, their molecular nature remains
elusive. The KATP channels in the sarcolemma membrane, first described in the
heart by Noma (197) in 1983, are widely distributed in many tissues and cell types including pancreatic β-cell, neuron, skeletal muscle, and smooth muscle
(11, 13). The major function of the KATP channel is to couple the cell metabolic
state to its membrane potential by sensing changes in intracellular adenine
nucleotide concentration (6). The sarcKATP channels are heterooctamers
consisting of two structurally unrelated proteins (2, 3, 20, 254, 257): four pore-
lining subunits that belong to the Kir6 subfamily of inwardly rectifying potassium
channels and four regulatory subunit sulfonylurea receptors (SUR) of the ATP-
binding cassette (ABC) superfamily. It is currently known that the cardiac
112 sarcKATP channel is composed of an octomeric complex of two types of
subunits, the Kir6.2 and the SUR2A subunit.
Whereas the mitoKATP channel has been characterized
pharmacologically (50, 87), its molecular nature remains unknown. Whether
Kir6.2 contributes to mitoKATP channels is under debate; the subunit has been
found in heart mitochondria (54, 147, 265, 319). We believe that much of the
controversy or difficulty regarding the existence of Kir6.2-containing KATP
channels in mitochondria may be due to the low level of basal KATP channels
present in mitochondria. All these studies concerning the existence of Kir6.2-
containing KATP in mitochondria were conducted under basal conditions that do not involve IPC or its signaling molecule(s), a critical step that may upregulate
protein transport to mitochondria and thus confer cardioprotection (26).
Although KATP channel regulation and implication in cardioprotection
have been studied by many groups for years, no study has ever explored the
trafficking aspect of KATP function in mitochondria. The present study utilizing
KATP-deficient COS-7 cells represents a novel approach to elucidation of the molecular basis of the KATP channels in mitochondria and their regulatory
mechanism. We report the novel finding that a protein kinase C (PKC) isozyme,
PKCε, promotes mitochondrial import of Kir6.2-containing KATP channels from
cytosol, assessed with a combination of immunofluorescence microscopy,
Western blot, fluorescence resonance energy transfer (FRET) analysis, and
mitochondrial functional studies.
113 2. MATERIALS AND METHODS
DNA constructs
The hemagglutinin (HA) epitope was introduced into mouse Kir6.2 cDNA
by a sequential overlap extension polymerase chain reaction (PCR) as
described previously (318). The epitope was inserted at position 102 of Kir6.2.
The 11 amino acids (98GDLYAYMEKGIT99) were inserted into Kir6.2 before the
HA epitope. Mammalian expression constructs were either in pcDNA3
(Invitrogen) or pEGFP/pECFP/pEYFP (Clontech). Mito-cyan fluorescent protein
(CFP) or Mito-yellow fluorescent protein (YFP) was constructed by subcloning the mitochondrial presequence (the NH2- terminal 12 amino acids of the presequence) from subunit IV of cytochrome-c oxidase into the pEGFP-N1, pECFP-N1, or pEYFP-N1 vector (Clontech). To generate green fluorescent protein (GFP), YFP, or CFP fusion proteins, full-length Kir6.2 was amplified by
PCR and ligated into pEGFP-N1, pECFP-N1, or pEYFP-N1. All constructs were verified by DNA sequencing.
Cell culture and transfection
COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM)-F-12 supplemented with 10% fetal bovine serum, 2 mM glutamine,
and penicillin- streptomycin (113, 115). Cells were grown on coverslips or petri
dishes depending on experimental purpose. Cells were transfected with HA-
tagged (Kir6.2-HA) or GFP-fused (Kir6.2-GFP) Kir6.2 and SUR2A (pCMV) KATP
channel subunits or vectors alone by using FuGENE6 (148). Two to three days 114 later, the cells were treated with phorbol 12- myristate 13-acetate (PMA, 100
nM), PMA plus the PKC inhibitor chelerythrine (10 µM), or PMA plus the
selective PKCε inhibitor peptide myristoylated PKCε V1-2 (PKCε V1-2, 10–20
µM) for 30–60 min before the experiments. 4α-PMA (100 nM) was used as negative control.
Isolation of cardiomyocytes
Adult ventricular myocytes were isolated from Wistar rats (250–300 g) by
enzymatic dissociation (112, 115). The animal procedure was approved by the
Ohio State University Institutional Laboratory Animal Care and Use Committee.
In brief, hearts were excised and retrogradely perfused via the aorta with
oxygenated (100% O2) Tyrode solution containing (mM) 126 NaCl, 5.4 KCl, 1.0
CaCl2, 1.0 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose at 37°C. The
perfusate was then changed to a Tyrode solution that was nominally Ca2+ free but otherwise had the same composition. The hearts were perfused with the same solution containing collagenase for 20 min. The digested cell suspension was gently centrifuged, and the pellet was resuspended in culture medium 199 with 2% fetal bovine serum and 1% penicillin-streptomycin at 37°C.
Cardiomyocytes were placed in 100-mm petri dishes and incubated at 37°C in a humidified 5% CO2-95% air mix. Cardiomyocytes were subjected to various
pretreatments before mitochondrial isolation.
115 Preparation of mitochondria and mitoplasts
Mitochondrial fractions and total cell homogenate were prepared from
cultured COS-7 cells transiently transfected with Kir6.2 and SUR2A, isolated by
differential centrifugation, and further purified by 30% Percoll ultracentrifugation.
