Hormonal Control and Pharmacology of Btrek-1 K+ Channels in Bovine Adrenal Zona Fasciculata Cells

Home , Niguldipine

HORMONAL CONTROL AND PHARMACOLOGY OF bTREK-1 K+ CHANNELS

IN BOVINE ADRENAL ZONA FASCICULATA CELLS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Haiyan Liu, M.S.

*****

The Ohio State University

2009

Dissertation Committee:

Professor John J. Enyeart, Adviser

Professor R. Thomas Boyd Approved by

Professor Jack A. Boulant

Professor Michael X. Zhu Adviser Biophysics Graduate Program

ABSTRACT

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K+ channels that belong to the two-pore/four-transmembrane segment (2P/4TMS) family of K+ channels. They set the resting membrane potential and function in coupling receptor activation at the membrane to depolarization-dependent Ca 2+ entry and cortisol secretion. Angiotensin II (ANG II) and adrenocorticotropic hormone (ACTH) inhibit bTREK-1 channels in AZF cells. However, the signaling pathways underlying the inhibition of bTREK-1 by ANG II and ACTH are partially understood.

In my thesis, I explored the signaling mechanisms by which ANG II and

ACTH inhibit bTREK-1 channels and bTREK-1 pharmacology from bovine AZF cells in whole cell patch clamp recordings. I found that the ATP-dependent inhibition of bTREK-1 by ANG II occurred through a novel mechanism that was independent of phospholipase C (PLC), protein kinase C (PKC) and phosphatidylinositol 4, 5 bisphosphate (PIP 2). These results indicate that under physiological conditions, ANG II inhibits bTREK-1 by two novel independent pathways that diverge proximal to the activation of PLC, which is different from that described in expression systems.

Next, ACTH, NPS-ACTH, and forskolin inhibited bTREK-1 channels in

AZF cells through cAMP by both PKA-dependent and independent signaling mechanisms which involved the activation of Epac2 (a guanine nucleotide exchange protein activated by cAMP). These results indicate that ACTH and cAMP inhibit bTREK-1 through parallel PKA-and Epac-dependent signaling mechanisms which may provide for failsafe membrane depolarization by ACTH.

Finally, I investigated five Ca 2+ channel antagonists as inhibitors of bTREK-1 channels and discovered that selected dihydropyridine (DHP) Ca 2+ channel antagonists such as amlodipine and niguldipine potenly inhibited native bTREK-1 channels. Overall, these results demonstrate that organic Ca 2+ antagonists rank as the most potent inhibitors of TREK-1 channels yet described.

Together, these studies described novel signaling mechanisms by which

ANG II and ACTH inhibit bTREK-1 channels in AZF cells and established the pharmacological profile of TREK-1 channels. These results provide important insights on the physiological roles of bTREK-1 channels in cortisol secretion and the potential target for drug development. .

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Dedicated to my family and parents

ACKNOWLEDGMENTS

I would like to express my sincere gratitude to my adviser, Dr. John

Enyeart, for always being a considerate mentor, for his intellectual support, constant guidance, passionate encouragement, and his patience throughout my whole graduate study.

I am grateful to Judy Enyeart for never hesitating to share her academic and technical expertise, for her countless assistance and encouragement in the lab and for her invaluable help for my life outside the lab.

I also wish to thank my committee members, Drs. R. Thomas Boyd, Jack

A. Boulant, and Michael X. Zhu for their precious time, stimulating discussions and advising.

I am greatly indebted to my husband, Dr. Rui Xiao, who has always been on my side and support me with his love throughout the whole time.

At last, I would like to thank my parents and my parents-in-law from the bottom of my heart. It is almost impossible to have my graduate study without their helping me taking care of my children.

VITA

October 25, 1977……………………………………Born-Xinxiang, Henan, China

1996-2000…………………………………………….B.S. in Biology, Beijing Normal

University

2000-2003………………………...... M.S. in Cell Biology, Beijing

Normal University

2003-2004……………………………………………Graduate student, Department

of Physiology, The University of

Texas Health Science Center at

San Antonio

2004-present….……………………………………..Ph.D. Candidate, Biophysics

Graduate program, the Ohio

State University

PUBLICATIONS

Research Publication

1. Liu H , Enyeart JA, Enyeart JJ. ACTH inhibits bTREK-1 K+ channels through multiple cAMP-dependent signaling pathways. J Gen Physiol. 132(2): 279-94, (2008).

2. Enyeart JA, Liu H , Enyeart JJ. Curcumin inhibits bTREK-1 K+ channels and stimulates cortisol secretion from adrenocortical cells. Biochem Biophys Res Commun. 370(4): 623-8, (2008). vi

3. Liu H , Enyeart JA, Enyeart JJ. Potent inhibition of native TREK-1 K+ channels by selected dihydropyridine Ca2+ channel antagonists. J Pharmacol Exp Ther. 323(1): 39-48. (2007).

4. Liu H , Enyeart JA, Enyeart JJ. Angiotensin II inhibits native bTREK-1 K+ channels through a PLC-, kinase C-, and PIP2-independent pathway requiring ATP hydrolysis. Am J Physiol Cell Physiol. 293(2): C682-95. (2007).

5. Liu H , Danthi SJ, Enyeart JJ. Curcumin potently blocks Kv1.4 potassium channels. Biochem Biophys Res Commun. 344(4): 1161-5. (2006).

6. Enyeart JJ, Danthi SJ, Liu H , Enyeart JA. Angiotensin II inhibits bTREK-1 K+ channels in adrenocortical cells by separate Ca2+- and ATP hydrolysis- dependent mechanisms. J Biol Chem. 280(35): 30814-28. (2005).

FIELDS OF STUDY

Major Field: Biophysics

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TABLE OF CONTENTS

page ABSTRACT ...... ii DEDICATIONS ...... iv ACKNOWLEDGMENTS ...... v VITA ...... vi LIST OF TABLES ...... xi LIST OF FIGURES ...... xii ABBREIVIATIONS ...... xiv

CHAPTERS:

Chapter 1 General introduction ...... 1

1.1. A general overview of two-pore-domain K + channels ...... 1 1.2. Biophysical properties of TREK-1 K + channels ...... 3 1.2.1. Ion permeation ...... 3 1.2.2. Time and voltage denpendency ...... 3 1.3. Modulation of TREK-1 K + channels ...... 4 1.3.1. Physical stimulators ...... 4 1.3.2. Chemical stimulators ...... 6 1.3.3 G-protein coupled receptor signaling pathways ...... 8 1.3.4 Other second messenger pathways ...... 11 1.4. Pharmacology of TREK-1 K + channels ...... 12 1.5. Physiological functions of TREK-1 K + channels ...... 13 1.5.1. Physiological functions in neurons ...... 13 1.5.2. Physiological functions in adrenal ...... 14

Chapter 2 Methods and materials ...... 20

2.1. Materials ...... 20 2.2. Isolation and culture of AZF cells ...... 21 2.3. Patch-clamp experiments ...... 22 2.4. Measurement of Epac2 mRNA ...... 24 2.5. PKA Assay ...... 25 2.6. Transient Transfection and Visual Identification of HEK293 Cells Expressing bTREK-1 ...... 25 viii

Chapter 3 Angiotensin II inhibits native bTREK-1 K + channels through a PLC, PKC, and PIP 2-independent pathway requiring ATP hydrolysis ...... 27

3.1. Introduction ...... 27 3.2. Results ...... 30 3.2.1. PLC and ANG II inhibition of bTREK-1 ...... 32 3.2.2. PKC and ANG II inhibition of bTREK-1 ...... 33 3.2.3. Other kinases ...... 36 3.2.4. Inorganic polyphosphates block ATP-dependent bTREK-1inhibition . 38 3.2.5. PIP 2 and bTREK-1 gating ...... 39 3.2.6. Activation of bTREK-1 by a high-affinity ATP analog ...... 40 3.2.7. PIP 2 and voltage-dependent bTREK-1 gating ...... 41 + 3.2.8. PIP 2 and ANG II-dependent inhibition of bTREK-1 K channels ...... 41 2+ 3.2.9. PIP 2 and Ca -dependent regulation of bTREK-1 ...... 42 3.3. Discussion ...... 43

Chapter 4 ACTH Inhibits bTREK-1 K + Channels through Multiple cAMP- dependent Signaling Pathways ...... 61

4.1. Introduction ...... 61 4.2. Results ...... 64 4.2.1. bTREK-1 Inhibition by ACTH and cAMP Is Voltage Independent ...... 66 4.2.2. NPS-ACTH Inhibits bTREK-1 by a PKA- and Ca 2+ -independent Mechanism ...... 67 4.2.3. Forskolin Inhibits bTREK-1 by a PKA-independent Mechanism ...... 69 4.2.4. Effect of 8-pCPT-2'-O-Me-cAMP on PKA Activity and bTREK-1 Current in Perforated Patch Recordings ...... 70 4.2.5. Inhibition of bTREK-1 by Intracellular Application of 8-pCPT-2'-O-Me- cAMP ...... 71 4.2.6. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP Is Independent of PKA ...... 72 4.2.7. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP Requires Hydrolyzable ATP ...... 73 4.2.8. 8-pCPT-2'-O-Me-cAMP Fails to Inhibit bTREK-1 when Epac2 Expression Is Suppressed by ACTH...... 75 4.2.9. 8-pCPT-2'-O-Me-cAMP Does Not Inhibit bTREK-1 Activity in Transfected HEK293 Cells ...... 76 ix

4.3. Discussion ...... 77

Chapter 5 Potent Inhibition of Native TREK-1 K + Channels by Selected Dihydropyridine Ca 2+ Channel Antagonists ...... 94

5.1. Introduction ...... 94 5.2. Results ...... 96 5.2.1. Inhibition of bTREK-1 by DHP Ca 2+ Channel Antagonists ...... 97 5.2.2. Amlodipine Reduces bTREK-1 Open Probability but Not Unitary Conductance ...... 98 5.2.3. Effects of Other Ca 2+ Channel Antagonists on bTREK-1 ...... 99 5.2.4. Amlodipine Inhibition of T-type Ca 2+ Current ...... 101 5.3. Discussion ...... 101

Chapter 6 Summary and conclusion ...... 115

BIBLIOGRAPHY ...... 120

LIST OF TABLES

TABLE 5.1 pharmacology of TREK-1 K + channels inhibition…..….…………….114

LIST OF FIGURES

Page

Figure 1.1 Structure and phylogenetic tree of K2P channels.….………………….18

Figure 1.2 The physiology role of bTREK-1 K + channel in adrenal………………19

Figure 3.1 ANG II inhibits bTREK-1 by a PLC-independent mechanism…….….52

Figure 3.2 Inhibition of bTREK-1 by PKC activation…………………....………….53

Figure 3.3 PKC- and ATP-dependent bTREK-1 inhibition by ANG II …..…...…..54

Figure 3.4 Calphostin C and ANG II inhibition of bTREK-1….……………………55

Figure 3.5 Effect of kinase inhibitors and inorganic polytriphosphate (PPPi) on ANG II inhibition of bTREK-1 …………………………………………….56

Figure 3.6 Activation of bTREK-1 channels by 6-PhEt-ATP, DiC 8PI(4,5)P 2, UTP, PPPi, and arachidonic acid (AA)..….……………………………………57

Figure 3.7 Effect of DiC 8PI(4,5)P 2 on ANG II inhibition and voltage dependence of bTREK-1……………..…….……………………………………………58

2+ Figure 3.8 Effect of DiC 8PI(4,5)P 2 on Ca -dependent expression and inhibition of bTREK-1 by ANG II…….. ……………….…………….…………………59

Figure 3.9 Model for ANG II inhibition of bTREK-1……….….…………………….60

Figure 4.1 Effect of PKA inhibitors on bTREK-1 inhibition by ACTH.……………84

Figure 4.2 bTREK-1 inhibition by ACTH and 8-pCPT-cAMP is voltage independent ………………………………………………….……………85

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Figure 4.3 Inhibition of bTREK-1 by NPS-ACTH is Ca 2+ and PKA independent …………….…………………………………………………86

Figure 4.4 Effect of PKA inhibitors on bTREK-1 inhibition by forskolin.…………87

Figure 4.5 Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings… …………….…………………………………………………88

Figure 4.6 Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2'-O-Me- cAMP …………………………………………………………….…………89

Figure 4.7 bTREK-1 inhibition by 8-pCPT-2'-O-Me-cAMP is independent of PKA ……….…………….……………………………………….…………90

Figure 4.8 Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP in twice-patched cells …...……………………………………………………………………91

Figure 4.9 Effect of suppression of Epac2 expression on bTREK-1 inhibition by 8- pCPT-2'-O-Me-cAMP and 8-br-cAMP ……….……………………….92

Figure 4.10 6-Bnz-cAMP but not 8-pCPT-2'-O-Me-cAMP inhibits bTREK-1 channels expressed in HEK293 cells ……….….…………………….93

Figure 5.1 Chemical structure of Ca 2+ channel and bTREK-1 K + channel antagonists …………..…………………………………………………..107

Figure 5.2 Concentration-dependent inhibition of bTREK-1 by amlodipine .….108

Figure 5.3 Amlodipine inhibition of bTREK-1 is voltage-independent and specific…………..………………………………………….……………..109

Figure 5.4 Effect of amlodipine on unitary bTREK-1 channel activity…………..110

Figure 5.5 Niguldipine inhibition of bTREK-1……………………………………...111

Figure 5.6 bTREK-1 inhibition by flunarizine, anandamide, and nifedipine..…..112

Figure 5.7 Inhibition of T-type Ca 2+ current by amlodipine….………………...... 113

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ABBREVIATIONS

2-APB, 2-amino-ethoxydiphenyl borate

4AP, 4-aminopyridine

5HT, serotonin

6-PhEt-ATP, N6-(2-Phenylethyl) adenosine-5´-O-triphosphate sodium salt

AA, arachidonic acid

AC, adenylate cyclase

ACTH, adrenocorticotropic hormone

AMP-PNP, 5'-adenylyl-imidodiphosphate

ANG II, Angiotensin II

AZF, adrenal zona fasciculata

AZG, adrenal zona glomerulosa

BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid

BIM, bisindolylmaleimide I

2+ 2+ [Ca ]i, concentration of intracellular free Ca

Calmodulin, CaM

CAPE, caffeic acid phenethyl ester

CDC, cinnamyl-1,3,4-dihydroxy-α-cyanocinnamate

CFTR, cystic fibrosis transmembrane conductance regulator

CH, chloral hydrate xiv

CNS, central nerve system

DAG, diacylglycerol

DHP, dihydropyridine

DMEM, Dulbecco’s Modified Eagle’s Medium

DMSO, dimethyl sulfoxide

DPBP, diphenylbutylpiperidine

DRG, dorsal root ganglion

EGTA, ethylene glycol tetraacetic acid

Epac, a guanine nucleotide exchange protein activated by cAMP

FBS, fetal bovine serum

GPCR, G-protein coupled receptor

HEK293 cell, human embryonic kidney 293 cell

IP 3, inositol 1, 4, 5-trisphosphate

I-V, current-voltage relationship

K2P, two pore domain K+ channel

LPs, lysophospholipids

LPC, lysophosphatidylcholine

MAP kinase, mitogen-activated protein kinase mGluRs, metabotropic glutamate receptors

NO, nitric oxide

NPS-ACTH, O -nitrophenyl, sulfenyl-adrenocorticotropin

P2Y, metabotropic purinergic receptors

PBS, phosphate buffered saline

PDBu, phorbol-12, 13-dibutyrate

PI 3K, phosphoinositide 3-kinases

PIP 2, Phosphatidylinositol (4, 5)-bisphosphate

PKA, protein kinase A

PKC, protein kinase C

PKD, protein kinase D

PKG, protein kinase G

PKI, protein kinase inhibitor peptide

PLC, phosphalipase C

PPPi, polytriphosphate

PUFAs, poly-unsaturated fatty acids

RMP, resting membrane potential

SDS , sodium dodecyl sulfate

TALK, TWIK-related Alkaline-activated K + channel

TASK, TWIK-related Acid-Sensitive K + channel

TCE, trichloroethanol

TEA, tetraethylammonium

THIK, TWIK-related Halothane-Inhibited K + channel

TRAAK, TWIK-Related Arachidonic Acid-activated K + channel

TREK-1, TWIK-RElated K + channel-1

TRESK, TWIK-RElated Spinal cord K + channel

TWIK, Tandem P domain Weak Inwardly rectifying K + channel

WT, wide type xvi

CHAPTER 1

GENERAL INTRODUCTION

TREK-1 (TWIK-Related K + channel-1) belongs to the two pore domain K +

(K 2P) channel superfamily. TREK-1 protein contains 411 amino acids and its mRNA is expressed in multiple regions of the brain such as hippocampus, hypothalamus and also in peripheral organs such as heart, stomach, intestine and bovine adrenal (Medhurst, 2001; Enyeart et al., 2002a). This channel sets the resting membrane potential (RMP) and can be modulated by a variety of factors including mechanical force, temperature, pH, fatty acids, phospholipids, and transmitters (Honoré, 2007).

1.1. General overview of two-pore-domain K + channels

In the mid 1990s, by searching the DNA database for sequences

+ homologous to the conserved pore sequence of K channels, K2P channels were identified as the third class of K + channels. These channels encode the background K+ conductance in variety of cells and drive the RMP close to the K + equilibrium potential to control cellular excitability.

+ Among all K channels, the K 2P channel is structurally unique. It is characterized by two pore-forming domains (2P) and four transmembrane 1

segments (M1-M4). It is also composed of an extended M1P1 extracellular loop and intracellular amino and carboxyl terminals (Figure 1.1a). Instead of forming

+ homo- or heterotetramers as one-pore-domain K (K 1P) channels, K 2P channels function as homo- or heterodimers (Figure 1.1b). At the pore region of K + channels, amino acid residues Glycine-Tyrosine-Glycine (GYG) or Glycine-

Phenylalanine-Glycine (GFG) are thought to be the signature sequences for K + selectivity. In dimeric K2P channels, each pore-forming domain contains one signature sequence in the selectivity filter. These signature sequences in the first pore (P1) and second pore (P2) domains are not necessarily identical (Honoré,

2007).

There are currently 15 members in the K2P channel family. They have been divided into six subgroups based on their functional properties (Figure 1.1c):

1)TWIK subgroup including TWIK-1, TWIK-2 and KCNK7 channels, which exhibits the weak inward rectification, 2)TREK subgroup including TREK-1,

TREK-2 and TRAAK channels, which can be activated by mechanical stimulation and lipids, 3)TASK subgroup including TASK-1, TASK-3 and TASK-5 channels, which are acid-inhibited channels, 4)TALK subgroup including TALK-1, TALK-2 and TASK-2 channels, which are alkaline-activated channels, 5)THIK subgroup including THIK-1 and THIK-2 channels, which can be inhibited by halothane,

6)TRESK subgroup including TRESK-1 channel which can be activated by Ca 2+ .

Although K2P channels share the same structural arrangement, they display diverse properties including sensitivity to pH, heat, membrane tension and free fatty acids, as well as volatile anesthetic and G-protein coupled receptor 2

(GPCR) agonists, indicating their diverse functions in RMP regulation, sensory transduction, heat and pain sensation, metabolic regulation and GPCR signaling.

1.2. Biophysical properties of TREK-1 K+ channels

1.2.1. Ion permeation

TREK-1 channels are highly K+ selective. The ion selectivity of these

+ + channels is Na << K with the permeability ratio (P Na /P K) of less than 0.03

(Honoré, 2007). Due to the high selectivity of K+ over Na +, the reversal potential of TREK-1 channels is close to the equilibrium potential of K +. Thus, TREK-1 channels display an outward rectification under physiological conditions and the activation of these channels will produce the efflux of K + and drive the membrane potential towards the K + equilibrium potential.

1.2.2. Time and voltage dependency

Because they function in setting the RMP, most K 2P channels lack time and voltage dependency. However, TREK-1 channels do display time and voltage dependency. Indeed, TREK-1 currents show an instantaneous component and a second time-dependent component in response to depolarization (Maingret et al., 2002; Enyeart et al., 1996). Deletion analysis shows that the time dependent gating is critically dependent on the cytoplasmic carboxyl terminal domain of TREK-1 channel (Maingret et al., 2002).

Moreover, TREK-1 channel displays an outward rectification in a symmetrical K + gradient instead of a linear current-voltage (IV) relationship due 3

to the inhibition by external Mg 2+ at negative potentials (Maingret et al., 2002). In addition, the cytoplasmic C-terminal domain of TREK-1 channel has been shown to be responsible for the voltage-dependent gating (Maingret et al., 2002).

Bockenhauer et al. (2001) showed that phosphorylation by protein kinase A (PKA) at Ser333 in the C-terminal converts rat hippocampal TREK-1 channel from a voltage-insensitive leak channel to a voltage-dependent channel.

1.3. Modulation of TREK-1 K+ channels

TREK-1 K+ channels can be regulated by a variety of modulators including physical stimuli, such as mechanical stimulation and temperature, chemical stimuli including free fatty acids, pH, lipids, nucleotides, and GPCR signaling.

1.3.1. Physical stimulators

1.3.1.1. Mechanical stimulation

TREK-1 K+ channels can be reversibly activated by membrane stretch

(Patel et al., 1998). In excised inside-out patch, membrane stretch enhances the channel activity at both negative and positive potentials (Maingret et al., 2002).

Additionally, activation of TREK-1 by membrane stretch can occur when actin cytoskeletons are disrupted. This suggests that this channel is modulated directly by the tension in the lipid bilayer produced by stretch (Lauritzen et al., 2005).

Deletion analysis shows that the C-terminal domain of TREK-1 channel is responsible for mechanical activation (Honoré et al., 2006).

1.3.1.2. Temperature

TREK-1 channels have been shown to be extremely sensitive to temperature (Maingret et al., 2000a). Rises in temperature reversibly activate

TREK-1 with a Q 10 of 7 between 32°C and 37°C (Maingret et al., 2000a). In excised patches, TREK-1 channel loses its response to temperature, suggesting that intact intracellular signaling pathways are required for its temperature sensitivity. In addition, partial deletion of its C-terminus greatly reduces TREK-1 channel sensitivity to temperature, indicating the important role of this region in temperature regulation (Maingret et al., 2000a).

1.3.1.3. Intracellular pH

Intracellular acidosis can activate both cloned and native TREK-1 in whole cell, cell-attached, and excised inside-out patch configurations (Enyeart et al.,

2002a; Maingret et al., 1999). When TREK-1 channel is closed at pH 7.3, lowering intracellular pH from 7.0 to 5.0 increases channel activity (Maingret et al., 1999). Again, the C-terminus is important for pH sensing, as deletion of its C- terminal domain abolishes intracellular acidification-evoked channel activation. In particular, a negatively charged glutamate residue (Glu306) has been shown to serve as its proton sensor (Honoré et al., 2002; Maingret et al., 1999).

1.3.2. Chemical stimulators

1.3.2.1. Polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), can reversibly activate TREK-1 channels in both native and expression systems

(Patel et al., 1998; Danthi et al., 2003). Importantly, this activation still exists in excised patches even in the presence of AA metabolism blockers, indicating a direct effect of PUFAs on TREK-1 channel (Patel et al., 2001; Danthi et al., 2003).

Moreover, studies have shown that PUFAs that have carbon lengths of 16~24, at least one double bond and negative charges can all activate TREK-1 (Patel et al.,

2001). Deletion and mutation at its C-terminal region impair the activation of

TREK-1 by AA, indicating a critical role of C-terminus for the sensitivity to PUFAs

(Patel et al., 1998; Patel et al., 2001).

1.3.2.2. Cellular Lipids

Lysophospholipids (LPs) activate TREK-1 channels both in vivo and in vitro (Maingret et al., 2000b; Danthi et al., 2003). For example, lysophosphatidylcholine (LPC) activates TREK-1 channel in whole cell and cell- attached configurations but not in excised patches, suggesting that the effect of

LPC activation is indirect and intracellular signaling components are required

(Maingret et al., 2000b). Deletion analysis indicates that the C-terminal region of

TREK-1 channel is required for LPC activation (Maingret et al., 2000b).

TREK-1 has also been reported to be activated by intracellular phospholipids such as PIP 2 (Chemin et al., 2005b; Chemin et al., 2005a). The 6

effect of phospholipids is dependent on their negative charged phosphate group.

Accordingly, the C-terminal region of TREK-1 channel which contains a cluster of positive charges is responsible for the interaction with phospholipids (Chemin et al., 2005b; Chemin et al., 2005a).

1.3.2.3. Nucleotides

Nucleotides such as ATP activate bovine TREK-1 channels through nonhydrolytic binding in bovine adrenal zona fasciculata (AZF) cells when applied intracellularly at physiological concentrations (Enyeart et al., 1997).

Similarly, intracellular ATP has also been shown to directly activate TREK-1 in rat ventricular myocytes at millimolar concentrations (Tan et al., 2002).In contrast, increasing pipette ATP concentration fails to increase the cloned TREK-1 current expressed in human embryonic kidney (HEK) 293 cells (Enyeart et al., 2002a).

In addition, UTP, GTP and the nucleotide diphosphates such as ADP,

GDP and UDP all significantly increase bTREK-1 currents in AZF cells (Enyeart et al., 1997). Moreover, the non-hydrolyzable ATP analog 5'-adenylyl- imidodiphosphate (AMP-PNP) and the inorganic polytriphosphate (PPPi) also strongly activate bTREK-1 channel in these cells (Xu and Enyeart, 2001).

1.3.2.4. Ca 2+

bTREK-1 channel has been shown to be inhibited by Ca 2+ and ionomycin, a Ca 2+ ionophore, in AZF cells (Gomora and Enyeart, 1998). In whole cell mode, increasing intracellular Ca 2+ concentration ([Ca 2+ ]i) from 20nM to 10µM greatly 7

reduces bTREK-1 currents. In inside-out patches, Ca 2+ dramatically and reversibly inhibits bTREK-1 activity. In addition, ionomycin potently inhibits bTREK-1 channel in the presence of calmodulin (CaM) inhibitory peptide or

AMP-PNP (Gomora and Enyeart, 1998). Taken together, these results suggest that the inhibition of bTREK-1 by Ca 2+ may be mediated through a CaM and protein kinase independent mechanism.

1.3.3. G protein-coupled receptor signaling pathways .

TREK-1 channels have been shown to be inhibited by agonists that bind to Gs- and Gq-coupled receptors in both expression and native systems (Chemin et al., 2003; Enyeart et al., 1996; Enyeart et al., 2005; Patel et al., 1998). Other

G-protein coupled receptors such as Gi-coupled receptors also regulate TREK-1 activity (Cain et al., 2008).

1.3.3.1. Gs-coupled signaling pathway

TREK-1 channel has been reported to be inhibited by activation of Gs- coupled receptors such as 5-hydroxytryptamine 4 (5-HT4) receptors and ACTH receptors (Enyeart et al., 1996; Patel et al., 1998). Activation of these receptors leads to accumulation of intracellular cAMP through activation of adenylate cyclase (AC). cAMP activates protein kinase A (PKA) which phosphorylates

TREK-1 channel at Ser333 in the C-terminal region (Bockenhauer et al., 2001).

In addition, Murhartian et al. (2005) have identified a second cAMP/PKA- dependent phosphorylation site (Ser300) in the C-terminus of TREK-1 channel. 8

Phosphorylation at both Ser333 and Ser300 are required for channel inhibition by receptor stimulation (Murhartian et al., 2005).

Bovine AZF cells express both bTREK-1 and Gs-coupled type 2 melanocortin receptor (MC 2R), a high affinity ACTH receptor (Enyeart et al., 2001;

Raikhinstein et al., 1994). Activation of the ACTH receptor has been shown to stimulate intracellular cAMP synthesis in these cells (Yamazaki et al., 1998).

ACTH potently inhibits bTREK-1 currents in AZF cells and cAMP mimics the action of ACTH (Mlinar et al., 1993a; Enyeart et al., 1996). Although bTREK-1 channels can be inhibited by cAMP through the well accepted PKA-dependent pathway, ACTH may also inhibit bTREK-1 channels through cAMP by a PKA- independent mechanism (Enyeart et al., 1996). In this thesis, the PKA- independent signaling pathway will be further investigated in chapter 4.

1.3.3.2. Gq-coupled signaling pathway

TREK-1 channels can also be inhibited by activation of Gq-coupled receptors such as group I metabotropic glutamate receptors (mGluRs) (Chemin et al., 2003), angiotensin II (ANG II) (AT1) receptors (Enyeart et al., 2005; Lopes et al., 2005), Muscarinic (M1) receptors (Lopes et al., 2005), and P2Y receptors

(Xu and Enyeart, 1999). Activation of Gq-coupled receptors stimulates intracellular phospholipase C (PLC) which hydrolyses PIP 2 into diacylglycerols

(DAG) and inositol 1,4,5-trisphosphate (IP 3). DAG activates protein kinase C

(PKC) which can phosphorylate TREK-1 channels at the C-ternimal domain while

2+ IP 3 stimulates Ca release from intracellular stores. 9

The involvement of these molecular components of Gq-coupled receptor signaling pathways in TREK-1 channel inhibition has been investigated by several studies (Chemin et al., 2003; Lopes et al., 2005; Murbartian et al., 2005).

First, U73122, a PLC inhibitor, prevents the agonist-induced inhibition of TREK-1 in expression system, suggesting that activation of PLC is sufficient to inhibit this channel (Chemin et al., 2003). Second, PIP 2 hydrolysis has been shown to mediate agonist-induced TREK-1 channel inhibition (Lopes et al., 2005).Third, it has been shown that DAG directly inhibits TREK-1 in both whole cell and inside- out modes (Chemin et al., 2003). However, conflicting results have been observed by Lopes and colleagues (2005), in which the direct application of DAG produces only minimal inhibition of TREK-1. Finally, sequential phosphorylation at Ser333 and Ser300 of TREK-1 by PKC is sufficient for TREK-1 inhibition

(Murbartian et al., 2005).

Notably, these studies have all been performed using cloned TREK-1 channel in expression systems. In bovine AZF cells which express AT1 receptors,

ANG II potently inhibit native bTREK-1 channels (Mlinar et al., 1993a; Enyeart et al., 2005). Previous study has discovered that ANG II inhibits bTREK-1 channel by separate Ca 2+ and ATP hydrolysis-dependent signaling pathways in AZF cells

(Enyeart et al., 2005). Although PLC has been shown to be required for Ca 2+ dependent inhibition of bTREK-1 by ANG II (Enyeart et al., 2005), the ATP hydrolysis-dependent pathway that mediates bTREK-1 inhibition by ANG II is still poorly understood. In Chapter 3, the ATP dependent pathway will be further discussed. 10

1.3.3.3. Gi-coupled signaling pathway

Activation of Gi-coupled receptors decreases intracellular cAMP concentration by inhibiting AC. Since activation of Gs-coupled signaling pathway leads to TREK-1 channel inhibition, stimulation of Gi signaling is expected to enhance TREK-1 activity. Indeed, the stimulatory effect of Gi signaling for TREK-

1 has been observed in several studies (Cain and Bushell, 2006; Cain et al.,

2008). Activation of Gi-coupled group II and III mGluRs including mGlu 2R and mGlu 4R increases TREK-1 activity in expression system (Cain et al., 2008).

Pharmacological and mutagenasis analysis reveals that this effect is mediated by dephosphorylation of Ser333 and Ser300 in the C-terminal domain resulting from the inhibition of PKA (Cain et al., 2008).

1.3.4. Other second messenger pathways

TREK-1 has also been shown to be regulated by nitric oxide (NO) through cGMP pathway in both native and expression systems (Koh et al., 2001). NO increases intracellular cGMP concentration by stimulating guanylyl cyclase. cGMP in turn activates protein kinase G (PKG) which phosphorylates Ser351 at the C-terminal domain of TREK-1 channel, leading to increased channel activity.

Point mutation of this Ser residue has been shown to prevent this stimulatory effect (Koh et al., 2001).

1.4. Pharmacology of TREK-1 K+ channels

TREK-1 K+ channel has unique pharmacological properties. It is resistant to all classic K1P channel blockers including tetraethylammonium (TEA) and 4- aminopyridine (4AP) whereas it can be blocked by multiple other pharmacological agents including antidepressant agents such as fluoxetine and norfluoxetine (Kennard et al., 2005), a neuroprotective agent, sipatrigine

(Meadows et al., 2001), and some Ca 2+ channel blockers such as penfluridol, fluspirilene and mibefradil (Gomora et al., 1999; Gomora and Enyeart, 1999b).

Importantly, TREK-1 can be activated by an important class of pharmacological agents, general anaesthetics (Patel et al., 1999; Gruss et al.,

2004a). General inhalational anesthetics such as chloroform, halothane diethyl ether and isoflurane have been shown to activate TREK-1 at clinical doses (Patel et al., 1999). Moreover, anesthetic gases such as nitrous oxide, xenon, and cyclopropane also activate TREK-1 at clinical concentrations (Gruss et al.,

2004a). In addition, non-volatile anaesthetics such as chloral hydrate (CH) and its active metabolite trichloroethanol (TCE) have been shown to reversibly activate TREK-1 (Harinath and Sikdar, 2004).

In addition to general anaesthetics, selected caffeic acid ester derivatives including Cinnamyl 1–3,4-dihydroxy- -cyanocinnamate (CDC) and caffeic acid phenethyl ester (CAPE) greatly enhance bTREK-1 activity in both whole cell and single channel levels in bovine AZF cells (Danthi et al., 2004).

1.5. Physiological functions of TREK-1 K+ channels

1.5.1. Physiological functions in neurons

TREK-1 K+ channels are distributed widely throughout the central nerve system (CNS). Since they set the RMP and are sensitive to a variety of factors such as PUFAs, intracellular pH and temperature, these channels may serve as metabolic sensors and play important roles in regulating the excitability of neurons.

1.5.1.1. Physiological role in neuroprotection

During brain ischemia and seizures, the level of intracellular free fatty acids and protons increases. PUFAs and intracellular acidosis will open TREK-1 channels and induce hyperpolarization, causing a reduction of Ca 2+ influx through voltage-gated Ca 2+ channels. Thus, activation of TREK-1 during ischemia may play a protective role. Interestingly, some TREK-1 activators such as PUFAs, lysophosholipids, NO, and xeon are neuroprotective agents

(Lauritzen et al., 2000; Blondeau et al., 2002; Gruss et al., 2004a). Consistently,

TREK-1 knockout mice display increased sensitivity to ischemia than wide type

(WT) mice and the neuroprotective action of PUFAs disappears in those knockout mice (Heurteaux et al., 2004), indicating that these neuroprotective agents may work through the activation of TREK-1 channel. Therefore, TREK-1 may provide a therapeutic target for neuroprotection.