Cells transfected with vector alone were always used as a negative control. For
intact mitochondria or mitoplast colocalization experiments, COS-7 cells were
transfected with Kir6.2-GFP and SUR2A. Both cardiomyocytes and COS-7 cells
were stained with the mitochondrial marker MitoTracker (250 nM) for 15 min before fractionation. Cells after treatment were collected in an ice-cold homogenizing buffer containing (mM) 250 sucrose, 5 HEPES, and 5 EDTA, with proteinase inhibitor cocktail. Two 15-s homogenization cycles were performed on ice. The homogenate was centrifuged at 1,000 g for 10 min to remove nuclei and debris. The supernatant was centrifuged at 8,500 g for 20 min. The pellet containing the mitochondrial fraction was resuspended in the homogenizing buffer and further centrifuged at 8,500 g for 20 min. The washed mitochondria were then resuspended. For Percoll purification, the crude mitochondrial suspension (0.5 ml) was laid on the top of 10 ml of a solution containing 30% Percoll, 0.25 M sucrose, 1 mM EDTA, and 10 mM HEPES (pH
7.4). A self-generating Percoll gradient was developed by centrifugation at
95,000 g for 30 min at 4°C. The mitochondrial band was collected with a
Pasteur pipette and washed in the homogenizing buffer. Mitoplasts were
prepared from intact mitochondria by osmotic shock (264). Briefly, mitochondria
were subjected to osmotic shock for 5 min in hypotonic solution containing 116 (mM) 5 sucrose, 5 HEPES and 1 EGTA. Mitoplasts were sedimented from this
solution by centrifugation at 3,920 g for 5 min and resuspended in hypertonic
solution containing (mM) 750 KCl, 100 HEPES, and 1 EGTA (pH 7.2 with
KOH).
Western blotting
Immunoblot analysis was carried out for the mitochondrial fraction and
total cell homogenate from COS-7 cells expressing Kir6.2 and SUR2A after
treatment. The purity of mitochondria was evaluated with antibody against the
mitochondrial marker protein prohibitin, the plasma membrane marker Na+-K+-
ATPase, and the endoplasmic reticulum marker calreticulin to ensure that there was no significant contamination of other membrane fractions in mitochondria.
Mitochondria and total cell homogenate were denatured in a sample buffer, electrophoresed on 8–10% SDS-polyacrylamide gels (148), and transferred onto nitrocellulose membranes (276). The transferred blots were blocked with
5% nonfat milk in Tris-buffered saline (TBST; 150 mM NaCl, 20 mM Tris _HCl,
0.05% Tween 20, pH 7.4) and incubated for 1 h at room temperature with the antibodies against HA epitope (Roche) or Kir6.2 or SUR2A (Santa Cruz
Biotechnology) in TBST. After being washed, the blots were reacted with peroxidase-conjugated secondary antibodies (Jackson Immunuolabs) for 45 min and developed with an enhanced chemiluminescence detection system.
The specificity of antibodies for Kir6.2 and SUR2A was tested by preincubation of antibodies with their antigen peptides and/or confirmed in non-transfected 117 COS-7 cells or transfected cells with different KATP subunit. Equal loading was
confirmed by staining with Ponceau S or using antibodies against α-tubulin.
Immunofluorescence microscopy
Mitochondria or mitoplasts from cells after treatments were fixed with 4%
formaldehyde in PBS for 30 min, blocked, permeabilized in 5% goat serum in
PBS with 0.1% Triton X-100 (30 min), and labeled with primary antibody against
HA tag or Kir6.2 for 2 h. Mitoplasts isolated from COS-7 cells transfected with
Kir6.2-GFP/SUR2A were fixed only. Cells were washed three times and
labeled with fluorescence-conjugated secondary antibody for 1 h (113). We
used anti-Kir6.2 or anti-HA primary antibody for labeling KATP channel subunit
Kir6.2 and the mitochondrion-specific stain MitoTracker (250 nM) for labeling mitochondria. The cells were incubated with MitoTracker for 15 min before isolation of mitochondria/mitoplasts. Immunofluorescence was visualized with a fluorescent microscope (Nikon) or a confocal scanning laser microscope
(Zeiss).
FRET measurements
COS-7 cells were transfected with Kir6.2-CFP/SUR2A or Mito-YFP or
cotransfected with both Kir6.2-CFP/SUR2A and Mito-YFP. Images were
acquired sequentially through CFP, YFP, and FRET filter channels. Filter sets
used were the donor CFP, the acceptor YFP, and FRET. A background value
was determined from a region in each image without any cells. The background 118 value was subtracted from the raw images before FRET calculations were
carried out. Corrected FRET (FRETC) was calculated for entire images or selected regions of images, such as individual mitochondria, by using the
equation FRETC = FRET - (0.5 x CFP) - (0.3 x YFP), where FRET, CFP, and
YFP correspond to background-subtracted images of cells coexpressing CFP
and YFP acquired through the FRET, CFP, and YFP channels, respectively.
The 0.5 and 0.3 values are the fractions of bleed through of CFP and YFP fluorescence, respectively, estimated from cells expressing either CFP or YFP fusion proteins. Mean FRETC values were calculated from mean fluorescence
intensities for each selected subregion. FRETC images are presented as a
quantitative pseudocolor image. Data were analyzed with ImagePro and
MetaMorph.
Mitochondrial membrane potential
The changes in mitochondrial membrane potential (∆ψm) were monitored with the dye 5,5’,6,6’-tetrachloro-1,1’,3,3’- tetraethylbenzimidazolcarbocyanine iodide (JC-1) (233). Cells were treated with
4α-PMA, PMA, PMA plus chelerythrine, or PMA plus PKCε V1-2 and then stained with JC-1 (5 µM) at 37°C for 15 min and rinsed three times with Tyrode solution. The mitoKATP opener diazoxide or diazoxide plus the mitoKATP inhibitor
5-hydroxydecanoic acid (5-HD) were added 20 min before the measurement.