1.5.1.2. Physiological role in depression

TREK-1 channels are highly expressed in the prefrontal cortex and hippocampus. Studies have shown that they play an important role in mood regulation (Heurteaux et al., 2006). Interestingly, TREK-1 knockout mice are resistant to depression in several models. Antidepressants, such as fluoxetine, paroxetine and amitriptyline suppress depression-related behaviors of WT mice whereas TREK-1 knockout mice show behaviors similar to that of WT treated with antidepressants (Heurteaux et al., 2006). Thus, TREK-1 may also provide a target for new antidepressants.

1.5.1.3. Physiological role in pain sensation

TREK-1 channel is expressed in the peripheral sensory system, particularly in small dorsal root ganglion (DRG) neurons (Alloui et al., 2006). As both heat and pressure can activate TREK-1, it may play a role in temperature regulation and touch sensation (Maingret et al., 2000a). Consistently, TREK-1 knockout mice display increased sensitivity to noxious heat and mechanical stimulus, (Alloui et al., 2006).

1.5.2. Physiological functions in adrenal

Adrenocortical cells include AZF cells which secret cortisol and adrenal zona glomerulosa (AZG) cells which secret aldosterone. In bovine adrenocortical cells, bTREK-1 sets the RMP and plays an important role in the physiology of corticosteroid secretion from these cells by linking membrane receptor activation 14

to depolarization-dependent Ca 2+ entry (Mlinar et al., 1993b; Enyeart et al., 1993;

Enyeart et al., 2002a).

1.5.2.1. Hormone control of bTREK-1 channels

The secretory function of adrenocortical cells is controlled by two important hormones, ANG II and ACTH. Both ANG II and ACTH have been shown to potently inhibit bTREK-1 in bovine adrenocortical cells (Enyeart et al.,

2002a; Enyeart et al., 2005; Enyeart et al., 2004). However, the mechanisms for hormone induced inhibition are only partially understood.

Bovine adrenocortical cells express Gq-coupled AT1 receptors. Activation of these receptors by ANG II results in PLC activation and the generation of IP 3

2+ and DAG. IP 3 further induces Ca release from intracellular stores while DAG activates PKC. However, it is not clear whether these signaling molecules are involved in bTREK-1 inhibition by ANG II in bovine adrenocortical cells. Previous study has discovered that ANG II inhibits bTREK-1 channels by separate Ca 2+ and ATP hydrolysis-dependent signaling pathways in AZF cells (Enyeart et al.,

2005). When both Ca 2+ and ATP-dependent pathways are available, ANG II is more potent and effective than when only a single pathway is available. The

ATP-dependent inhibition by ANG II can be eliminated by replacing pipette ATP by AMP-PNP or UTP, indicating that ATP hydrolysis is required for this inhibition

(Xu and Enyeart, 2001). However, the kinase or ATPase that mediates ANG II inhibition through this ATP hydrolysis-dependent pathway has not been identified.

In addition, the Ca 2+ dependent inhibition of bTREK-1 by ANG II is blunted in the 15

presence of the PLC antagonist U73122, IP 3 receptor antagonist 2-amino- ethoxydiphenyl borate (2-APB) or the absence of external Ca 2+ (Enyeart et al.,

2005), suggesting the involvement of both intracellular and extracellular Ca 2+ in

ANG II-mediated inhibition.

Bovine adrenocortical cells also express Gs-coupled MC 2 receptors.

Activation of these receptors by ACTH rapidly increases intracellular cAMP level in these cells. Thus, ACTH potently inhibits bTREK-1 currents in AZF cells while cAMP mimics the inhibitory effect by ACTH (Mlinar et al., 1993a; Enyeart et al.,

1996). Surprisingly, application of multiple PKA antagonists fails to prevent the inhibition of bTREK-1 by ACTH and cAMP. Indicating that a PKA-independent mechanism is recruited by ACTH and cAMP to inhibit bTREK-1 (Enyeart et al.,

1996). In addition to PKA, a second cAMP-binding protein, Epac (guanine nucleotide exchange factors activated by cAMP), has been identified (de Rooij et al., 1998). It is possible that the cAMP-dependent and PKA-independent inhibition of bTREK-1 by ACTH in AZF cells is mediated through Epac. Taken together, ACTH inhibits bTREK-1 channel through cAMP by both PKA- dependent and independent mechanisms.

1.5.2.2. bTREK-1 and corticosteroid secretion

Because they function in setting the RMP, inhibition of TREK-1 channels leads to membrane depolarization. Early studies demonstrate that activation of both ANG II and ACTH receptors is coupled to membrane depolarization through inhibition of bTREK-1 channels in both bovine AZF and AZG cells (Enyeart et al., 16

2004; Mlinar et al., 1995). Membrane depolarization leads to a secondary increase in intracellular Ca 2+ through T-type Ca 2+ channels, the major Ca 2+ channel expressed in these cells (Enyeart et al., 1993; Mlinar et al., 1993b), which triggers corticosteroid secretion.

Indeed, at concentrations that inhibit bTREK-1 channels, ANG II and

ACTH stimulate cortisol and aldosterone secretion from these cells (Enyeart et al., 1993; Enyeart et al., 2004). ANG II-stimulated secretion can be blocked by selective antagonists of T-type Ca 2+ channels, indicating that T-type Ca 2+ channels play an important role in ANG II-stimulated cortisol and aldosterone release from these cells (Enyeart et al., 1993; Spat et al., 1996). Therefore, bTREK-1 couples membrane depolarization to hormone-stimulated cortisol and aldosterone secretion (Fig 1.2).

The aim of this thesis is to further explore the signaling mechanisms that inhibit native bTREK-1 K+ channels by ANG II and ACTH in AZF cells and the pharmacology of this channel. I have found that:

1): ANG II inhibits bTREK-1 K + channels through a mechanism that is independent of PLC, PKC, and PIP 2 but requires ATP hydrolysis.

2): ACTH inhibits bTREK-1 K + channels by multiple cAMP -dependent signaling mechanisms.

3): Selected DHP Ca 2+ channel antagonists potently inhibit native bTREK-1 K+ channels. 17

Figure 1.1 Structure and phylogenetic tree of K 2P channels (Honore, 2007).

ANG II ACTH

R R

Gq Gs

bTREK-1

Membrane Depolarization

2+ T-type Ca Channel Open

Cortisol/ Aldosterone Secretion

Figure 1.2 The physiological role of bTREK-1 K + channel in adrenal.

CHAPTER 2

MATERIALS AND METHODS

2.1. Materials

Tissue culture media, antibiotics, fibronectin, and fetal bovine sera (FBS) were obtained from Invitrogen (Carlsbad, CA). Coverslips were from Bellco

(Vineland, NJ). Enzymes, Phosphate-buffered saline (PBS), 1,2 bis-(2- aminophenoxy)ethane-N,N,N',N''-tetraacetic acid (BAPTA), MgATP, Na 2ATP,

GDP-β-S, ACTH (1-24), ANG II, PPPi, collagenase, DNase, H-89, nystatin, 8- pCPT-cAMP, AMP-PNP, Rp-cAMPs, EGTA and nifedipine (C17 H18 N2O6) were obtained from Sigma (St. Louis. MO). U73122, U0126 and anandamide

(C 22 H37 NO 2) were purchased from Tocris Bioscience (Ellisville, MO). Edelfosine, wortmannin, SB203580, phorbol-12, 13-dibutyrate (PDBu), bisindolylmaleimide I

(BIM), calphostin C, PKI (6–22) amide and PKI (14–22) myristolated were obtained from EMD Biosciences, Inc. (San Diego, CA). DiC 8PI (4, 5) P2 was purchased from Echelon Biosciences (Salt Lake City, UT). Curcumin, amlodipine

(C 20 H25 ClN 2O5), niguldipine (C 36 H39 N3O6), and flunarizine (C 26 H26 F2N2) were obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). N6-(2-

Phenylethyl)adenosine-5´-O-triphosphate, sodium salt (6-phEt-ATP, Biolog

#P-012) and 8-pCPT-2'-O-Me-cAMP (Biolog #C041) were purchased from

Axxora, LLC (San Diego, CA). Human full-length cDNA for Epac2 (cAMP-GEFII, clone ID# 4823935) was purchased from Open Biosystems. p3-CD8 clone was provided by B. Seed (Massachusetts General Hospital, Boston, MA). SignaTect cAMP-dependent protein kinase (PKA) assay system was from Promega. . [ 32 P]

ATP was purchased from Perkin Elmer. NPS-ACTH was custom synthesized by

Celtek Peptides.

2.2. Isolation and culture of AZF cells

Bovine adrenal glands were obtained from steers (age 2–3 yr) at a local slaughterhouse. Isolated AZF cells were obtained and prepared as previously described (Enyeart et al., 1997). After isolation, cells were either resuspended in

DMEM/F12 (1:1) with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and the antioxidants 1µM tocopherol, 20 nM selenite, and 100 µM ascorbic acid

(DMEM/F12+) and plated for immediate use, or resuspended in FBS/5% dimethyl sulfoxide (DMSO), divided into 1-ml aliquots and stored in liquid nitrogen for future use. For patch-clamp experiments, cells were plated in DMEM/F12+ in 35- mm dishes containing 9 mm 2 glass coverslips. To ensure cell attachment, coverslips were treated with fibronectin (10µg/ml) at 37°C for 30 min then rinsed with warm, sterile PBS immediately before adding cells. Cells were maintained at

37°C in a humidified atmosphere of 95% air-5% CO2.

2.3. Patch-clamp experiments

Patch-clamp recordings of K + channel currents were made in the whole cell configuration from bovine AZF cells. The standard external solution consisted of 140 mM NaCl, 5mM KCl, 2 mM CaCl 2, 2 mM MgCl 2, 10 mM HEPES, and 5 mM glucose, with pH adjusted to 7.3 using NaOH. The standard pipette solution consisted of (in mM): 120 KCl, 2 MgCl 2, 10 HEPES, and 0.2 GTP, with pH titrated to 6.8 using KOH. The patch pipette solution was maintained at pH 6.8 to enhance the expression of bTREK-1. The buffering capacity of the pipette solutions was varied by adding combinations of Ca 2+ and BAPTA or EGTA using the Bound and Determined software program (Brooks and Storey, 1992). Low- and high-capacity Ca 2+ -buffering solutions contained 0.5 mM EGTA and 11 mM

BAPTA, respectively. The low -capacity Ca 2+ -buffering solution was nominally

Ca 2+ -free. [Ca 2+ ]i was buffered to 22 nM in the high-capacity buffering solution.

Nucleotides, including MgATP, NaUTP, and AMP-PNP were added to pipette or bath solutions as noted in the text. The standard external and pipette solutions used for single-channel recording from outside-out patches were identical to those used for whole-cell recordings.

For perforated patch recordings, the pipette solution contained 130 mM

KCl, 2 mM MgCl 2, and 20 mM HEPES, with pH adjusted to 6.8 using KOH. The pipette tip was filled with this solution and backfilled with this same solution supplemented with 120 µg/ml nystatin. Nystatin stock solutions (30 mg/ml) were made fresh daily in DMSO. Perforated patch recordings were made as previously described (Horn and Marty, 1988). 22

Patch-clamp recordings of T-type Ca 2+ currents were made in the whole- cell configuration. The standard pipette solution was 120 mM CsCl, 1 mM CaCl 2,

2 mM MgCl 2, 11 mM BAPTA, 10 mM HEPES, and 1 mM MgATP, with pH titrated to 7.2 with CsOH. The external solution consisted of 117 mM TEA-Cl, 5 mM CsCl,

10 mM CaCl 2, 2 mM MgCl 2, 5 mM HEPES, and 5 mM glucose, with pH adjusted to 7.3 with TEA-OH. All solutions were filtered through 0.22-µm cellulose acetate filters.

AZF cells were used for patch-clamp experiments 2–12 h after plating.

Typically, cells with diameters <15 µm and capacitances of 8–15 pF were selected. Coverslips were transferred from 35-mm culture dishes to the recording chamber (volume: 1.5 ml), which was continuously perfused by gravity at a rate of 3–5 ml/min. Drugs were applied externally by bath perfusion controlled manually by a six-way rotary valve. Patch electrodes with resistances of 1.0 –2.0

MΩ were fabricated from Corning 0010 glass (World Precision Instruments,

Sarasota, FL). K + currents were recorded at room temperature (22–24°C) following the procedure of Hamill et al. using a List EPC 7 patch-clamp amplifier.

Pulse generation and data acquisition were done using a personal computer and PCLAMP software with Digidata 1200 interface (Axon Instruments,

Burlingame, CA). Currents were digitized at 2–10 KHz after filtering with an 8- pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using summed scaled hyperpolarizing steps of 1/2 to 1/4 pulse amplitude. Data were analyzed using

PCLAMP (CLAMPFIT 9.2) and SigmaPlot (ver. 10.0) software (Systat Software,

Inc., Point Richmond, CA). p values were calculated using Student's t test.

2.4. Measurement of Epac2 mRNA

RNeasy columns (QIAGEN) that had been treated with RNase-free DNase

(QIAGEN) to remove genomic contamination were used to extract total RNA from

AZF cells that had been cultured for 48h both with and without ACTH (2 nM) or confluent HEK293 cells. Poly (A)+ mRNA was extracted from total RNA using a

Poly(A) Pure kit (Ambion). 7 µg of total or poly (A)+ mRNA were separated on a denaturing 8% formaldehyde, 1.0% agarose gel, and transferred to a nylon membrane (Gene Screen Plus, NEN). The RNA was fixed to the membrane by

UV cross-linking using a Stratalinker (Stratagene). Northern blots were prehybridized in heat-sealable plastic bags for 2 h at 42°C in ULTRAhyb (Ambion) and then hybridized with a [α 32P]dCTP-labeled 700-bp XHO1 fragment of h-

Epac2 DNA overnight at 42°C in minimal volume of ULTRAhyb. After 18–24 h, membrane was washed twice at room temperature in 2x SSPE for 15 min, twice at 40°C in 1x SSPE, 1% SDS for 30 min, with a final wash at 40°C with 0.1x

SSPE, 1% SDS for 15 min before exposing to a storage phosphor screen for 4–

12 h. Northern autoradiogram was imaged using a Typhoon 9200 variable mode phosphorimager.

2.5. PKA Assay

PKA activity was measured with a SignaTECT cAMP-dependent protein kinase assay kit (Promega). This kit uses PKA-dependent phosphorylation of biotinylated peptides as a measure of PKA activity. AZF cells were plated on 60- mm fibronectin-treated dishes in DMEM/F12+ at a density of ~4 x 106 cells/dish.

After 24 h, the serum-supplemented media was removed and replaced with either control media (DMEM/F12+) or the same media containing myristolyated

PKI (14–22) and H-89. At the end of the incubation period, cells were washed two times with ice-cold PBS and suspended in 500 µl of cold extraction buffer (25 mM Tris-HCl pH 7.4, 0.5 mM EGTA, 10 mM β-mercaptoethanol, 0.5 mM

Pefabloc-SC [Roche Applied Science], and protease inhibitors with EDTA

[Complete Mini protease inhibitor cocktail tablet, 1 per 10 ml lysis solution, Roche

Applied Science]). Lysates were homogenized using a cold Dounce homogenizer then centrifuged for 5 min at 4°C at 14,000 g. 5-µl samples of lysate supernatant were assayed using the SignaTECT cAMP-dependent protein kinase assay system. Each experimental condition was assayed in quadruplicate.

2.6. Transient Transfection and Visual Identification of HEK293 Cells Expressing bTREK-1

For patch clamp recording of bTREK-1 currents, HEK293 cells were cotransfected with a mixture of pCR®3.1-Uni-bTrek-1 and an expression plasmid

(p3-CD8) for the α-subunit of the human CD8 lymphocyte surface antigen at a

5:1 ratio using Lipofectamine (Life Technologies). Cells were visualized 1–2 d 25

post transfection after a15-min incubation with anti-CD8 antibody-coated beads

(Dynal Biotech Inc.) as previously described (Jurman et al., 1994).

CHAPTER 3

ANGIOTENSIN II INHIBITS NATIVE bTREK-1 K + CHANNELS THROUGH A

PLC-, KINASE C-, AND PIP 2-INDEPENDENT PATHWAY REQUIRING ATP

HYDROLYSIS

The discovery presented in this chapter has been published in Am J

Physiol Cell Physiol. (Liu et al., 2007a).

3.1. Introduction

Bovine adrenocortical cells, including cortisol-secreting AZF cells and aldosterone-secreting AZG cells, express bTREK-1 leak- type K + channels that belong to the mechanogated, thermo- and fatty acid-sensitive subgroup of two- pore/ four-transmembrane spanning (2P/4TMS) K + channels (Enyeart et al., 2004;

Enyeart et al., 2002a; Maingret et al., 2000a; Mlinar et al., 1993a; Patel and

Honore, 2001). bTREK-1 K + channels function pivotally in the physiology of corticosteroid secretion by coupling hormonal signals originating at the cell membrane to depolarization-dependent Ca 2+ entry (Enyeart et al., 2004; Enyeart et al., 2005; Enyeart et al., 1996; Enyeart et al., 1993; Enyeart et al., 2002a;

Mlinar et al., 1993a; Mlinar et al., 1995). bTREK-1 channels set the RMP of

bovine adrenocorticalcells and are inhibited by ANG II and ACTH at concentrations identical to those which trigger membrane depolarization and corticosteroid secretion (Enyeart et al., 2004; Enyeart et al., 2005; Enyeart et al.,

1996; Mlinar et al., 1993a).