The observations were made with a fluorescence microscope. Solution
changes in this protocol were made by aspirating and replacing the contents of 119 the recording chamber. One hundred areas were selected from each image,
and the average intensity for each region was quantified by MetaMorph. The
ratio of JC-1 aggregate (red fluorescence) to monomer (green fluorescence)
intensity for each region was calculated after background subtraction. A
decrease in this ratio was interpreted as decrease of ∆ψm, whereas an
increase in the ratio was interpreted as gain in ∆ψm (277). Cells lacking red fluorescence were considered severely damaged and were excluded from analysis.
Statistics
Group data are presented as means ± SE. Unpaired t-test was used to
compare between groups. Multiple group means were compared by ANOVA
followed by post hoc least significant difference (LSD) test. Differences with a
two-tailed P<0.05 were considered statistically significant.
3. RESULTS
Localization of Kir6.2 in isolated mitoplasts
To determine whether PKC induces import of cardiac KATP to mitochondria, we took advantage of the COS-7 cell line, which lacks native KATP
channels (131), to deliver genes encoding cardiac KATP channel subunits Kir6.2
and SUR2A. We first examined whether the PKC activator PMA promotes the
mitochondrial import of recombinant cardiac KATP channels. 4α-PMA was used
120 as a negative control. To avoid colocalization of Kir6.2 with the mitochondrial
marker MitoTracker due to close apposition between Kir6.2-containing vesicles
and mitochondria, we performed colocalization experiments in mitoplasts
(devoid of outer membrane) freshly isolated from COS-7 cells transfected with
SUR2A and Kir6.2 with GFP fused to its COOH terminus. The cells were
pretreated with 4α-PMA, PMA, or PMA plus PKCε V1-2 for 60 min and were
stained with MitoTracker before isolation of mitochondria. Mitoplasts were
obtained by subjecting intact mitochondria to hypotonic shock (see
MATERIALS AND METHODS for details, section 2). About 20–30 images in the same treatment group (2 or 3 coverslips) from each experiment were analyzed.
Three independent experiments were conducted. Without PMA (100 nM, data not shown) or with 4α-PMA (100 nM) treatment, only a small portion of fluorescent Kir6.2 was localized in mitoplasts that were stained with
MitoTracker. PMA treatment significantly increased mitoplast localization of
Kir6.2 (Fig. 4.1). Quantification analysis revealed that the activation of PKC by
PMA increased the number of Kir6.2-positive mitoplasts by ~100% (Fig. 4.1C;
205.78 ± 8.84% vs. 4α-PMA, P<0.01). To identify which PKC isozyme is involved in PKC-induced increase in mitochondrial localization of Kir6.2, we studied the effect of the selective PKCε peptide inhibitor PKCε V1-2. PKCε has been shown to modulate KATP channels (125) and is linked to IPC (223). As
expected, the effect of PMA on mitochondrial localization of Kir6.2 was completely prevented by PKCε V1-2 (10 µM; 110.39 ± 10.33%). These results
121 indicate that Kir6.2-containing KATP channels are localized in mitochondria at low levels under normal conditions and can be enhanced by the activation of
PKC isozyme PKCε.
122
Figure 4.1. Immunofluorescence microscopy of mitochondrial localization of Kir6.2 in mitoplasts isolated from COS-7 cells. A: transmitted and fluorescent images of a mitoplast (mitochondria devoid of outer membrane) labeled with the mitochondrial marker MitoTracker. Scale bar: 3 µm. B: COS-7 cells were transfected with sulfonylurea receptor (SUR)2A/Kir6.2-green fluorescent protein (GFP), and mitoplasts were prepared after COS-7 cells were treated with agents as indicated. Results are representative of 3 independent experiments. About 20–30 images in the same treatment group (2 or 3 coverslips) from each experiment were analyzed. Scale bar: 5 µm. PMA, phorbol 12-myristate 13-acetate; PKCε, protein kinase C isoform ε. C: % of mitoplasts (MitoTracker positive) with Kir6.2-positive fluorescence are shown
(data were normalized to the control without treatment). **P < 0.01 vs. 4α-PMA.
123
Figure 4.1. Immunofluorescence microscopy of mitochondrial localization of Kir6.2 in mitoplasts isolated from COS-7 cells.
124 FRET analysis of Kir6.2 in mitochondrial inner membrane
Although our observation that Kir6.2 is localized in mitoplasts indicates
that Kir6.2 is present in mitochondrial inner membrane, there is a possibility that
part of the outer membranes of mitochondria may still be attached to mitoplasts.
To further define whether KATP channels are indeed localized in mitochondrial
inner membrane, we tested in COS-7 cells whether CFP fused to the COOH
terminus of Kir6.2 is sufficiently close to Mito-YFP that is targeted into the
mitochondrial matrix to yield FRET (90). If KATP channels are in the outer
membrane of mitochondria, the fluorescent protein fused to the cytoplasmic
domain of Kir6.2 would be exposed to the cytosol. If these channels actually
reside in the mitochondrial inner membrane, their cytoplasmic domains should
be exposed to the matrix enclosed by the mitochondrial inner membrane—the
equivalent of the cytosol for these endosymbiotic organelles. As shown in Fig.