The metabolic and ionic signaling mechanisms that regulate bTREK-1 activity and couple ACTH, ANG II, and nucleotide receptors to bTREK-1 channel gating are partially understood. In early studies, TREK-1 channels were shown to be activated by membrane stretch, high temperatures, and low pH (Maingret et al., 2000a; Maingret et al., 1999). More recently, TREK-1 has been reported to be activated by membrane phospholipids, including PIP2 (Chemin et al., 2005b;

Lopes et al., 2005). Native bTREK-1 channels in bovine AZF cells are activated by intracellular ATP, and other nucleotide triphosphates through nonhydrolytic binding (Enyeart et al., 1997; Xu and Enyeart, 2001). ATP has also been reported to activate TREK-1 channels in rat ventricular myocytes (Tan et al.,

2002). Overall, the gating of bTREK-1 channels by PIP2 and ATP may resemble

+ that of several other channels, including K ATP and M-type K channels, and cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channels. The activity of each of these channels can be regulated by PIP 2 and ATP at physiological concentrations by binding to identical or related sites on the channel (Himmel and Nagel, 2004; MacGregor et al., 2002; Nichols, 2006; Shyng and Nichols,

1998).

The signaling pathways that couple receptors for ANG II to bTREK-1 inhibition are also complex and incompletely understood. ANG II inhibits bTREK- 28

1 K + current and depolarizes bovine AZF cells through activation of a losartan- sensitive AT1 receptor (Enyeart et al., 2005; Mlinar et al., 1995). Although AT1 receptors in adrenocortical cells activate multiple signaling pathways (Hunyady and Catt, 2005; Smith et al., 1999; Tian et al., 1998), the primary transduction mechanism is the Gq-mediated activation of PLCβ, leading to the production of

IP 3 and DAG from PIP 2. These PIP 2-derived second messengers activate effectors, including PKC and Ca 2+ channels of the endoplasmic reticulum

(Hunyady and Catt, 2005; Spat and Hunyady, 2004). Further, the PLC-modulated hydrolysis of PIP 2 leads to the depletion of this membrane-associated phosphoinositide (Xu et al., 2003). It is not clear which of these PLC-dependent signaling mechanisms mediates bTREK-1 inhibition by ANG II in bovine adrenocortical cells.

In this regard, studies describing the modulation of native or cloned neuronal TREK-1 channels by Gq-coupled receptors have implicated PKC, DAG, and PIP 2 as mediators, but the results are conflicting. Specifically, although the reports indicate that TREK-1 channels can be activated by PIP 2, the importance of PLC-mediated PIP 2 degradation to TREK-1 inhibition is controversial. Although one study reported that the inhibition of TREK-1 through PLC-coupled receptors is mediated through the hydrolysis-dependent depletion of PIP 2, a second study found that Gq-coupled receptors inhibit these K + channels through the activation of PKC, independently of PIP 2 hydrolysis (Lopes et al., 2005; Murbartian et al.,

2005). A third study reported that TREK-1 inhibition through PLC-coupled

receptors was mediated directly by DAG independently of PKC activation or PIP 2 depletion (Chemin et al., 2003).

Nearly all of the studies of TREK-1 modulation by Gq-coupled receptors were done using cloned channels expressed in cell lines. The relevance of these studies to modulation of native TREK-1 channels is uncertain. Recently, we reported that ANG II inhibited native bTREK-1 K + channels in AZF cells by separate Ca 2+ - and ATP hydrolysis-dependent mechanisms (Enyeart et al.,

2005). The Ca 2+ -dependent inhibition of bTREK-1 requires activation of PLC.

The signaling mechanism for the ATP hydrolysis-dependent inhibition of bTREK-

1 by ANG II has not been determined.

In this chapter, I discovered that ATP-dependent inhibition of bTREK-1 by

ANG II occurs by a mechanism that is different from that described for TREK-1 inhibition through Gq-coupled receptors in other cells or expression systems.

Specifically, this novel pathway is independent of PLC, PKC, PIP 2 hydrolysis, and DAG. Overall, these results indicate that, under physiological conditions,

ANG II inhibits bTREK-1 K + channels and depolarizes bovine adrenocortical cells by parallel ATP- and Ca 2+ -dependent mechanisms that have not been described for TREK-1 inhibition in other cells.

3.2. Results

Bovine AZF cells express two different types of K + channels. These include voltage-gated rapidly inactivating K v1.4 channels and noninactivating background bTREK-1 channels (Enyeart et al., 2002a; Mlinar et al., 1993a; 30

Mlinar and Enyeart, 1993). In whole cell patch-clamp recordings, bTREK-1 K + current amplitude typically increases spontaneously with time to a steady-state value and is enhanced by acidifying the pipette solution or by adding ATP or other nucleotide triphosphates (Enyeart et al., 1997; Mlinar et al., 1995; Xu and

Enyeart, 2001).

The absence of time- and voltage-dependent inactivation allows bTREK-1

K+ current to be isolated in whole cell recordings using either of two voltage clamp protocols. When voltage steps of several hundred millisecond duration are applied from a holding potential of -80 mV, bTREK-1 K + current can be measured near the end of a voltage step when the transient K v1.4 current has fully inactivated (Fig. 3.1, A–C, left traces). Alternatively, bTREK-1 K + current can be selectively activated by an identical voltage step applied immediately after a 10-s prepulse to -20 mV has fully inactivated K v1.4 channels (Fig. 3.1, A–C, right traces).

In a previous study, we showed that ANG II inhibits bTREK-1 K + channels by separate Ca 2+ - and ATP hydrolysisdependent pathways (Enyeart et al., 2005).

The ATP-dependent pathway can be studied in isolation by strongly buffering the

[Ca 2+] in the pipette to low concentrations with 11 mM BAPTA. Under these conditions, when ATP in the pipette is replaced by a nonhydrolyzable nucleotide, such as AMP-PNP or UTP, ANG II-dependent inhibition of bTREK-1 is nearly eliminated (Enyeart et al., 2005) (Figs. 3.1, A and D).

3.2.1. PLC and ANG II Inhibition of bTREK-1

The ineffectiveness of ANG II in the absence of hydrolyzable ATP could suggest that activation of a PLC-dependent kinase is required for bTREK-1 inhibition. Experiments were done to characterize the ATP-dependent pathway for ANG II inhibition of bTREK-1. Both the Ca 2+ - and ATP hydrolysis-dependent inhibition of bTREK-1 are mediated through an AT1-type ANG II receptor

(Enyeart et al., 2005; Mlinar et al., 1993a; Mlinar et al., 1995). Activation of AT1 receptors in AZF cells is coupled to PLC activation through the GTP binding protein Gq (Spat and Hunyady, 2004). ANG II-dependent inhibition of bTREK-1 through the Ca 2+ -dependent pathway is blunted by the PLC antagonist U73122

(Enyeart et al., 2005; Thompson et al., 1991). To determine whether the ATP- dependent inhibition of bTREK-1 required the activation of PLC, whole cell recordings were made in the presence of either U73122 or a second PLC antagonist, the ether lipid analog edelfosine (Powis et al., 1992). When K + currents were recorded with pipette solutions containing U73122 (3 µM), inhibition of bTREK-1 by ANG II (10 nM) was not reduced. With standard pipette solutions, ANG II (10 nM) inhibited bTREK-1 by 78.6 ± 5.2% ( n =12), compared with 79.7 ± 6.9% ( n= 4) in the presence of U73122 (Fig. 3.1, B and D).

In a second series of experiments, AZF cells were preincubated for 30 min with edelfosine (10 µM) before recording bTREK-1 currents with standard pipette solution. Preincubation of cells with edelfosine failed to reduce bTREK-1 inhibition by ANG II (Fig. 3.1, C and D). In other experiments, K + currents were recorded from cells that had been preincubated with edelfosine using pipettes 32

containing edelfosine (10 µM). Under these conditions, ANG II inhibited bTREK-1 by 84.5± 4.4% (n= 4), a value not significantly different from control (Fig. 3.1D).

3.2.2. PKC and ANG II inhibition of bTREK-1

In AZF cells, ANG II-mediated activation of PLC leads to the synthesis of

DAG, which, in turn, activates PKC (Hunyady and Catt, 2005; Spat and Hunyady,

2004). The failure of U73122 and edelfosine to suppress ATP-dependent inhibition of bTREK-1 suggests that neither PLC nor PKC is necessary for ANG

II-mediated inhibition of bTREK-1 through this pathway. However, these results do not exclude the possibility that activation of PKC alone would be sufficient to inhibit bTREK-1.

Phorbol esters such as PDBu activate PKC at nanomolar concentrations

(Castagna et al., 1982). It was discovered that PDBu (100 nM) inhibited bTREK-1 by 31.7 ± 9.0% ( n = 10) with pipette solutions containing 5 mM MgATP (Fig. 3.2,

A and C). The activation of PKC by DAG is facilitated by Ca 2+ (Oancea and

Meyer, 1998). However, increasing [Ca 2+] in the pipette solution from 20 nM to

200 nM failed to enhance the inhibition of bTREK-1 by PDBu (100 nM) (Fig.

3.2C).

To determine whether PDBu-mediated inhibition of bTREK-1 was specific and involved activation of PKC, these experiments were repeated with pipette solutions containing UTP rather than ATP. UTP enhances the activity of bTREK-

1 but is not a substrate for protein kinases (Enyeart et al., 1997). In the presence

of UTP, PDBu (100 nM) was much less effective at inhibiting bTREK-1 (Fig. 3.2,

B and C).

At higher concentrations, PDBu inhibited bTREK-1 more effectively, but a large fraction of this inhibition was independent of PKC. As shown in Fig. 3.2C,

PDBu (1 µM) inhibited bTREK-1 by 67 ± 4.4% ( n= 3) with MgATP in the pipette solution, compared with 51.3 ± 7.4% ( n= 3) in the presence of UTP. Thus, depending on its concentration, PDBu inhibits bTREK-1 by PKC-dependent and - independent mechanisms.

Experiments with PDBu showed that native bTREK-1 could be partially inhibited by activation of PKC. If the ATP-dependent actions of ANG II on bTREK-1 were mediated solely by PKC, then inhibition of this current by PDBu and ANG II should be similar and less than additive. However, when AZF cells were sequentially superfused with PDBu (100 nM), followed by ANG II (10 nM), inhibition by these two agents was additive, and ANG II was far more effective

(Fig. 3.3A). In these experiments, PDBu inhibited bTREK-1 by 21.0 ± 4.9% ( n= 3), while ANG II (10 nM) inhibited the remaining current by 80.5 ± 7.0% ( n= 3).

BIM is a potent PKC antagonist with an IC 50 of less than 10 nM (Toullec et al., 1991). Experiments in which cells were sequentially superfused with PDBu and ANG II, while recording K + currents with pipettes containing BIM provided convincing evidence that ATP-dependent inhibition of bTREK-1 by ANG II does not require activation of PKC. With pipette solutions containing BIM (5 µM),

PDBu (100 nM) failed to produce any measurable inhibition of bTREK-1.

Subsequent superfusion of the same cells with ANG II (10 nM) inhibited bTREK- 34

1 by 77.6 ±10% ( n= 3) (Fig. 3.3B). These results indicated that BIM was an effective inhibitor of PKC in our experiments. They further demonstrated that in cells where PDBu has been rendered ineffective by specific inhibition of PKC,

ANG II retained its effectiveness.

The PKC-independent inhibition of bTREK-1 by ANG II was confirmed in additional experiments in which either PDBu or ANG II was applied to cells while recording bTREK-1 with pipettes containing BIM (1 or 5 µM). With 5 µM BIM in the pipette, the inhibition of bTREK-1 by PDBu was reduced from its control value of 31.7 ± 9.0% ( n= 10) to 12.4 ± 6.2% ( n= 9) (Fig. 3.3D). In contrast, with pipette solutions containing 1 or 5 µM BIM, ANG II inhibited bTREK-1 by 76.9 ±

7.4% ( n= 7) and 67.7 ± 5.5% ( n = 13), respectively (Fig. 3.3, C and D). Thus, even at the higher concentration, BIM only slightly blunted ANG II inhibition of bTREK-1.

TREK-1 resembles M-type (KCNQ2/3) K + channels in that both can be associated with an AKAP150 anchoring protein within a signaling complex (Hoshi et al., 2003; Sandoz et al., 2006). The PKC-dependent inhibition of KCNQ channels observed upon activation of a muscarinic receptor is suppressed by calphostin C, which acts at the DAG binding site of the enzyme, but not by BIM, which acts at the catalytic site (Hoshi et al., 2003; Shen et al., 2005). In this regard, calphostin C was particularly effective at suppressing KCNQ1 inhibition at low agonist concentrations, thereby affecting a pronounced rightward shift in the dose-response curve (Hoshi et al., 2003). This raised the possibility that at low

concentrations, ANG II inhibits bTREK-1 through activation of PKC, while at higher concentrations the activation of additional pathways masked this effect.

We found that calphostin C was ineffective at suppressing ANG II-mediated inhibition of bTREK-1, even at low ANG II concentrations (Fig. 3.4). With pipettes containing calphostin C (1 µM), ANG II inhibited bTREK-1 with an IC 50 of 95 pM, a value slightly lower than that previously observed under the control condition

(Mlinar et al., 1993a). Overall, these results provide convincing evidence that over a wide range of concentrations, ANG II effectively inhibits bTREK-1 K + channels through an ATP-dependent pathway that does not involve activation of

PKC.

3.2.3. Other kinases

In addition to PKC, ANG II activates a number of other protein kinases in adrenocortical cells. These include p38 and p42/44 MAPKs, phosphatidylinositol-

3 (PI 3) kinase, and protein kinase D (PKD) (Hunyady and Catt, 2005; Romero et al., 2006; Smith et al., 1999; Tian et al., 1998; Toullec et al., 1991). Experiments were done to determine whether the ATP hydrolysis-dependent inhibition of bTREK-1 by ANG II occurred through activation of one of these protein kinases.

U0126 is a potent inhibitor of the MAPK family members MEK-1 and MEK-2

(Davies et al., 2000; Favata et al., 1998). By inhibiting MEKs, U0126 suppresses activation of MAPKs, including p42 MAPK and p44 MAPK (Chabre et al., 1995;

Hunyady and Catt, 2005; Tian et al., 1998). Whole cell recordings were made with pipette solutions containing U0126 at concentrations of 10 and 30 µM. At 36

either concentration, U0126 failed to significantly reduce ANG II inhibition of bTREK-1 (Fig. 3.5, A and B).

A second MAPK inhibitor SB203580 inhibits p38 MAPK with an IC 50 of

~20 nM (Startchik et al., 2002). Application of SB203580 (10 µM) to the cytoplasm directly through the pipette also failed to blunt ANG II-mediated inhibition of bTREK-1 (Fig. 3.5, A and B). With SB203580 in the pipette solution,

ANG II inhibited bTREK-1 by 90.4 ± 3.13% ( n= 4), compared with the control value of 78.6 ± 5.2% ( n= 12).

Activation of PI 3 kinase in bovine adrenocortical cells by ANG II is linked to the activation of other kinases, including Raf-1 (Smith et al., 1999). PI 3 kinase is selectively inhibited by wortmannin with an IC 50 of ~3 nM (Ui et al., 1995).

However, wortmannin failed to blunt ATP-dependent inhibition of bTREK-1 by

ANG II (Fig. 3.5, A and B). In the presence of wortmannin (100 nM), ANG II (10 nM) inhibited bTREK-1 by 79.4 ± 8.3% ( n= 3).

ANG II stimulates cortisol and aldosterone secretion from H295R human adrenocortical cells by activation of PKD (Romero et al., 2006). PKD is inhibited by curcumin with an IC 50 of 4.1 µM (Uhle et al., 2003). The addition of curcumin

(20 µM) to the pipette solution also failed to suppress the inhibition of bTREK-1 by ANG II (Fig. 3.5, A and B).

At 10 nM, ANG II activates multiple kinases in bovine adrenal cortical cells, including PKC, MAP42, and PI 3 kinase (Hoshi et al., 2003). The failure of individual kinase antagonists to suppress inhibition of bTREK-1 could indicate that inhibition by ANG II is mediated through multiple kinases, which act 37

synergistically. However, the addition of multiple kinase antagonists including

U0126, BIM, and wortmannin to patch electrodes failed to significantly affect inhibition of bTREK-1 by ANG II (Fig. 3.5B).

3.2.4 Inorganic polyphosphates block ATP-dependent bTREK-1inhibition.

The bTREK-1 K + channel resembles the CFTR Cl - channel in its complex regulation by protein kinases, nucleotides, and polyphosphates (Baukrowitz et al.,

1994;Combeau and Carlier, 1988;Xu and Enyeart, 2001). The gating of CFTR channels is coupled to an ATP hydrolysis cycle, which is blocked by inorganic phosphates, locking CFTR in the open state (Baukrowitz et al., 1994;Catalano et al., 1986;Gunderson and Kopito, 1995).