4.2, FRETC was minimal in all regions of cells that were not stimulated with
PMA (Fig. 4.2A), indicating that there was no significant amount of Kir6.2 localized in mitochondria. In particular, FRET signals were not observed in the mitochondria enriched with Mito-YFP or in the Golgi area containing a large amount of Kir6.2-CFP. This validates the accuracy of our method of correction for the non-FRET component of the FRET images. Incubation of cells with PMA at 37°C for 30–60 min led to rapid import of Kir6.2 protein into mitochondria and exhibited FRET signals due to energy transfer from CFP at the COOH terminus of Kir6.2 to YFP in the mitochondrial matrix. To compare FRET efficiencies, the
FRETC was normalized to the intensity of Kir6.2-CFP after background 125 subtraction. The FRET signal between Mito-CFP and Mito-YFP was comparable to that between Kir6.2-CFP and Mito-YFP after PMA treatment for
60 min, indicating that by then the KATP channels targeted to the mitochondria
had fully exposed the CFP at the COOH terminus of Kir6.2 to the matrix. Similar
FRET signals were observed in cells expressing Mito-YFP, SUR2A, and Kir6.2
with CFP fused to its NH2 terminus (data not shown). For dipole-dipole coupling
to induce energy transfer from donor to acceptor, the fluorophore at the NH2 or
COOH terminus of Kir6.2 must be within 6–10 nm of the fluorophore within the mitochondrial matrix (90, 219). These findings strongly suggest that PKC activation causes insertion of Kir6.2-containing KATP channels into the
mitochondrial inner membrane.
126
Figure 4.2. A: Proximity of Kir6.2-cyan fluorescent protein (CFP) (left) to
Mito-yellow fluorescent protein (YFP) in mitochondrial matrix (center)
yielded fluorescence resonance energy transfer (FRET) signals (right, in
quantitative pseudocolor) in cells treated with PMA (100 nM) for 60 min
(bottom) but not in cells without PMA treatment (top). FRETC, corrected FRET.
B: FRET values were normalized by dividing FRETC by the mean intensity of
CFP after background subtraction. The FRET signal between Mito-YFP and
Mito-CFP provides calibration for a strong signal produced by 2 matrix
fluorescent proteins.
127
Figure 4.2. Proximity of Kir6.2-cyan fluorescent protein (CFP) (left) to Mito- yellow fluorescent protein (YFP) in mitochondrial matrix (center) yielded fluorescence resonance energy transfer (FRET) signals
128 Alterations of Kir6.2 protein in mitochondria
To assess the effect of PKC activation on Kir6.2 protein level, immunoblot analysis was carried out for Percoll-purified mitochondria fraction and total cell homogenate prepared from COS-7 cells treated with 4α-PMA,
PMA, PMA plus chelerythrine, or PMA plus PKCε V1-2 for 60 min. Enrichment of mitochondria was established by labeling with anti-prohibitin, a molecular marker of mitochondria. With anti-HA antibody, which is known as a very good antibody, a single band at 37 KDa was detected in the mitochondrial fraction as well as total cell homogenate (Fig. 4.3, A and B). Anti-HA antibody did not show any nonspecific band in our study. Interestingly, the band in the mitochondrial fraction from COS-7 cells treated with PMA for 60 min exhibited a more intense signal than that from COS-7 cells without PMA treatment or with 4α-PMA treatment. The PMA effect was significantly eliminated not only by the general
PKC inhibitor chelerythrine (10 µM) but also by the selective PKCε inhibitor peptide PKCε V1-2 (10 µM). However, there was no difference in the Kir6.2 band in total cell homogenate between cells treated with 4α-PMA and PMA. No
Kir6.2 band was detected from cells without transfection. Similar results were obtained with antibody against Kir6.2 (data not shown). Figure 4.3B shows average Kir6.2 band intensity normalized to control from three independent experiments. PMA induces almost 100% increase in Kir6.2 protein level in the mitochondrial fraction compared with that in the control group (216.11 ±
20.29%), which is consistent with the data in mitochondrial localization of
Kir6.2, but has no effect on total cellular Kir6.2 protein level. When we tried a 129 similar experiment with anti-SUR2A antibody, there was a single faint band of
SUR2A at a molecular mass of 150 KDa in the mitochondrial fraction under control conditions, which was not affected by PMA pretreatment (Fig. 4.3C).
This result indicates that only a minimum amount of SUR2A exists in
mitochondria, if there is any. However, we did not detect SUR2A with antibody
against SUR2, which is similar to the report by Lacza et al., (147) showing that
SUR2 was not present in mitochondria with the same antibody (purchased from
the same company). This discrepancy is likely due to the differential sensitivities of two different antibodies. The specificity of anti-SUR2A antibody
was confirmed in nontransfected COS-7 cells and COS-7 cells transfected with
SUR2B showing no cross-reactivity with SUR2B. We noted that the anti-SUR2A
antibody is very specific and more sensitive than the anti-SUR2 antibody.
These results suggest that Kir6.2 is present in mitochondria and PKCε
activation by PMA further increases Kir6.2 protein level in mitochondria. This
increase may not be due to increased protein synthesis for Kir6.2 since Kir6.2
in the total cellular fraction was not changed after PMA treatment.
130
Figure 4.3. Immunoblot analysis of Kir6.2 protein in mitochondria. COS-7 cells were transfected with Kir6.2-hemagglutinin (HA)/SUR2A. A: total cell homogenate (cellular fraction, CF) and mitochondrial fraction (MF) of COS-7 cells were prepared after cells were treated with 4α-PMA, PMA, PMA + chelerythrine (Che), or PMA + PKCε V1-2. These proteins were separated on an 8–10% SDS-polyacrylamide gel and immunoblotted with antibodies against
HA (top) and prohibitin (bottom). Thirty micrograms of protein for each lane was loaded for cellular fraction, whereas 20 µg of protein was loaded for mitochondrial fraction analysis. B: signal intensity of Kir6.2 in cellular and mitochondrial fractions was quantified as values normalized to control from 3 independent experiments with Image J software. C: similar Western blot analysis is shown for SUR2A in mitochondrial fraction with antibody against
SUR2A.
131
Figure 4.3. Immunoblot analysis of Kir6.2 protein in mitochondria 132 Kir6.2-dependent changes in mitochondrial membrane potential
To determine whether Kir6.2-containing KATP channels in mitochondria
are functional, we measured the changes in ∆ψm in intact COS-7 cells
transfected with Kir6.2 and SUR2A, using the potential-sensitive dye JC-1.
About 100 regions were selected from each image, which contains 10–20 cells,
and 5 images were taken from each well. Three independent experiments were
conducted, and each experiment had triplicate wells of the same treatment. The
average intensity for each image was quantified after background subtraction.
The green fluorescence of monomer indicates low potential (depolarized), and
the red fluorescence of J-aggregate indicates high potential (hyperpolarized).
Since KATP channels are usually not active because of inhibition by matrix ATP,
the mitochondrion-selective KATP opener diazoxide was used to activate KATP in mitochondria. Diazoxide has been shown to confer the cardioprotection of IPC by selectively activating mitoKATP channels (86, 199, 292). If Kir6.2/SUR2A-
containing KATP channels are localized in mitochondrial inner membrane and
involved in the protection of IPC, this channel is expected to be sensitive to
activation by diazoxide. The KATP-dependent changes were verified by the
mitochondrial KATP-selective inhibitor 5-HD. In the absence of diazoxide, the JC-
1 ratio (J-aggregate/monomer) in cells pretreated with PMA for 60 min was not
significantly different from the control cells pretreated with 4α-PMA (Fig. 4.4B).
However, diazoxide (100 µM), within 15 min of application, decreased the JC-1
ratio significantly in PMA-treated cells (potential decreased, green fluorescence
increased; Fig. 4.4A) compared with that in 4α-PMA-pretreated cells with 133 diazoxide (Fig. 4.4; 53.04 ± 10.22% vs. 97.87 ± 9.24%, PMA + diazoxide vs.
4α–PMA + diazoxide, P 0.01). When diazoxide was added to the solution 5 min
after the application of 5-HD, the diazoxide-induced decrease in ∆ψm in cells
treated with PMA was inhibited not only by 5-HD (500 µM; 83.79 ± 6%) but also by pretreatment with PKCε V1-2 (20 µM; 82.61 ± 4.56%). Either 5-HD application or PKCε V1-2 pretreatment alone did not have any effect on ∆ψm
(data not shown). These data show that ∆ψm in cells pretreated with PMA was
not different from that in cells with 4α-PMA but could be decreased by activation of KATP channels in mitochondria with the mitoKATP opener diazoxide after PMA
pretreatment, an effect that was inhibited by the mitoKATP inhibitor 5-HD.
Furthermore, the diazoxide effect in PMA-treated cells was not seen in nontransfected COS-7 cells, indicating that the effect of diazoxide on ∆ψm was
due to activation of Kir6.2-containing KATP channels in mitochondrial inner
membrane. The finding that diazoxide induces KATP-dependent depolarization
of ∆ψm after activation of PKCε by PMA pretreatment but not in the control
group with 4α-PMA treatment suggests that K+ influx caused by diazoxide
under basal conditions may not be sufficient to cause significant changes in
∆ψm under our experimental conditions. This result does not exclude the
possibility that other mitochondrial functions may be altered by diazoxide under
normal conditions or in isolated mitochondria where no translocation occurs.
Furthermore, the observation that PMA pretreatment alone did not change ∆ψm
suggests that KATP channels in mitochondrial inner membrane are normally
inhibited by matrix ATP even though PMA may increase the number of 134 functional Kir6.2-containing KATP channels in mitochondria. It should be noted that PMA was used only for pretreatment and was washed out before measurement of ∆ψm with diazoxide.
135
Figure 4.4. PMA-induced KATP-dependent changes in mitochondrial
membrane potential (∆ψm). A: COS-7 cells transfected with SUR2A/Kir6.2-HA
were incubated with mitochondrial potential-sensitive dye JC-1 (5 µM) for 15
min after various pretreatments as indicated. Diaz, diazoxide. Scale bar: 20 µm.
B: ∆ψm expressed as a ratio of J-aggregate to monomer fluorescence in different treatments. Three independent experiments were conducted, and each experiment had triplicates of the same treatment where ~15 images were collected. The average fluorescent intensity for each image was calculated after background subtraction. Data from different treatment groups were normalized to the control group without PMA treatment. **P <0.01 vs. 4α-PMA.
136
Figure 4.4. PMA-induced KATP-dependent changes in mitochondrial
membrane potential (∆ψm)
137 Localization of Kir6.2 in isolated mitochondria from cardiomyocytes
To exclude the possibility that overexpression of recombinant KATP channels contributes to our findings, we performed similar colocalization experiments in isolated mitochondria from rat adult cardiomyocytes. Because of very tightly packed myofibrils around mitochondria and other organelles in cardiomyocytes, it is very difficult to study subcellular localization of membrane proteins in isolated cardiomyocytes. We therefore performed colocalization experiments in isolated mitochondria. Cardiomyocytes were subjected to pretreatment with 4α-PMA, PMA, or PMA plus PKCε V1-2 for 60 min before mitochondrial isolation. To avoid bias in our analysis, 30 images were randomly collected from the triplicates of the same treatment in each independent experiment. Two to five independent experiments depending on treatments were conducted, and ~1,000 mitochondria were counted for statistical analysis.
Anti-Kir6.2 antibody was used to detect native Kir6.2 protein. As Fig. 4.5A shows, PMA increased mitochondrial localization of Kir6.2, as indicated by increased yellow staining in the merged image. The pretreatment of PMA alone with PKCε V1-2 blocked the PMA effect. Quantitative analysis by normalizing
Kir6.2-positive mitochondria to the control without PMA treatment showed that
PMA significantly increased the number of Kir6.2-positive mitochondria (Fig.