Native bTREK-1 channels are activated by the polytriphosphate PPPi (Xu and Enyeart, 2001). In the present study, we found that PPPi blocked ATP- but not Ca 2+ -dependent inhibition of bTREK-1. When K + currents were recorded with pipette solutions containing PPPi (2 mM) and MgATP (1 mM), ANG II was ineffective as an inhibitor of bTREK-1, reducing this current by only 10.0 ± 5%

(n= 3) (Fig. 3.5A and B). The suppression of ATP-dependent inhibition of bTREK-

1 by PPPi was specific. When K + currents were recorded with PPPi containing pipette solutions that supported activation of the Ca 2+ but not the ATP-dependent pathway, ANG II inhibited bTREK-1 by 46.7 ± 7.8% ( n= 6), a value similar to that obtained previously in the absence of PPPi (Enyeart et al., 2005) (Fig. 3.5B).

3.2.5 PIP 2 and bTREK-1 gating.

The membrane phosphoinositide PIP 2 regulates the gating of a number of

K+ channels, including members of the 2P/4TMS family of leak channels (Chemin et al., 2005b;Lopes et al., 2005;MacGregor et al., 2002;Nichols, 2006). While several studies have reported that TREK-1 activity is increased by PIP 2, only one of these has found that receptor-mediated PIP 2-depletion mediates inhibition of

TREK-1 K + channels (Chemin et al., 2005b;Lopes et al., 2005;Murbartian et al.,

2005). Nearly all of the conflicting results were obtained in studies of cloned

TREK-1 channels expressed in cell lines.

We did experiments to determine whether PIP 2 could modulate the activity of native bTREK-1 in AZF cells. At concentrations as low as 1 µM, the water- soluble PIP 2 derivative DiC 8PI (4,5)P 2 has been reported to markedly activate neuronal TREK-1 channels (Chemin et al., 2005b). At 40 µM, DiC 8PI(4,5)P 2 increased bTREK-1 current density modestly from a control value of 35.2 ± 6.0 pA/pF ( n= 9) to 50.6 ± 11.7 pA/pF ( n= 11) (Fig. 3.6A).

These results demonstrate that when whole cell recordings are made with pipettes containing ATP (1 mM), DiC 8PI(4,5)P 2 triggers only a slight increase in bTREK-1 activity. The limited effectiveness of DiC 8PI(4,5)P 2 could indicate that, under the conditions of our experiments, a large fraction of bTREK-1 channels are open with a high probability, even in the absence of this phosphoinositide, thereby limiting its effectiveness. This possibility was tested using AA, which markedly activates bTREK-1 channels (Danthi et al., 2003;Enyeart et al., 2002a).

In these experiments, recordings were made with pipette solutions containing DiC 8PI(4,5)P 2 (40 µM). When the bTREK-1 amplitude reached a stable maximum, AZF cells were superfused with AA (10 µM). AA proved to be far more effective than DiC 8PI(4,5)P 2 as an activator of bTREK-1 (Fig. 3.6B). In four experiments, bTREK-1 current density reached a maximum of 23.1± 3.4 pA/pF ( n= 4) in the presence of DiC 8PIP 2. Subsequent superfusion of AA increased bTREK-1 density nearly 10-fold to 209 ± 47 pA/pF ( n= 4) (Fig. 3.6B).

These results show that DiC 8PIP 2 activates a very small percentage of available bTREK-1 channels in AZF cells.

3.2.6 Activation of bTREK-1 by a high-affinity ATP analog.

ATP and other nucleotide triphosphates activate native bTREK-1 K + channels at millimolar concentrations. ATP analogs have been synthesized that bind with high affinity to ATP-binding proteins. One of these is 50 times more potent than ATP as an activator of CFTR chloride channels (Zhou et al., 2005).

We found that this same ATP analog, 6-PhEt-ATP, was significantly more effective than DiC 8PI(4,5)P 2 as an activator of bTREK-1. Specifically, with pipettes containing 6-PhEt-ATP (50 µM), bTREK-1 current density reached a value more than twice that observed with DiC 8PI(4,5)P 2 (Fig. 3.6A). At much higher concentrations, UTP and PPPi were both more effective than

DiC 8PI(4,5)P 2 as an activator of bTREK-1.

3.2.7 PIP 2 and voltage-dependent bTREK-1 gating.

Native bTREK-1 K + channels display limited voltage-dependent gating in whole-cell recordings (Enyeart et al., 1997). PIP 2 has been reported to enhance the activity of cloned bTREK-1 channels by shifting the voltage-dependent activation in the hyperpolarizing direction so that channel open probability is increased at more negative potentials (Lopes et al., 2005).

Experiments were done to determine whether PIP 2 altered the voltage- dependent gating of native bTREK-1 channels in AZF cells. In whole cell recordings, ramp voltage steps were applied between test potentials of +60 and -

100 mV, with or without DiC 8PI(4,5)P 2 (40 µM) in the pipette solution. Fig. 3.6C shows averaged, normalized current traces derived from control and

DiC 8PI(4,5)P 2-treated cells. The currents were nearly superimposable over the entire range of potentials. Importantly, no significant difference in the relative amplitude of bTREK-1 currents was observed at more negative potentials where a hyperpolarizing shift would be most evident.

+ 3.2.8 PIP 2 and ANG II-dependent inhibition of bTREK-1 K channels.

Experiments were done to determine whether the ATP-dependent inhibition of bTREK-1 involved the depletion of membrane-associated PIP 2. To explore this possibility, cells were superfused with ANG II (10 nM) while

+ recording K currents with pipette solutions containing DiC 8PI(4,5)P 2 (40 µM), thereby delivering an unlimited supply of this phosphoinositide directly to the cytoplasm. DiC 8PI(4,5)P 2 failed to reduce ANG II-dependent inhibition of bTREK- 41

1 (Fig. 3.7A). With DiC 8PI(4,5)P 2 in the pipette solution, ANG II inhibited bTREK-

1 by 86.1 ± 3.6% ( n= 3), compared with the control value of 78.6 ± 5.2% ( n= 12).

Activation of PLC-coupled receptors has been reported to inhibit TREK-1 by shifting the voltage dependence of TREK-1 activation to more positive potentials through a mechanism involving PIP 2 hydrolysis and depletion (Lopes et al., 2005). Consequently, TREK-1 inhibition in response to receptor activation was voltagedependent and much less effective at positive potentials.

To determine whether ANG II-dependent inhibition of bTREK-1 is voltage dependent, bTREK-1 currents were activated by voltage ramps between +100 and -100 mV, before and after superfusing cells with ANG II (10 nM). In these experiments, bTREK-1 was inhibited equivalently over the range of voltages where it could be accurately measured (Fig. 3.7B). In particular, ANG II effectively inhibited bTREK-1 at +100 mV. If ANG II functioned through a depolarizing shift in voltage-dependent activation, it would have been much less effective at reducing bTREK-1 at this positive potential. Thus, bTREK-1 inhibition by ANG II through the ATP-dependent pathway is apparently not mediated by modulating voltage-dependent gating.

2+ 3.2.9 PIP 2 and Ca -dependent regulation of bTREK-1.

In contrast to the ATP-dependent inhibition of bTREK-1, Ca 2+ - dependent inhibition of this channel by ANG II is suppressed by the PLC antagonist U73122

(Enyeart et al., 2005). Experiments were done to determine whether the Ca 2+ - and PLC-dependent inhibition of bTREK-1 by ANG II required PIP 2 depletion. 42

When K + currents were recorded with pipette solutions designed to allow the selective activation of the Ca 2+ -dependent pathway (2 mM UTP, 0.5 mM EGTA), inclusion of DiC 8PI(4,5)P 2 in the pipette again failed to blunt ANG II-dependent inhibition of bTREK-1 (Fig. 3.8, A and B). In the presence of DiC 8PI(4,5)P 2 (40

µM), ANG II inhibited bTREK-1 by 67.2 ± 5.0% ( n= 6), compared with the control

2+ value of 57.8 ± 4.0% ( n= 4). The failure of DiC 8PI(4,5)P 2 to alter Ca -dependent inhibition of bTREK-1 indicates that endogenous PIP2 depletion does not contribute to this response. In these same experiments with pipette solutions containing 2 mM UTP, DiC 8PI(4,5)P 2 also failed to increase bTREK-1 current density compared with the control value (Fig. 3.8C).

3.3. Discussion

It was discovered that ANG II inhibits native bTREK-1 K+ channels in bovine AZF cells by an unusual ATP hydrolysis dependent mechanism that does not require or involve the primary signaling pathway by which this peptide exerts most of its known effects in the adrenal cortex. Specifically, ATP-dependent inhibition of bTREK-1 occurs independently of PLC activation, PIP 2 depletion,

PKC activation, or the release of intracellular Ca 2+. The ATP-dependent inhibition of bTREK-1 is also insensitive to antagonists of the other major protein and lipid kinases activated by ANG II in adrenocortical cells. Whether ATP-dependent inhibition of bTREK-1 occurs through an unidentified kinase or an ATPase remains to be determined.

When combined with the findings of a previous study (Enyeart et al., 2005), the results reported here demonstrate that the modulation of AZF bTREK-1 K + channels, including their activation by phosphoinositides and nucleotide triphosphates and inhibition through Gq-coupled receptors, is distinctly different from that reported for native and cloned TREK-1 channels from other species and organs. With regard to inhibition, the novel ATP and Ca 2+ -dependent mechanisms allow for complete inhibition of bTREK-1 in nearly every cell, providing an effective fail-safe means for membrane depolarization (Fig. 3.9).

Our results do not, however, rule out the possibility of a contribution from PKC in bTREK-1 inhibition by ANG II. Overall, they demonstrate that the modulation of native bTREK-1 channel activity in AZF cells is complex, and mediated through multiple signaling pathways.

3.3.1 PLC- and PKC-independent inhibition of bTREK-1 by ANGII.

The ATP hydrolysis-dependent inhibition of bTREK-1 by ANG II is unusual because of its apparent complete independence from the classical PLC-coupled signaling pathways by which ANG II mediates most of its responses in adrenocortical cells (Hunyady and Catt, 2005;Spat and Hunyady, 2004). In this regard, U73122 and edelfosine are two of the most potent and effective PLC antagonists available. Each has been shown to effectively suppress PLC-coupled

K+ channel inhibition at the concentrations used in our study (Favata et al.,

1998;Horowitz et al., 2005; Kobrinsky et al., 2000; Zaika et al., 2006). Further, in whole cell patch-clamp experiments where Ca 2+ was buffered to 20 nM with 11 44

mM BAPTA, it is doubtful that ANG II could activate PLCβ even in the absence of the PLC antagonists. Activation of PLCβ is strictly Ca 2+-dependent and is blocked, provided that [Ca 2+]i is maintained below resting levels (Horowitz et al., 2005;

Rhee, 2001). Overall, we have provided convincing evidence that the ATP- dependent inhibition of bTREK-1 by ANG II bypasses PLC.

ANG II-mediated activation of PLC is the major mechanism for PKC activation in adrenocortical cells. Because ANG II inhibited bTREK-1 in the apparent absence of PLC activation, it was not surprising that inhibition was not blocked by the PKC antagonist BIM. BIM is among the most potent and specific

PKC antagonists available, with a reported IC 50 as low as 10 nM (Toullec et al.,

1991). However, because BIM is a competitive inhibitor of ATP-binding to PKC, its potency varies inversely with the ATP concentration. When ATP is present at millimolar concentrations, the IC 50 for BIM increases to concentrations as high as

1 µM or more (Toullec et al., 1991). Therefore, it was critical to establish that BIM effectively inhibited PKC when 5 mM ATP was present in the pipette. Accordingly, experiments with PDBu showed that this phorbol ester significantly inhibited bTREK-1 and that this response was completely eliminated by BIM at concentrations that did not affect the inhibition by ANG II. Overall, these results indicate that BIM effectively suppressed PKC-dependent bTREK-1 inhibition and that inhibition by ANG II, at least at this high concentration (10 nM), occurred through a separate mechanism.

Experiments with calphostin C were done to address the possibility that bTREK-1 channels in AZF cells are bound, along with PKC, to AKAP150- 45

anchoring protein, an association that renders an antagonist like BIM, but not calphostin C, less effective at suppressing PKC activation (Hoshi et al., 2003;

Sandoz et al., 2006). Calphostin C produced a distinct rightward shift in the dose- response curve for Gq-coupled receptor-dependent inhibition of AKAP150- associated KCNQ2/3 K + channels (Hoshi et al., 2003). In contrast, calphostin C did not affect the potency of ANG II as an inhibitor of bTREK-1. Thus, PKC does not contribute to TREK-1 inhibition by ANG II regardless of concentration.

3.3.2 Other kinases.

Antagonists of other kinases activated by ANG II in bovine AZF cells were also ineffective at suppressing the inhibition of bTREK-1 by ANG II. Although each of these antagonists was used at concentrations many fold higher than their reported IC 50 s, it is possible that the targeted enzymes were not completely inhibited under the conditions of our experiments. However, at the concentrations utilized, each of the inhibitors has been shown to effectively inhibit the specified kinase in intact cells (Cuenda et al., 1995; Davies et al., 2000; Favata et al., 1998;

Uhle et al., 2003; Ui et al., 1995).

The failure of individual antagonists to alter bTREK-1 inhibition by ANG II suggested the possibility that this response was mediated through the activation of multiple kinases, each of which was sufficient to produce inhibition. However, the ineffectiveness of U0126, BIM, and wortmannin applied together through the pipette in suppressing bTREK-1 inhibition by ANG II argues against this possibility. At the very least, this result indicates that simultaneous inhibition of 46

the three major kinases activated by ANG II in these cells does not blunt the

ANG II response.

In a previous study, we found that antagonists of other ANG II-activated kinases, including tyrosine kinases and JAK2 kinases, were ineffective at suppressing bTREK-1 inhibition by ANG II (Xu and Enyeart, 2001). These results may indicate that ANG II inhibits bTREK-1 by an, as yet, unidentified kinase.

Alternatively, the possibility that the ATP hydrolysis-dependent inhibition of bTREK-1 is mediated by an ATPase cannot be excluded. The remarkable activation of bTREK-1 by 6-PhEt-ATP and by nucleotide triphosphates and inorganic phosphates resembles that of CFTR, wherein the gating of the Cl - channel is regulated through a cycle of ATP binding and hydrolysis (Baukrowitz et al., 1994; Gadsby et al., 2006; Ikuma and Welsh, 2000; Xu and Enyeart, 2001;

Zhou et al., 2005).

3.3.3 Modulation of TREK-1 channel gating by PIP 2 and ATP.

ATP and PIP 2 have been reported to comodulate the gating of many ion channels. In addition to the CFTR Cl - channel, members of each of the major classes of K + channels, including voltage-gated channels, inward rectifiers, and

4TMS/2P background channels, are reportedly modulated by this nucleotide and phosphoinositide (Delmas and Brown, 2005; Himmel and Nagel, 2004; Lopes et al., 2005; MacGregor et al., 2002; Simmons and Schneider, 1998; Suh and Hille,

2003). In some cases, these modulators compete for identical or related sites on the channel (MacGregor et al., 2002). Interestingly, for all of these channels, 47

gating is controlled by the synthesis and hydrolysis of these phosphate- containing molecules.

In the present study, DiC8PI(4,5)P 2 (40 µM) was a relatively weak activator of bTREK-1 when included in the patch electrode with 1 mM NaATP. By comparison, under the same conditions, 6-PhEt-ATP (50 µM) produced a fourfold greater increase in bTREK-1 activity. The activation of bTREK-1 by 6-PhEt-ATP resembles its activation of CFTR Cl - channels, wherein it binds with more than

50-fold higher affinity than ATP (Zhou et al., 2005). The ATP binding site associated with bTREK-1 channels has not been identified.

In contrast to our results, PIP 2 was reported to robustly activate native neuronal or transfected TREK-1 channels by interacting with a cluster of positively charged amino acids on the carboxy terminal domain (Chemin et al.,

2005b; Lopes et al., 2005). These seemingly conflicting results could be due to differing experimental conditions. In our experiments, bTREK-1 was measured in whole cell recordings with 1 mM NaATP in the pipette, while in the above- mentioned studies bTREK-1 was recorded from excised patches in the absence of ATP. The relative ineffectiveness of DiC 8PI(4,5)P 2 as an activator of bTREK-1 suggests that, under physiological conditions where ATP is present at millimolar concentrations, fluctuations in PIP 2 may be less important in regulating bTREK-1 activity in AZF cells.

3.3.4 bTREK-1 inhibition and PIP 2 hydrolysis.

Overall, our results do not support a role for PLC-dependent PIP 2 hydrolysis and depletion in any of the separate pathways by which ANG II inhibits bTREK-1 channels. First, the ATP-dependent inhibition of bTREK-1 can occur under conditions where PLC activation has been suppressed by strong Ca 2+ buffering and the PLC antagonists U73122 and edelfosine. Further, PLC-coupled

PIP 2 hydrolysis does not require the hydrolysis of ATP. Therefore, ANG II- stimulated PIP 2 depletion would not be prevented by substituting UTP in the patch pipette. However, the ATP-dependent inhibition of bTREK-1 by ANG II is eliminated in the absence of hydrolyzable ATP. Finally, the ATP-dependent inhibition of bTREK-1 is not affected by adding DiC 8PI(4,5)P 2 to the recording pipette.

Although the Ca 2+-dependent inhibition of bTREK-1 by ANG II proceeds through a PLC-dependent mechanism, this pathway was also unaffected by

2+ including DiC 8PIP 2 in the pipette. Ca -dependent inhibition of bTREK-1 appears

2+ to involve IP 3-stimulated Ca release, rather than PIP 2 hydrolysis and depletion

(Enyeart et al., 2005).