4.5B; 240.31 ± 13.6% vs. 4α-PMA, P<0.01). This PMA effect was almost completely prevented by pretreatment with the PKC inhibitor chelerythrine (10
µM; data not shown) as well as the selective PKCε inhibitor peptide PKCε V1-2
(10 µM; 120.02 ± 9.77% vs. PMA group, P<0.01). These results are consistent 138 with our observation in COS-7 cells transiently transfected with Kir6.2/SUR2A and support the scenario that Kir6.2-containing KATP channels are localized in mitochondria and this mitochondrial localization was further enhanced by PKC activation.
139
Figure 4.5. Immunofluorescence microscopy of mitochondrial localization
of Kir6.2 in mitochondria isolated from rat adult cardiomyocytes. A: fluorescent images of mitochondria with Kir6.2 labeled with anti-Kir6.2 antibody.
The intact mitochondria were prepared after treatment of rat cardiomyocytes with agents as indicated. Results are representative of 2–5 independent experiments depending on the treatments, and each experiment had triplicates
of the same treatment with 30 images taken. Scale bar: 10 µm. B: normalized
% of mitochondria with Kir6.2-positive fluorescence over control. **P < 0.01 vs.
4α-PMA.
140
Figure 4.5. Immunofluorescence microscopy of mitochondrial localization of Kir6.2 in mitochondria isolated from rat adult cardiomyocytes
141 4. DISCUSSION
The present study points to a novel trafficking mechanism for dynamic
regulation of KATP channel number: PKC-induced import of KATP channels into mitochondria. We show that the KATP channel pore-forming subunit Kir6.2 is localized in mitochondria, using both cardiomyocytes and KATP channel deficient
COS-7 cells transiently transfected with Kir6.2 and SUR2A. Most importantly,
we demonstrate that activation of PKC isozyme PKCε significantly increases
the number of functional Kir6.2-containing KATP channels in mitochondrial inner
membrane, possibly by promoting import of Kir6.2 to mitochondria from cytosol.
One likely scenario is that KATP channels are simultaneously targeted to not only cell surface but also mitochondrial inner membrane after their synthesis in the cytosol and PKC activation enhances the import of these channels to
mitochondria. The specific pharmacological and biophysical properties of KATP
channels at the plasma membrane and mitochondrial inner membrane may
differ depending on the environment in which they reside and the regulatory
factors with which they are associated (55).
The function of ion channels depends critically on not only channel
activity but also the number of functional channels. Our observation that
activation of PKCε by PMA increases mitochondrial localization of Kir6.2- containing KATP channels points out a novel trafficking mechanism in regulation of KATP channel function. However, this finding does not exclude the possibility
that PKC may directly interact with the channel. Actually, PMA has been shown
to activate cell surface Kir6.2/SUR2A-containing KATP channels (112, 163) as 142 well as mitoKATP channels (125). Under our experimental conditions, PMA was
washed out before we tested diazoxide-induced changes in ∆ψm. If there is any
residue of PMA, it may not be enough to activate KATP channels and cause
alterations in ∆ψm. The present study mainly attempted to address whether KATP
channels translocated by PKC activation are functional.
Our data and that of others (54, 147, 265, 319) show that there is a basal
level of Kir6.2-containing KATP channels in mitochondria. It is therefore likely
that K+ channel openers or stimulators such as PMA may directly activate these channels under normal conditions or in isolated mitochondria where no translocation occurs. In the present study, diazoxide alone did not change ∆ψm
in control cells without PMA or pretreated with 4α-PMA. One explanation is that
K+ influx induced by diazoxide in COS-7 cells may not be high enough to cause
significant changes in ∆ψm but may produce changes in other mitochondrial
functions that we did not measure, such as matrix volume. On the other hand,
our observation that diazoxide caused changes in ∆ψm after PMA treatment
indicates that the KATP channels translocated by PMA are functional by
increasing K+ influx. However, our data do not imply that the same functional
consequence in mitochondria (∆ψm) caused by diazoxide-induced opening of
KATP channels would occur in native cardiomyocytes. We employed COS-7
cells with expressed recombinant KATP channels, which may be different from
cardiomyocytes in terms of the extent of K+ influx. We therefore do not believe
that our data conflict with that of others showing no significant changes in ∆ψm
by diazoxide induced opening of mitoKATP (50, 146) from isolated heart 143 mitochondria. Nevertheless, the purpose of the JC-1 experiment was not to
define ultimate changes in mitochondrial function by diazoxide but rather to
study whether the translocated Kir6.2-containing KATP channels are functional.
Diazoxide was used as a tool to activate KATP channels since KATP channels are
normally inactive.
Although it is generally assumed that the Kir6.2/SUR2Acontaining KATP
channel is localized exclusively at the sarcolemma there is evidence that Kir6.2 or SUR2A is present in mitochondria. Several groups have reported that Kir6.2
has been found more or less in isolated heart mitochondria (54, 147, 265, 319).
One study has reported that Kir6.2 is not part of the components of mitoKATP
channels in the rabbit heart (256). The discrepancy may be due to the relatively
low level of Kir6.2-containing KATP channels in mitochondria under normal
conditions. The technical difficulties in dissecting a small amount of membrane-
targeted proteins localized in mitochondria with conventional
immunofluorescence microscopy may contribute to the inconsistency,
especially in adult cardiomyocytes with tightly packed myofibrils and
suborganelles. Differential sensitivity of antibodies against individual KATP
subunits may also likely be a reason for the discrepancy. With regard to the
SUR subunit, less is known about its presence in mitochondria because of the
difficulty in getting antibody specific to SUR2A or SUR2B. We tested anti-
SUR2A antibody, recently available commercially, and found a faint band of
SUR2A present in mitochondria of COS-7 cells by Western blot, which is
consistent with some reports (55, 265). Similar to another study (147), we did 144 not detect a SUR2 band with anti-SUR2 antibody in mitochondria. Because of
the lack of series of studies with different approaches, we cannot make a
conclusion at the present time that SUR2A is indeed localized in mitochondria.