3.3.5 Comparison to modulation of other TREK-1 channels.

The combined results of this and a previous study have shown that ANG II can produce near-complete inhibition of bTREK-1 in AZF cells by two pathways that are completely independent of PKC, DAG, and PIP2 hydrolysis (Enyeart et al., 2005). In this regard, considerable controversy exists regarding the 49

modulation of other TREK-1 channels by these three signals. Murbartian et al. reported that Gq-coupled receptors inhibit TREK-1 in transfected HEK293 cells through activation of PKC and phosphorylation of a specific amino acid. They also found that PDBu (1 µM) robustly inhibited TREK-1. However, at this concentration, a large fraction of bTREK-1 inhibition by PDBu may be unrelated to PKC activation.

In contrast to these findings, Chemin et al. report that TREK-1 inhibition through PLC-coupled receptors is not mediated through DAG activation of PKC, but through direct inhibition of the channel by DAG (50 µM). However, lipid soluble diacylglycerol analogs nonspecifically affect the function of many ion channels when used at micromolar concentrations (Hockberger et al., 1989).

Finally, Lopes et al. report that inhibition of cloned bTREK-1 channels by

ACh through PLC-coupled muscarinic receptors is not mediated through PKC or

DAG, but through PIP 2 hydrolysis and depletion. In this study, it was reported that PIP 2 converts TREK-1 from a voltage-gated outwardly rectifying channel to an open leak channel by inducing a large negative shift in the voltage dependence of activation. Conversely, receptor-mediated PIP 2 hydrolysis inhibited bTREK-1 through a marked depolarizing shift in activation voltage

(Lopes et al., 2005). Under the condition of our experiments on native bTREK-1 channels, PIP 2 did not change the voltage-dependent rectifying properties of bTREK-1. Further, inhibition of bTREK-1 by ANG II was voltage independent, a result consistent with our finding that this response is not mediated through PIP 2 hydrolysis. 50

Overall, studies exploring the modulation of native and cloned TREK-1 channels through PLC-coupled receptors demonstrate that inhibition of these channels occurs through activation of multiple signaling pathways, involving one or more kinases, Ca 2+, phospholipids, and perhaps, ATPases. Given the rich and complex network of modulators, it is not surprising that studies on native TREK-1, as well as cloned channels expressed in oocytes and cell lines, have produced varied and conflicting results.

In recent years, multiple studies have demonstrated that G protein- coupled receptors modulate ion channels through cell type- and species-specific signaling complexes that include scaffolding proteins, enzymes, guanine nucleotide exchange factors, and other nucleotides (Hoshi et al., 2003; Hoshi et al., 2005). It is highly unlikely that the native signaling complexes are present in the cell lines where modulation of transfected channels is often studied. The emerging picture highlights the importance of studying receptor- mediated modulation of a channel in its native cell.

Fig. 3.1. ANG II inhibits bTREK-1 by a PLC-independent mechanism . The inhibition of bTREK-1 K + currents by ANG II was measured in whole cell patch- clamp recordings using pipette solutions that permitted or blocked selective activation of the ATP hydrolysis-dependent pathway with or without the addition of U73122 (3 µM) or edelfosine (10 µM). K + currents were recorded from bovine AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of -80 mV with or without a 10-s prepulse to -20 mV. When bTREK-1 reached a stable amplitude, cells were superfused with saline containing ANG II (10 nM). A–C: K + current traces recorded with (right traces) and without (left traces) depolarizing prepulses. bTREK-1 amplitude recorded with (o) or without (●) prepulses are plotted against time. Numbers on traces correspond to those on the plot. Pipette solution: 5 mM NaUTP ( A) or 5 mM MgATP ( B, C). D: Summary of experiments as in ( A–C). Bars indicate the percentage of bTREK-1 blocked by ANG II under control conditions, with U73122 (3 µM) or edelfosine (10 µM) in the pipette solution with edelfosine in both external and pipette solutions. Values are means ± SE for the indicated number of determinations . 52

Fig. 3.2. Inhibition of bTREK-1 by PKC activation . The inhibition of bTREK-1 by PKC activator phorbol 12,13 dibutyrate (PDBu) was measured using pipette solution that supported or blocked activation of the ATP- dependent pathway. K + currents recorded from bovine AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of -80 mV with or without 10-s prepulses to -20 mV. After bTREK-1 reached a stable amplitude, cells were superfused with PDBu (100 nM or 1 µM). A and B: K + currents were recorded with (right traces) or without (left traces) depolarizing prepulses. bTREK-1 amplitude recorded with (o) or without (●) prepulses are plotted against time at right. Numbers on traces correspond to those on plot. Pipette solutions contained 5 mM MgATP ( A) or 2 mM uridine triphosphate (UTP; B). C: summary of experiments as in ( A and B) with pipette solutions containing nucleotides and Ca 2+ as indicated. Bars indicate percentage of bTREK-1 blocked by PDBu (100 nM or 1 µM). Values are means ± SE for the indicated number of determinations. Statistical analysis was performed using the Student’s paired t-test. * P < 0.05 compared with 5 mM MgATP .

Fig. 3.3. PKC- and ATP-dependent bTREK-1 inhibition by ANG II . Whole cell K + currents from bovine AZF cells were recorded at 30-s intervals in response to voltage steps to +20 mV, applied from a holding potential of -80 mV, with or without 10-s prepulses to -20 mV. After bTREK-1 reached a stable value, cells were superfused with saline containing PDBu (100 nM) or ANG II (10 nM). Pipettes contained standard solution (11 mM BAPTA, 5 mM MgATP) or the same solution supplemented with 1 or 5 µM BIM. A–C: K + currents recorded with (right traces) or without (left traces) depolarizing prepulses. bTREK-1 amplitude recorded with (o) or without (●) prepulses are plotted against time at right. Numbers on traces correspond to those on plot. D: summary of experiments as in A–C: bars indicate percentage of bTREK-1 remaining after steady state block by ANG II or PDBu. Values are means ± SE for the indicated number of determinations. Statistical analysis was performed using the Student’s paired t-test. * P < 0.02 compared with 100 nM PDBu.

Fig. 3.4. Calphostin C and ANG II inhibition of bTREK-1. Whole cell K + currents were recorded at 30-s intervals from bovine AZF cells in response to voltage steps to +20 mV applied from -80 mV with or without 10-s prepulses to -20 mV. Pipettes contained standard saline (11 mM BAPTA, 5 mM MgATP) supplemented with calphostin C (1 µM). A: TREK-1 current amplitudes recorded with (o) or without (●) depolarizing prepulses are plotted against time. B: inhibition curve. bTREK-1, expressed as a percentage of control, plotted against the ANG II concentration. Data were fit with an equation of the form: I/Imax= 1/[1+(B/Kd) x], where B is the ANG II concentration, Kd is the equilibrium dissociation constant, and x is the Hill coefficient. Values are means ± SE of the indicated number of determinations.

Fig. 3.5. Effect of kinase inhibitors and inorganic polytriphosphate (PPPi) on ANG II inhibition of bTREK-1. Whole cell patch-clamp recordings were made from bovine AZF cells using pipette solutions designed for selective activation of the ATP- (11 BAPTA, 5 mM MgATP) or Ca 2+ -dependent pathway (0.5 mM EGTA, 5 mM NaUTP) with or without the addition of kinase inhibitors or PPPi. K + currents were activated by voltage steps to +20 mV applied at 30-s intervals from a holding potential of -80 mV, with or without a 10-s prepulse to - 20 mV. When bTREK-1 reached a stable amplitude, cells were superfused with saline containing ANG II (10 nM). A and B: effect of kinase inhibitors and PPPi. K + currents were recorded with pipette solutions as described above and no further addition or U0126 (10 or 30 µM), SB203580 (10 µM), wortmannin (100 nM), curcumin (20 µM), U0126 (30 µM) +BIM (5 µM), U0126 (30 µM) + BIM (5 µM) + wortmannin (100 nM), or PPPi (2 mM). A: current amplitudes with (o) and without (●) prepulse are plotted against time. B: summary of results from experiments as in A. Bars indicate the percentage of bTREK-1 blocked by ANG II under the indicated conditions. Values are means ± SE for the indicated number of determinations.

Fig. 3.6. Activation of bTREK-1 channels by 6-PhEt-ATP, DiC 8PI(4,5)P 2, UTP, PPPi, and arachidonic acid (AA). + The effect of UTP, DiC 8PI(4,5)P 2, PPPi, and AA on bTREK-1 K channel expression was measured in whole cell recordings from AZF cells. A: effect of 6-PhEt-ATP, + DiC 8PI(4,5)P 2, UTP, and PPPi. K currents were recorded in response to voltage steps to +20 mV, applied at 30-s intervals with pipettes containing 1 mM NaATP (control) or the same solution supplemented with 50 µM 6-PhEt-ATP, 40 µM DiC 8PI(4,5)P 2, 2 mM UTP, or 2 mM PPPi. Left : bTREK-1 current amplitudes are plotted against time with pipettes containing control saline (●), 6-PhEt-ATP (■) or DiC 8PI(4,5)P2 ( ), as indicated. Right : Summary of bTREK-1 current densities (expressed in pA/pF). Values are means ± SE of indicated number of determinations. B: effect of DiC 8PI(4,5)P 2 and AA on bTREK-1 expression. Whole cell recordings of K + currents were made with pipette solutions containing DiC 8PI(4,5)P 2 (40 µM). When bTREK-1 current reached a stable amplitude, cells were superfused with AA (10 µM) as indicated. bTREK-1 current amplitudes are plotted against time. C: effect of DiC8PI(4,5)P 2 on voltage-dependent bTREK-1 gating. bTREK-1 K + currents were recorded at 30-s intervals in the absence or presence of DiC 8PI(4,5)P 2. When bTREK-1 current reached a stable amplitude, ramp voltage protocols were applied between test voltages of +60 to -140 mV at 0.5 mV/ms. Averaged current traces from three control and three DiC 8PI(4,5)P 2-treated cells are displayed. Statistical analysis was performed using the Student’s paired t-test. * P < 0.002 compared with the control value. 57

Fig. 3.7. Effect of DiC 8PI(4,5)P 2 on ANG II inhibition and voltage dependence of bTREK-1. Whole cell K + currents were recorded from AZF cells with pipette solution that permitted selective activation of the ATP dependent pathway, supplemented with DiC 8PI(4,5)P 2 (40 µM). After bTREK-1 current reached a stable amplitude, cells were superfused with ANG II (10 nM). A: K + currents were activated by voltage steps to +20 mV applied at 30-s intervals from a holding potential of -80 mV with (bottom traces) or without (top traces) depolarizing prepulses to -20 mV. Numbers on traces correspond to those on plot of current amplitudes at right. B: ANG II inhibition is voltage independent. Current traces recorded in response to ramp voltage steps between -100 and 100 mV at 0.5 mV/s before and after superfusing cell with ANG II (10 nM).

2+ Fig. 3.8. Effect of DiC 8PI(4,5)P 2 on Ca - dependent expression and inhibition of bTREK-1 by ANG II. Whole-cell K+ currents were recorded from AZF cells using pipette solutions that permitted selective activation of the Ca2+-dependent pathway by ANG II (i.e., 2 mM UTP + + 0.5 mM EGTA) with or without the addition of DiC 8PI(4,5)P 2 (40 µM). K currents were activated by voltage steps to +20 mV applied at 30-s intervals in the absence (●) or presence (o) of a 10-s depolarizing prepulse to -20 mV. A: DiC 8PI(4,5)P 2 and ANG II + inhibition. K currents recorded with pipette containing DiC 8PI(4,5)P 2 (40 µM). After bTREK-1 current reached a stable amplitude, cell was superfused with ANG II (10 nM). Numbers on current traces correspond to those on plot of bTREK-1 amplitude at right. B: summary of experiments as in A, with or without DiC 8PI(4,5)P 2. Values are means ± SE of indicated number of determinations. C: effect of DiC 8PIP 2 on bTREK-1 current density. bTREK-1 current density, expressed as pA/pF, determined from recordings in the absence or presence of DiC 8PI(4,5)P 2 (40 µM). Values are means ± SE of indicated number of determinations.

Fig. 3.9. Model for ANG II inhibition of bTREK-1.

CHAPTER 4

ACTH INHIBITS bTREK-1 K + CHANNELS THROUGH MULTIPLE cAMP-

DEPENDENT SIGNALING PATHWAYS

The discovery presented in this chapter has been published in J Gen.

Physiol. (Liu et al., 2008). Haiyan Liu performed most experiments in this chapter except that Judy Enyeart provided measurements of PKA activity in figure 4.3C and 4.7C and Northern blots in figure 4.9A and 4.10D.

4.1. Introduction

Cortisol secretion from the AZF occurs under the control of the pituitary peptide ACTH (Simpson and Waterman, 1988). The molecular mechanisms that couple ACTH receptor activation to cortisol production are only partially understood. Early studies established that cAMP was the principal intracellular messenger for ACTH in AZF cells (Sala et al., 1979; Richardson and Schulster,

1973; Grahame-Smith et al., 1967; Haynes and Berthet, 1957). ACTH stimulates cAMP synthesis in AZF cells, while cAMP mimics the steroidogenic actions of

ACTH (Simpson and Waterman, 1988; Waterman, 1994; Roesler et al., 1988;

HAYNES, Jr. et al., 1959). Accordingly, bovine AZF cells express a high

affinity, MC2-R melanocortin receptor coupled to adenylate cyclase through Gs

(Penhoat et al., 1989; Raikhinstein et al., 1994).

Although cAMP appears to function as the primary intracellular messenger,

Ca 2+ may also act pivotally in ACTH-stimulated corticosteroid secretion. At concentrations that produce little or no measurable increase in cAMP synthesis,

ACTH has been reported to increase intracellular Ca 2+ concentration and stimulate cortisol secretion in bovine AZF cells (Kimoto et al., 1996; Yanagibashi et al., 1990). Similarly, the NPS-ACTH stimulates large increases in [Ca 2+ ]i and corticosteroid secretion at concentrations that trigger no measurable increases in cAMP synthesis in either rat or bovine AZF cells (Moyle et al., 1973; Yamazaki et al., 1998). Overall, these results suggest that cAMP and Ca 2+ are dual messengers that are both required to mediate the full steroidogenic response to

ACTH.

In this regard, a role for electrical events and depolarization-dependent

Ca 2+ entry in ACTH-stimulated cortisol secretion has been established (Enyeart et al., 1993; Mlinar et al., 1993a). Specifically, bovine AZF cells express bTREK-

1 leak-type K + channels, which set the RMP (Mlinar et al., 1993a; Enyeart et al.,

2002a). ACTH receptor activation is coupled to membrane depolarization and

Ca 2+ entry through the inhibition of bTREK-1 channels (Mlinar et al., 1993a;

Enyeart et al., 1993; Enyeart et al., 1996). Accordingly, organic Ca 2+ antagonists inhibit T-type Ca 2+ channels at concentrations that also inhibit ACTH-stimulated cortisol secretion (Enyeart et al., 1993). The NPS-ACTH–stimulated increase in

[Ca 2+ ]i in bovine AZF cells is also inhibited by organic Ca 2+ antagonists at 62

concentrations that inhibit T-type Ca 2+ channels (Yamazaki et al., 1998). Thus, bTREK-1 appears to link ACTH receptor activation to cortisol secretion through depolarization-dependent Ca 2+ entry.

The signaling mechanisms by which ACTH inhibits bTREK-1 channels are incompletely understood. They may involve multiple cAMP- and Ca 2+ -dependent pathways. Specifically, neuronal TREK-1 channels are inhibited by cAMP through PKA-dependent phosphorylation of a carboxyl-terminal serine that is also present in bTREK-1 channels (Patel et al., 1998; Honoré, 2007). Although native bTREK-1 channels can also be inhibited by this mechanism, ACTH and cAMP inhibit bTREK-1 channels in AZF cells in the presence of any of several PKA antagonists (Enyeart et al., 1996).

While all of the cAMP-dependent actions of ACTH in AZF cells were previously thought to be mediated by PKA, alternative signaling pathways for cAMP-mediated responses are likely present in these cells. Specifically, two cAMP-activated guanine nucleotide exchange factors, Epac1 and Epac2 (also known as cAMP-GEFI and cAMP-GEFII), have been identified and implicated in the regulation of proteins including ion channels (Kawasaki et al., 1998; de Rooij et al., 1998; Holz et al., 2008). While Epac1 is expressed in many tissues, Epac2 is robustly expressed in selected areas of the brain and the adrenal glands of rats and humans (Kawasaki et al., 1998). This raises the possibility that cAMP- dependent inhibition of bTREK-1 in the adrenal gland is mediated through separate PKA- and Epac2-dependent mechanisms.

In addition, studies with NPS-ACTH raise the possibility that ACTH might inhibit bTREK-1 by a Ca 2+ -dependent mechanism that is independent of both cAMP and PKA. In this regard, ANG II which also stimulates cortisol secretion from bovine AZF cells, inhibits bTREK-1 in these cells by separate Ca 2+ - and

ATP-dependent pathways (Enyeart et al., 2005; Gomora and Enyeart, 1998; Liu et al., 2007a). Overall, our results and those of other investigators suggest that

ACTH may regulate bTREK-1 channels by multiple cAMP- and Ca 2+ -dependent pathways.

In this regard, Epac1 and Epac2 possess cAMP binding domains that lack a specific glutamate residue present in the binding domain of PKA (Enserink et al., 2002). Using this information and rational drug design, novel cAMP analogues were developed that, at appropriate concentrations, selectively activate the Epac proteins (Enserink et al., 2002; Christensen et al., 2003). We have used one of these, 8-pCPT-2'-O-Me-cAMP, along with other agents involved in cAMP metabolism to further characterize the signaling pathways by which ACTH and cAMP inhibit bTREK-1 in bovine AZF cells. The goal of this study was to determine whether ACTH and cAMP could inhibit bTREK-1 by

Epac2 as well as PKA-dependent pathways.

4.2. Results

Bovine AZF cells express two types of K + channels: a voltage-gated, rapidly inactivating K v1.4 channel, and a two pore domain, four transmembrane- spanning segment (2P/4TMS) bTREK-1 background K + channel (Mlinar et al., 64

1993a; Mlinar and Enyeart, 1993;Enyeart et al., 2000; Enyeart et al., 2002a). In whole cell patch clamp recordings, bTREK-1 amplitude typically increases with time to a steady-state maximum. The absence of time- and voltage-dependent inactivation allows bTREK-1 K + currents to be isolated in whole cell recordings using either of two voltage clamp protocols. When voltage steps of several hundred milliseconds duration are applied from a holding potential of –80 mV, bTREK-1 K + current can be measured near the end of a voltage step when the

+ Kv1.4 K current has fully inactivated (Fig. 4.1, A–C, left traces). Alternatively, bTREK-1 current can be selectively activated by an identical voltage step applied immediately after a 10-s prepulse to –20 mV has fully inactivated K v1.4 channels

(Fig. 4.1, A–C, right traces).

In whole cell recordings from bovine AZF cells, we previously showed that

ACTH inhibits bTREK-1 current by a mechanism that was insensitive to PKA antagonists H-89 and a synthetic inhibitory peptide PKI (5–24) when either was added to the recording pipette (Enyeart et al., 1996). This result indicated that

ACTH could inhibit bTREK-1 by a PKA-independent pathway. In the present study, we measured the inhibition of bTREK-1 with the addition of a second PKI inhibitory peptide, PKI (6–22) amide, to the pipette solution, either alone or in combination with H-89. PKI (6–22) amide is a synthetic peptide patterned after a portion of the naturally occurring PKA inhibitory peptide and inhibits PKA with a reported IC 50 of 1.7 nM (Glass et al., 1989). When added to cytoplasmic extracts from AZF cells, PKI (6–22) amide (4 µM) and H-89 (10 µM) completely inhibited

PKA activated by maximally effective concentrations of cAMP (see Fig. 4.7 C). 65

In contrast, when applied directly to the cytoplasm of AZF cells through the patch electrode, these PKA inhibitors failed to alter the potency or effectiveness of ACTH as an inhibitor of bTREK-1. ACTH inhibits bTREK-1 with an IC 50 of 4.1 pM (Mlinar et al., 1993a). With standard pipette solution, ACTH

(200 pM) inhibited bTREK-1 almost completely (95.2 ± 2.4%, n = 4) (Fig. 4.1, A and D). The addition of PKI (6–22) amide (2 or 4 µM) to the pipette solution did not blunt ACTH-induced inhibition of bTREK-1 (Fig. 4.1 D). PKI (6–22) amide in combination with H-89 (5 or 10 µM) also failed to significantly reduce ACTH- mediated inhibition of bTREK-1 (Fig. 4.1, B and D). The PKA inhibitors were also ineffective at reducing bTREK-1 inhibition by ACTH at a concentration of 20 pM where <2% of all receptors would be activated (Buckley and Ramachandran,

1981; Raikhinstein et al., 1994) (Fig. 4.1, C and D).

4.2.1. bTREK-1 Inhibition by ACTH and cAMP Is Voltage Independent

These results provide further proof that ACTH inhibits bTREK-1 by a PKA- independent mechanism. In this regard, cAMP acting through PKA was reported to inhibit hippocampal TREK-1 channels by a mechanism that converted TREK-1 from a voltage-insensitive leak channel into a voltage-gated outward rectifier

(Bockenhauer et al., 2001). When phosphorylated by PKA, TREK-1 channel open probability was markedly reduced only at negative potentials.

To determine whether ACTH or cAMP converted native bTREK-1 channels into voltage-gated channels, we measured bTREK-1 inhibition by

ACTH (200 pM) or the membrane-permeable cAMP analogue 8-pCPT-cAMP 66

over a wide range of test potentials. When bTREK-1 was activated by voltage steps between –60 and +40 mV, ACTH selectively inhibited this current almost completely at every test potential (Fig. 4.2 A). Similarly, when bTREK-1 currents were measured in response to voltage ramps between –100 and +100 mV,

ACTH totally inhibited this current, even at the most positive test potential (Fig.

4.2 A).

In similar experiments, the membrane-permeable cAMP analogue 8- pCPT-cAMP (300 µM) inhibited bTREK-1 with equal effectiveness over the same range of test voltages (Fig. 4.2 B). These results indicate that neither ACTH nor cAMP inhibit bTREK-1 solely by converting it to a voltage-gated channel through

PKA phosphorylation. Overall, these findings are consistent with the hypothesis that ACTH inhibits bTREK-1 through separate PKA-dependent and -independent mechanisms.

4.2.2. NPS-ACTH Inhibits bTREK-1 by a PKA- and Ca 2+-independent Mechanism

Although our results are consistent with a model wherein ACTH inhibits bTREK-1 through parallel cAMP-dependent paths, they do not exclude the possibility that inhibition could also occur through a separate Ca 2+ -dependent mechanism.

NPS-ACTH reportedly increases [Ca 2+ ]i in bovine AZF cells with little or no increase in cAMP synthesis (Yamazaki et al., 1998). This peptide was used to determine if ACTH could inhibit bTREK-1 by a Ca 2+ -dependent pathway that is independent of cAMP. In this regard, AngII inhibits bTREK-1 by parallel Ca 2+ - 67

and ATP hydrolysis–dependent signaling pathways that can be activated separately using two different pipette solutions (Enyeart et al., 2005; Liu et al.,

2007a). The standard pipette solution containing 5 mM MgATP with Ca 2+ strongly buffered by 11 mM BAPTA facilitates selective activation of an ATP hydrolysis- dependent pathway. The Ca 2+ -dependent pathway can be selectively activated with a pipette solution containing UTP instead of ATP, and 0.5 mM EGTA instead of 11 mM BAPTA (Enyeart et al., 2005).

It was discovered that NPS-ACTH inhibited bTREK-1 only through an ATP hydrolysis–dependent pathway. With pipette solution designed to permit selective activation of a Ca 2+ -dependent pathway, NPS-ACTH (1 nM) failed to significantly inhibit bTREK-1. In contrast, AngII (10 nM) in these same experiments inhibited bTREK-1 by 72.1 ± 4.2% (n = 3) (Fig. 4.3 A). With pipette solution that allowed selective activation of the ATP-dependent pathway, NPS-ACTH reversibly and completely inhibited bTREK-1 with an IC 50 of 20.0 pM (Fig. 4.3, B and C).

These results suggested that NPS-ACTH may inhibit bTREK-1 by the same pathways as ACTH, although less potently. In this regard, the activation of

PKA by ACTH in AZF cells serves as a remarkably sensitive measure of cAMP synthesis. In bovine AZF cells, ACTH activates PKA with an EC 50 of 1.4 pM

(Enyeart and Enyeart, 1998). We found that NPS-ACTH activated PKA in AZF cells, but much less potently, with an EC 50 of 336 pM (Fig. 4.3 C). Although less potent than ACTH, NPS-ACTH was equally effective as a PKA activator at maximum concentrations (unpublished data).

These results show that NPS-ACTH increases cAMP synthesis in AZF cells to a level sufficient to activate PKA. However, they also indicate that bTREK-1 inhibition by NPS-ACTH occurs at concentrations that produce minimal activation of PKA. Accordingly, we found that bTREK-1 inhibition by NPS-ACTH was not affected by including PKA inhibitors in the patch pipette (Fig. 4.3 D).

Overall, these experiments showed that NPS-ACTH increases cAMP synthesis in

AZF cells, but can inhibit bTREK-1 by a PKA- and Ca 2+ -independent mechanism.

4.2.3. Forskolin Inhibits bTREK-1 by a PKA-independent Mechanism

Although all of the actions of ACTH in the adrenal cortex may require the synthesis of cAMP, the failure of PKA inhibitors to suppress bTREK-1 inhibition by ACTH could be indicative of a cAMP-independent, rather than a PKA- independent, action of cAMP. The di-terpene forskolin directly activates AC, increasing intracellular cAMP (Awad et al., 1983). If cAMP inhibits bTREK-1 in

AZF cells solely through activation of PKA, then including PKA inhibitors in the recording pipette should block bTREK-1 inhibition by forskolin. Under control conditions, forskolin (2.5 µM) inhibited bTREK-1 current by 76.9 ± 9.3% (n = 4)

(Fig. 4.4 A). The addition of PKI (6–22) amide and H-89 to the pipette solution, at concentrations that completely inhibited PKA, failed to significantly reduce bTREK-1 inhibition by forskolin (Fig. 4.4, B and C). These findings are consistent with the hypothesis that ACTH and cAMP inhibit bTREK-1 by the same PKA- independent mechanism.

4.2.4. Effect of 8-pCPT-2'-O-Me-cAMP on PKA Activity and bTREK-1 Current in

Perforated Patch Recordings

8-pCPT-2'-O-Me-cAMP has been reported to be a relatively selective activator of Epac proteins. It binds to Epac1 with 100-fold higher affinity than the cyclic nucleotide binding domain of the PKA regulatory subunit (Enserink et al.,

2002; Christensen et al., 2003). When applied to cells in culture at concentrations up to 100 µM, 8-pCPT-2'-O-Me-cAMP has been reported to activate Epac1, but not PKA (Enserink et al., 2002). However, in experiments on bovine AZF cells, we found that at concentrations >30 µM, 8-pCPT-2'-O-Me-cAMP produced significant increases in PKA activity. When applied externally at 100 µM, this cAMP analogue increased PKA activity approximately threefold over the control value (unpublished data).

These PKA activity measurements demonstrated that 8-pCPT-2'-O-Me- cAMP could be used in bovine AZF cells as a selective Epac activator at concentrations up to 30 µM. To determine whether 8-pCPT-2'-O-Me-cAMP could inhibit bTREK-1 independently of PKA, AZF cells were superfused with this agent while recording bTREK-1 currents with the nystatin perforated patch technique

(Horn and Marty, 1988). By using this technique, we hoped to minimize cell dialysis allowing 8-pCPT-2'-O-Me-cAMP to reach a higher intracellular concentration. However, in these experiments, 8-pCPT-2'-O-Me-cAMP (30 µM) inhibited bTREK-1 by only 10.2 ± 6.4% (n = 5), while ACTH (200 pM) inhibited bTREK-1 under the same conditions by 83.8 ± 2.8% (n = 7) (Fig. 4.5, A and C).

The ineffectiveness of 8-pCPT-2'-O-Me-cAMP in perforated patch recordings suggested that ACTH and cAMP do not inhibit bTREK-1 through activation of Epac2. However, the membrane-permeable cAMP analogue 8- pCPT-cAMP, which potently activates PKA and Epac2 (Christensen et al., 2003), also failed to inhibit bTREK-1 when applied at a concentration of 30 µM in perforated patch recordings (Fig. 4.5, B and C). In contrast, at 10-fold higher concentrations, 8-pCPT-cAMP inhibited bTREK-1 by 91.2 ± 6.7 (n = 3) (Fig. 4.5,

B and C). These findings indicate that even in perforated patch recordings, cyclic nucleotides applied externally fail to reach intracellular concentrations that are comparable to those present in the bath solution.

4.2.5. Inhibition of bTREK-1 by Intracellular Application of 8-pCPT-2'-O-Me-cAMP

Because of the limited effectiveness of the cAMP analogues when applied extracellularly at low concentrations and the associated uncertainty of their intracellular concentrations, we measured the effects of 8-pCPT-2'-O-Me-cAMP on bTREK-1 K + channel activity when it was applied directly to the cytoplasm through the pipette. When applied through the pipette at concentrations from 1 to

30 µM, 8-pCPT-2'-O-Me-cAMP potently and effectively suppressed the time- dependent growth of bTREK-1 with an IC 50 of 0.63 µM (Fig. 4.6, A–D). Notably, at a concentration of 1 µM, 8-pCPT-2'-O-Me-cAMP reduced bTREK-1 current density by 63.7% from a control value of 45.2 ± 5.9 pA/pF (n = 15) to 16.4 ± 4.4 pA/pF (n = 6). Using this low concentration of 8-pCPT-2'-O-Me-cAMP in the pipette, bTREK-1 amplitude initially grew, but then declined to a steady-state 71

value (Fig. 4.6 B). At higher concentrations, the initial increase in bTREK-1 amplitude was absent and inhibition of bTREK-1 activity was nearly complete

(Fig. 4.6, C and D). The inhibition of bTREK-1 expression by 8-pCPT-2'-O-Me- cAMP was specific. This agent did not alter the expression of the voltage-gated

Kv1.4 current in these cells (Fig. 4.6 E).

When applied intracellularly through the patch pipette, 8-pCPT-2'-O-Me- cAMP inhibited bTREK-1 at concentrations similar to those that activate Epac in vitro (EC 50 = 2.2 µM). By comparison, 10–20-fold higher concentrations were required to activate PKA holoenzyme (Enserink et al., 2002). Accordingly, we found that 8-pCPT-2'-O-Me-cAMP activated PKA in AZF cell lystates only at concentrations significantly higher than those required to inhibit bTREK-1 current

(Fig. 4.7 C).

4.2.6. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP Is Independent of PKA

The suppression of bTREK-1 expression in AZF cells by 8-pCPT-2'-O-Me- cAMP at concentrations that induce little or no activation of PKA indicate that this enzyme does not mediate the inhibition. Accordingly, 8-pCPT-2'-O-Me-cAMP was effective in inhibiting bTREK-1 activity in the presence of PKA antagonists.

In the experiments illustrated in Fig. 4.7 (A and B), AZF cells were preincubated for at least 30 min with the cell-permeable PKA inhibitor myristolyated PKI(14–22) amide (4 µM) and H-89 (5 or 10 µM) before recording

K+ currents with pipettes containing PKI (6–22) amide (4 µM) and H-89 (5 or 10

µM) with no further addition or with 8-pCPT-2'-O-Me-cAMP at several 72

concentrations. The PKA inhibitors failed to suppress bTREK-1 inhibition by 8- pCPT-2'-O-Me-cAMP at concentrations from 5 to 30 µM (Fig. 4.7, A and B). In contrast, when PKI (6–22) amide and H-89 were added to AZF cell cytoplasmic extracts, PKA activation by 8-pCPT-2'-O-Me-cAMP (1–30 µM) or cAMP (5 µM) was completely inhibited to levels below control values (Fig. 4.7 C). Overall, 8- pCPT-2'-O-Me-cAMP effectively suppressed bTREK-1 channel activity under conditions where PKA had been completely eliminated.

Adenosine-3'-5'-cyclic monophosphorothioate, Rp isomer (Rp-cAMPS) competitively inhibits cAMP activation of PKA, but not Epac, in living cells (Poppe et al., 2008; Holz et al., 2008). The presence of 500 µM Rp-cAMPS in the pipette solution failed to blunt the complete inhibition of bTREK-1 by 8-pCPT-2'-O-Me- cAMP (30 µM), providing further evidence for an Epac2-dependent inhibition of bTREK-1 (Fig. 4.7, A and D).

4.2.7. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP Requires Hydrolyzable

ATP

The inhibition of bTREK-1 by ACTH and 8-pCPT-cAMP requires hydrolyzable ATP (Enyeart et al., 1996). If 8-pCPT-2'-O-Me-cAMP–mediated inhibition of bTREK-1 also proceeds through an ATP hydrolysis-dependent mechanism, then substitution of the nonhydrolyzable ATP analogue AMP-PNP for ATP in the pipette solution should eliminate inhibition by the Epac2 activator.

Accordingly, when pipette solutions contained 2 mM AMP-PNP in place of ATP,

8-pCPT-2'-O-Me-cAMP (30 µM) failed to suppress bTREK-1 expression in whole cell recordings (Fig. 4.7 D).

Inhibition of bTREK-1 in Twice Patched Cells by 8-pCPT-2'-O-Me-cAMP

In whole cell recordings, intracellularly applied 8-pCPT-2'-O-Me-cAMP suppressed bTREK-1 expression, even when the AZF cell had been preincubated with PKA inhibitors, and the patch electrode contained PKA inhibitors. To further demonstrate that 8-pCPT-2'-O-Me-cAMP inhibited bTREK-1 under conditions where PKA activity had been totally blocked in advance, AZF cells were sequentially patched with a pipette containing the PKA antagonists, followed by one containing these antagonists as well as 8-pCPT-2'-O-Me-cAMP.

Control experiments showed that AZF cells could often be consecutively patched with two pipettes without compromising the recording of bTREK-1 currents. In the experiment illustrated in Fig. 4.8 A, the cell was consecutively patched by two pipettes containing standard pipette solution. bTREK-1 current amplitude remained relatively constant upon voltage clamping the cell with the second pipette. In contrast, when the second pipette contained 8-pCPT-2'-O-Me- cAMP (15 µM), bTREK-1 was rapidly inhibited (Fig. 4.8 B). Similar results were obtained in each of four experiments.