The contribution of Kir6.1 to mitoKATP channels is also under debate
(184, 268). The Kir6.1/SUR2B-based channels do not exhibit sensitivity to the inhibition by intracellular ATP (310), whereas the mitoKATP channels recorded in
mitochondria inner membrane from liver and reconstituted lipid bilayer from
purified heart mitochondria are very sensitive to ATP, a characteristic feature of
the KATP channel pore-forming subunit Kir6.2 (56). Although Kir6.1 protein level
was increased by ischemia, this only occurred after prolonged ischemia (60 min
of ischemia followed by 24–72 h of reperfusion). Sublethal ischemia (15–30 min
followed by 24 h of reperfusion), which is equivalent to the time course of PMA
treatment in the present study, did not induce Kir6.1 expression (5). It is
therefore less likely that Kir6.1 would contribute to functional mitoKATP channels
even though it was found in mitochondria (268). Furthermore, knockout of
Kir6.1 gene in intact mice does not disrupt mitoKATP opening (184).
Nevertheless, the present study focuses on the regulatory mechanism of Kir6.2- containing KATP channels in mitochondria and does not exclude the possibility
that other protein(s) may form the part of KATP channel complex in mitochondria.
The dual distribution at both plasma membrane and mitochondria has
been reported for other ion channel proteins, such as Kv1.3 (271), the Ca2+- activated BK potassium channels (264), the voltage-dependent anion channels
(20, 63), and connexin43 (26, 158). Interestingly, connexin43 in mitochondria 145 has recently been shown to be increased by IPC, possibly through
phosphorylation (26). These channels lack the NH2-terminal mitochondrial
targeting sequence but are targeted to mitochondrial inner membrane by
unknown mechanisms. Many mitochondrial proteins of the inner membrane
such as mitochondrial carriers lack the NH2-terminal targeting sequence but
instead contain sorting and targeting information throughout the mature protein,
which is hardly recognizable (32, 58). The targeting information seems to
comprise several distant amino acids spread throughout the entire protein
(278). Similarly, Kir6.2 does not exhibit a classic NH2-terminal mitochondrial
targeting presequence according to the prediction from TargetP (71) and may
contain unidentified internal targeting sequences. Although specific
posttranslational protein modifications and differential splicing have been
hypothesized to account for organelle protein targeting, the mechanisms
responsible for the simultaneous targeting of a protein to different cellular compartments are not known and are beyond the focus of the present study.
Import of most nucleus-encoded proteins into mitochondria is highly
regulated and mediated by mitochondria targeting sequence with the aid of a handful of distinct complexes including molecular chaperones (137). Both heat shock protein (HSP)70 and HSP90, which are linked to cardioprotection, have been shown to facilitate mitochondrial protein import to mitochondria (154, 278,
286, 288, 314, 315). HSP90 and HSP70 specifically interact with the mitochondrial protein import receptor TOM70 at the outer membrane and are required for translocation of precursor proteins. Furthermore, PKC has been 146 shown to be involved in HSP-mediated cardioprotection (45, 302). The present
finding points to an important mechanism in Kir6.2-containing KATP channel-
mediated cardioprotection: PKCε- induced mitochondrial import. It should be
noted that the present study was not aimed at defining the molecular identity of putative mitoKATP but rather at studying the regulatory mechanism involved in
Kir6.2-containing KATP channel trafficking. This observation does not exclude
the possibility that a distinct mitoKATP or other KATP component(s) in
mitochondria may exist. It has been reported that succinate dehydrogenase
may be a part of a mitoKATP macromolecular supercomplex that contributes to
cardioprotection (9). A Ca2+-activated K+ channel has also been found in
mitochondrial inner membrane and linked to cardioprotection (308).
The evidence supporting a protective role for the Kir6.2- containing KATP
channels has been provided by studies using KATP-deficient COS-7 cells and
Kir6.2-knockout mice. By cotransfection of Kir6.2/SUR2A genes, Jovanovic et
al., (131) demonstrated that delivery of Kir6.2 and SUR2A genes into COS-7
cells resulted in protection against hypoxia-reoxygenation injury as a result of
inhibition of intracellular Ca2+ loading. Studies in Kir6.2-knockout mice (99, 269) showed a failure of IPC to reduce infarct size and preserve contractile recovery in Kir6.2-deficient mice. The activation of PKC has also been linked to cardioprotection (223) and KATP channels (163, 291). A number of possibilities
have been suggested as to how opening the mitoKATP results in cardioprotection, such as reduced Ca2+ overload in mitochondria by
147 depolarizing, mild uncoupling and reduced free radical production, and moderate swelling and increased ATP production (50).
It is known that movement of key proteins into mitochondria during apoptosis is essential for the regulation of apoptosis (42). Mitochondria are increasingly recognized as key players in cell survival (92). Most of the mitochondrial proteins are nuclear encoded, synthesized in the cytosol, and transported to mitochondria by translocases (137, 166, 220). Given that mitochondria are a critical site for cardioprotection; our observations indicate that the dynamic regulation of protein trafficking to mitochondria represents a novel mechanism in cardioprotection of IPC.
ACKNOWLEDGMENTS
We are grateful to S. Seino for providing Kir6.2 and SUR2A clones and to R. Y. Tsien for the kind gifts of Mito-CFP and Mito-YFP constructs. We thank
Douglas Pfeiffer for comments on the mitochondrial aspect of the study. We also thank Chen Gu and Ofer Wiser for technical help on the FRET assay and
Lily Jan for valuable support and suggestions. This study was supported in part by a research grant from the American Heart Association.
148
CHAPTER 5
CONCLUSIONS AND FUTURE DIRECTIONS
The novel findings of my dissertation work are:
1) KATP channels in the heart are predominantly present in cholesterol-rich
membrane microdomains called as caveolae
2) This caveolar localization is essential for adenosine activation of KATP
channels
3) Caveolin-3 has an inhibitory effect on activation of KATP channels
4) PKC activation induces import of Kir6.2 containing KATP channels to
mitochondria.
The cellular plasma membrane is not homogenous throughout. It is
extensively compartmentalized based on specific lipid and protein composition
(rafts/caveolae). These membrane microdomains can segregate and
concentrate specific signaling pathway components for rapid and more efficient
communication between extracellular environment and intracellular
homeostasis. For ion channels, their subcellular-localization, trafficking and
local environment can profoundly affect their function and properties. ATP-
sensitive potassium channels link the cellular metabolism with the membrane 149 excitability in various cell-types. In the heart, not only they shorten APD in
response to ischemia, their cytoprotective role has gained increasing
recognition in recent years. Traditionally classified just as a plasma membrane
protein, these KATP channels have been shown to be present in other subcellular organelles. Their localization can alter their response to various
stimuli. With this theme in mind, my dissertation work aimed to investigate
three related areas: (1) To characterize KATP channel subcellular localization on
plasma membrane caveolae and mitochondria; (2) Regulation of KATP channel
function by these membrane caveolae and associated protein caveolin-3; (3)
Trafficking of KATP channels to mitochondria in response to PKC stimulation.
The overall project was broadly divided into two parts based on membrane
microdomain or mitochondrial localization and these parts were separately
investigated.
We first studied the specific membrane microdomain localization of KATP channel using different techniques to rule out any confounding factors related to specific techniques utilized for the investigation. We report for the first time that cardiac KATP channels, consisting of Kir6.2/SUR2A are predominantly
concentrated in cholesterol-rich caveolar microdomains of the plasma
membrane. To understand the functional significance of this localization, we
employed adenosine which is one of the main KATP channel regulator under
ischemic conditions. Since adenosine receptors are also present in caveolae,
localization of both KATP channel and adenosine in the same compartment
signifies that probably this spatial closeness is important for regulation of KATP 150 channel function. And indeed, our patch clamp studies confirmed that intact
caveolar structure is important for adenosine activation of KATP channel. Since
many of the other KATP channel modulators (besides adenosine) are also
present in caveolae, it would be interesting to know if those regulators also
follow the same paradigm of caveolae dependent signaling similar to
adenosine.
Further, our discovery of caveolin-3 mediated inhibition of KATP current
using a simplified system revealed another interesting yet paradoxical aspect of
KATP channel regulation by caveolae. Activation of only 1% of total membrane
KATP channel can shorten the APD by 50%. Since excessive KATP channel
activation would be detrimental for the heart leading to silencing of electrical
activity and arrhythmias. Caveolae dependent control of both activation and
inhibition of KATP channel seems quite logical and brings forth the fact that KATP channels in the heart are very tightly regulated.
It is well known that KATP channels play an important role in cardioprotection and ischemic preconditioning (93). Importance of caveolin proteins and
caveolae mediated signal transduction in cardioprotection is just beginning to
unravel (213, 215). It would be interesting to determine the relative contribution
of caveolar and non-caveolar KATP channels to cardioprotection. Nevertheless,
there are several other related questions which we wish to investigate in our
future studies. Most important of these is, how are KATP channels targeted to
caveolae? Secondly, what is the kinetics and dynamics of KATP channel stay in
caveolae? Identifying the stimuli and mechanisms which increase the levels of 151 KATP channels in caveolae, and increase their residence time in caveolae will
have significant therapeutic implications. Lastly, how does the caveolin-3
protein mediate its inhibitory effect on KATP channel? We identified that caveolin-3 scaffolding domain is important in this interaction, but we don’t know which subunit of KATP channels it binds to.
In our effort to characterize the sub-cellular localization of cardiac KATP
channels, we detected a small amount of Kir6.2 in mitochondria of cardiac
myocytes and transfected COS-7 cells under basal conditions. But it does not
affect the mitochondrial function significantly. We showed that this localization
of channel protein in mitochondria can be significantly increased by specific
stimuli namely activation of PKCε here. Further, KATP channels translocated to
mitochondria were shown to be functional and affected the mitochondrial
potential when pretreated with PMA. This corroborates the statement made
above that KATP channel localization in a cardiac myocyte is very dynamic and a
controlled process which should be investigated further in detail under various
physiological conditions. In a latter collaborative study, I found that indeed
hypoxic preconditioning (one of the activator of endogenous cardioprotective mechanism similar to IPC) enhances Kir6.2 targeting to mitochondria in a heat-
shock protein 90 (HSP90)-dependent manner.
Collectively, our elucidation of caveolar and mitochondrial localization;
and regulation of KATP channels will significantly enhance our knowledge of the
complexity of regulation of this metabolic sensor. This will be potentially useful
in understanding the endogenous cardioprotective strategies employed by the 152 heart and their alteration in some pathological conditions. Therapeutic strategies can be further designed to enhance these cardioprotective regulatory mechanisms. The hypothetical model based on the findings of this thesis work is shown in fig. 5.1.
153
Figure 5.1. Hypothetical model for the targeting of KATP channels to
plasma membrane, caveolar microdomains and mitochondria. After
synthesis in the endoplasmic reticulum, KATP channels (Kir6.2/SUR2A) are
targeted to plasma membrane. Caveolae help in compartmentalizing KATP channels with its modulator adenosine A1 receptor. Caveolin-3 can bind to and
inhibit KATP channel independent of its localization to caveolae also. PKC
activation induces import of Kir6.2 into mitochondria. Abbreviations: Cav-3 =
caveolin-3, TIM/TOM = protein translocation machinery of mitochondria.
154
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