Finally, when cells were first voltage clamped with pipettes containing PKI

(4 µM) and H-89 (10 µM) to preinhibit PKA, subsequent voltage clamp of the cell with patch electrodes containing the PKA inhibitors as well as 8-pCPT-2'-O-Me- cAMP produced near complete inhibition of bTREK-1 (Fig. 4.8 C).

4.2.8. 8-pCPT-2'-O-Me-cAMP Fails to Inhibit bTREK-1 when Epac2 Expression

Is Suppressed by ACTH

In a previous study, we demonstrated that prolonged treatment of AZF cells with ACTH enhances the expression of bTREK-1 mRNA and K + channels

(Enyeart et al., 2003). We now report that, under similar conditions, ACTH markedly suppresses the expression of Epac2 mRNA. In the experiment illustrated in Fig. 4.9 A, AZF cells were treated with ACTH (2 nM) for 48 h before isolating mRNA. Northern analysis showed that Epac2-specific mRNA was markedly reduced, compared with its time-matched control.

When AZF cells were exposed to ACTH (10 nM) for 72 or 96 h before recording K + currents, 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 expression. In the experiments illustrated in Fig. 4.9 B, K + currents were recorded from ACTH-treated cells with pipettes containing either standard solution plus

PKA inhibitors, or the same solution supplemented with 8-pCPT-2'-O-Me-cAMP

(5 µM). The maximum bTREK-1 current densities were nearly identical in the absence or presence of the Epac activator. In contrast, when K + currents were recorded from ACTH-treated cells using pipettes containing 8-br-cAMP, which activates PKA as well as Epac2, bTREK-1 was nearly completely inhibited (Fig.

4.9 B).

In other experiments, AZF cells were treated for 72–96 h with ACTH (10 nM) before patch clamping them with pipettes containing PKA inhibitors followed by PKA inhibitors plus 8-pCPT-2'-O-Me-cAMP (5 µM). Under these conditions, 8- pCPT-2'-O-Me-cAMP (5 µM) failed to produce any inhibition of bTREK-1 current 75

in each of four cells (Fig. 4.9 C). In fact, maximum bTREK-1 density was slightly increased from 26.1 ± 3.0 to 29.6 ± 7.9 pA/pF in the presence of the Epac activator. In contrast, when the experiment was repeated with cells that had not been pretreated with ACTH, 8-pCPT-2'-O-Me-cAMP inhibited bTREK-1 by 88.1 ±

3.1% (n = 4) (Fig. 4.9 D).

4.2.9. 8-pCPT-2'-O-Me-cAMP Does Not Inhibit bTREK-1 Activity in Transfected

HEK293 Cells

Epac2 is robustly expressed in a limited number of tissues, including the adrenal glands of rats and humans (Kawasaki et al., 1998). However, the distribution of Epac2 expression within the adrenal has not been determined.

Specifically, it hasn't been shown that Epac2 is expressed in adrenal cortical cells, rather than neural crest–derived adrenal chromaffin cells. In Northern blots of mRNA from bovine AZF cells, Epac2 was readily detected. By comparison, little or no Epac2 mRNA could be detected in HEK293 cells (Fig. 4.10 D).

Experiments were done to determine whether 8-pCPT-2'-O-Me-cAMP could inhibit the activity of cloned bTREK-1 channels expressed in HEK293 cells where

Epac2 is poorly expressed. In contrast to its effect in bovine AZF cells, the presence of 8-pCPT-2'-O-Me-cAMP in the pipette solution at 5 or 15 µM failed to alter the functional expression of bTREK-1 in transfected HEK293 cells (Fig. 4.10,

A and C).

Although Epac2 may be absent from HEK293 cells, these cells do express functional PKA (Rich et al., 2007). N 6-benzoyl-cAMP (6-Bnz-cAMP) is a cAMP 76

analogue that selectively activates PKA over Epac (Christensen et al., 2003).

When 6-Bnz-cAMP (15 µM) was included in the pipette solution, the activity of transfected bTREK-1 K + channels in HEK293 cells was markedly suppressed from a control value of 141.2 ± 25.8 pA/pF (n = 19) to 34.5 ± 6.4 pA/pF (n = 9)

(Fig. 4.10, B and D). These results show that 8-pCPT-2'-O-Me-cAMP fails to inhibit bTREK-1 activity in cells that poorly express Epac2, while these same channels are inhibited in response to specific activation of PKA.

4.3. Discussion

The major findings of this study are that ACTH, NPS-ACTH, and cAMP inhibit bTREK-1 K + channels in bovine AZF cells by multiple cAMP-dependent signaling pathways that likely involve the activation of both PKA and Epac2. bTREK-1 is among the first K + channels identified thus far that is inhibited by parallel cAMP-dependent pathways. ATP-sensitive K + channels of pancreatic β cells are also inhibited by cAMP through separate PKA- and Epac-dependent mechanisms (Light et al., 2002);(Kang et al., 2008). The convergent inhibition of bTREK-1 K + channels by these two cAMP-dependent mechanisms provides for efficient fail-safe depolarization of AZF cells by ACTH.

4.3.1. PKA-independent Inhibition of bTREK-1 by ACTH and Forskolin

Experiments in which PKI (6–22) amide and H-89 failed to blunt bTREK-1 inhibition by ACTH or forskolin provide convincing evidence that inhibition of this channel by cAMP can occur through a PKA-independent mechanism. PKI (6–22) 77

amide was used at concentrations ~1,000–2,000 times the reported IC 50 of 1.7 nM (Glass et al., 1989). H-89, which competes with ATP for its binding site on

PKA, was used at 100–200 times its reported IC 50 of <50 nM (Hidaka et al.,

1991). When applied together to AZF cell cytoplasmic extracts, these agents eliminated the activation of PKA by 8-pCPT-2'-O-Me-cAMP or cAMP. When applied together to the cytoplasm through the pipette solution, these agents should have completely blocked PKA activation by ACTH and forskolin, yet bTREK-1 inhibition was not affected.

The voltage-independent inhibition of bTREK-1 by ACTH and 8-pCPT- cAMP in the presence of PKA antagonists provided additional evidence that this response was not mediated through PKA. Previous studies showed that inhibition of neuronal TREK-1 channels by PKA-dependent phosphorylation occurred through a rightward shift in the voltage-dependent activation of these channels

(Bockenhauer et al., 2001; Maingret et al., 2002). Consequently, open probability was not reduced at positive test voltages. In AZF cells, bTREK-1 was effectively inhibited by ACTH and 8-pCPT-cAMP regardless of test potential, suggesting the involvement of a separate PKA-independent mechanism.

4.3.2. Ca 2+- and PKA-independent Inhibition of bTREK-1 by NPS-ACTH

NPS-ACTH reversibly inhibited bTREK-1 current with an IC 50 of 20 pM.

Although this peptide was reported to trigger step-like and repetitive spike-like increases in [Ca 2+ ]i in bovine AZF cells (Yamazaki et al., 1998), it failed to inhibit bTREK-1 through a Ca 2+ -dependent pathway similar to that activated by ANG II. 78

In this regard, ANG II-stimulated increases in [Ca 2+ ]i are mediated mainly by Ca 2+ released from intracellular sites, while ACTH-mediated increases in [Ca 2+ ]i occur in response to Ca 2+ influx through voltage-gated Ca 2+ channels (Hunyady and

Catt, 2005; Yamazaki et al., 1998). In whole-cell patch clamp experiments where cells are voltage clamped at –80 mV, ACTH-stimulated influx through voltage- gated channels would be eliminated, preempting a Ca 2+ -dependent inhibition of bTREK-1. Under physiological conditions, Ca 2+ entry through voltage-gated channels could contribute to ACTH-stimulated bTREK-1 inhibition and membrane depolarization.

NPS-ACTH did increase the concentration of cAMP in AZF cells to a level sufficient to activate PKA. However, similar to ACTH, the inhibition of bTREK-1 by NPS-ACTH appeared not to be mediated by PKA. First, NPS-ACTH inhibited bTREK-1 half-maximally at a concentration 16-fold lower than that required for half-maximal activation of PKA. Second, TREK-1 inhibition by NPS-ACTH was insensitive to PKA inhibitors. Overall, these results are consistent with a mechanism where, similar to ACTH, NPS-ACTH can inhibit bTREK-1 by a PKA- independent action of cAMP.

4.3.3. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP

8-pCPT-2'-O-Me-cAMP potently and effectively inhibited bTREK-1 activity when applied intracellularly through the patch pipette in whole cell recordings.

Several lines of evidence strongly indicate that this inhibition was mediated through Epac2. First, bTREK-1 expression was inhibited half-maximally with an 79

IC 50 of <1 µM. This result is in excellent agreement with that reported for the potency of this agent in activating Epac1 and Epac2, where binding affinities and

EC 50 s of 1–5 µM have been reported (Rehmann et al., 2003; Enserink et al.,

2002); (Bos, 2003). In contrast, 8-pCPT-2'-O-Me-cAMP activates PKA only at

10–20-fold higher concentrations, and is far less effective than cAMP in this respect (Bos, 2003; Enserink et al., 2002).

Further, we showed that 8-pCPT-2'-O-Me-cAMP inhibited bTREK-1 completely at concentrations that produced little or no measurable increase in

PKA activity in AZF cells. This agent also effectively inhibited bTREK-1 activity in cells where PKA had been inhibited by preincubating cells with PKA inhibitors and with these inhibitors included in the patch pipette at concentrations 100–

1,000 times their reported EC50s. Third, 8-pCPT-2'-O-Me-cAMP inhibited bTREK-1 in twice-patched cells where PKA was first preinhibited by direct application of the two PKA inhibitors to the cytoplasm through the patch pipette.

Finally, 8-pCPT-2'-O-Me-cAMP was ineffective at inhibiting bTREK-1 channels when Epac2 expression had been suppressed by prolonged exposure of AZF cells to ACTH, or in HEK293 cells that express little or no Epac2. In contrast, cAMP derivatives that activate PKA retained their effectiveness in these two systems.

It isn't known whether cAMP might also inhibit bTREK-1 in other cells through PKA- and Epac2-dependent signaling pathways. Since the expression of

Epac2 is limited, other cells expressing both Epac2 and bTREK-1 have not yet

been identified. It is possible that TREK-1 inhibition through Epac2 could occur in selected CNS neurons that express both of these proteins.

4.3.4. Signaling Mechanism for bTREK-1 Inhibition by 8-pCPT-2'-O-Me-cAMP

The mechanism by which 8-pCPT-2'-O-Me-cAMP inhibits bTREK-1 K + channels after activation of Epac2 is unknown. This Epac-selective cAMP

+ analogue inhibits K ATP K channels of pancreatic β cells and cell lines through an

Epac2-dependent mechanism, wherein activated Epac2 interacts with the sulfonylurea receptor 1, a subunit of this inwardly rectifying K + channel to reduce channel activity (Kang et al., 2006). Further, it has recently been shown that

+ Epac-selective cAMP activators function by sensitizing K ATP K channels to inhibition by ATP, shifting the IC 50 20-fold (Kang et al., 2008). bTREK-1 inhibition by this Epac-selective cyclic nucleotide occurs through a different mechanism, since inhibition is blocked when AMP-PNP replaces ATP in the pipette solution, indicating a requirement for ATP hydrolysis. Specific protein kinases activated by

Epac2 have not been identified.

The failure of membrane-permeable 8'-substituted cAMP derivatives, including 8-pCPT-cAMP and 8-pCPT-2'-O-Me-cAMP to inhibit bTREK-1 when superfused externally at low concentrations in perforated patch recordings was unexpected. In perforated patch recording, whole cell dialysis is presumably limited when compared with standard whole cell recording. In standard whole cell recording, we previously found that 8-pCPT-cAMP inhibited bTREK-1 channels with an IC 50 of 167 µM, a concentration much higher than that required to 81

activate PKA in intact AZF cells (Enyeart et al., 1996). Similar high concentrations of 8-pCPT-cAMP were also required to inhibit bTREK-1 in perforated patch recordings.

It seems likely that the reduced potency of cyclic nucleotides applied externally in perforated patch recordings stems from limited transport across the cell membrane. Their potency as bTREK-1 inhibitors increased markedly when they were directly applied to the cytoplasm through the patch pipette. Similar

+ results have been seen in a study of the inhibition of K ATP K channels by Epac- selective cAMP analogues in pancreatic β cells (Kang et al., 2006).

Overall, the selectivity of 8-pCPT-2'-O-Me-cAMP as an Epac-specific activator may have been overstated in the initial studies of this and similar compounds (Enserink et al., 2002; Christensen et al., 2003; Rehmann et al.,

2003). It is clear that when applied externally to intact cells at concentrations >50

µM, these agents produce significant activation of PKA and possibly other cAMP- activated proteins. Results obtained from experiments where these agents were used at concentrations of 100 µM or more in the absence of PKA inhibitors should be interpreted with caution.

Although we have found that bovine AZF cells express Epac2 and that an

Epac-selective cAMP analogue inhibits bTREK-1 at low concentrations under conditions where PKA is inhibited, and fails to inhibit bTREK-1 in cells when

Epac2 expression has been suppressed, the possibility remains that 8-pCPT-2'-

O-Me-cAMP inhibits bTREK-1 through a cAMP-binding protein different than

PKA or Epac2. PKA- and Epac-independent actions of cAMP have been 82

observed in neurons and endocrine cells (Ivins et al., 2004; Gambaryan et al.,

2006).

Our results demonstrate that ACTH inhibits bTREK-1 K + channels in bovine AZF cells through activation of multiple cAMP-dependent signaling pathways. The convergent inhibition of bTREK-1 through separate PKA- and

Epac2-dependent signaling pathways provides an efficient, reliable mechanism for membrane depolarization and Ca 2+ entry.

The inhibition of bTREK-1 by parallel cAMP-dependent pathways resembles that for ANG II inhibition of these same channels by separate Ca2+ - and ATP hydrolysis–dependent pathways (Liu et al., 2007a; Enyeart et al., 2005).

In both instances, nature has developed fail-safe mechanisms for corticosteroid secretion in response to stress by activation of multiple convergent signaling pathways leading to membrane depolarization.

Fig. 4.1. Effect of PKA inhibitors on bTREK-1 inhibition by ACTH. Whole cell K + currents were recorded from AZF cells in response to voltage steps applied from –80 mV at 30-s intervals with or without depolarizing prepulses to –20 mV. Pipettes contained standard solution or the same solution supplemented with PKI (6–22) amide (2 or 4 µM) alone or in combination with H-89 (5 or 10 µM). After bTREK-1 reached a stable maximum, cells were superfused with ACTH (1–24) (200 pM). (A–C) K + current traces recorded with (right traces) and without (left traces) depolarizing prepulses, and corresponding plot of bTREK-1 amplitudes with (open circles) and without (closed circles) depolarizing pulses. Numbers on traces correspond to those on plots. (D) Summary of experiments as in A–C. Bars indicate mean ± SEM of bTREK-1 inhibition by 20 or 200 pM ACTH with or without PKA inhibitors as indicated .

Fig. 4.2. . bTREK-1 inhibition by ACTH and 8-pCPT-cAMP is voltage independent. bTREK-1 was permitted to grow to a stable maximum before whole cell K + currents were activated in response to voltage steps or voltage ramps before and after superfusion of ACTH or 8-pCPT-cAMP. Voltage steps were applied at 30-s intervals in 10-mV increments from a holding potential of –80 mV to test potentials from –60 to +40 mV. Voltage ramps were applied at 100 mV/s to potentials between +100 and –100 mV from a holding potential of 0 mV. (A) Effect of ACTH: K + currents were recorded in response to voltage steps or voltage ramps before and after steady-state block by ACTH (200 pM). Top: current traces in response to voltage steps before and after ACTH. Bottom left: bTREK-1 amplitudes plotted against test potential before (closed circles) and after (open circles) ACTH. Bottom right: bTREK-1 current traces in response to ramp voltages before and after ACTH. (B, left) bTREK-1 amplitudes plotted against test potential in control saline (closed circles) and after 8-pCPT-cAMP (open circles). (B, right) bTREK-1 current traces in response to ramp voltages before and after 8-pCPT-cAMP . 85

Fig. 4.3. Inhibition of bTREK-1 by NPS-ACTH is Ca 2+ and PKA independent. The inhibition of bTREK-1 in bovine AZF cells by NPS-ACTH was measured in whole cell patch clamp recordings using pipette solutions that permitted or blocked activation of Ca 2+ , ATP, or PKA-dependent signaling. K + currents were recorded at 30-s intervals in response to voltage steps to +20 mV from a holding potential of –80 mV. After currents reached a stable maximum, cells were superfused with NPS-ACTH. (A and B) Effect of NPS-ACTH on bTREK-1 through Ca 2+ - and ATP hydrolysis–dependent pathways. bTREK-1 current amplitudes with (open circles) and without (closed circles) depolarizing prepulses are plotted against time. Pipette solutions contained (A) 2 mM UTP, 0.5 mM EGTA, (B) 5 mM MgATP, 11 mM BAPTA. NPS-ACTH and AngII were superfused at indicated times. (C) Concentration dependence of bTREK-1 inhibition and PKA activation by NPS-ACTH were measured in AZF cells and cell, respectively. Data were B fit with an equation of the form (dotted line) I/I MAX = 1/[1 + (X/IC 50 ) ], where X is the NPS- ACTH concentration, and B is the Hill coefficient. IC 50 is the concentration that reduces bTREK-1 by 50%. Values are mean ± SEM of indicated number of determinations, (solid B line) PKA activity = 1/[1+(X/EC 50 ) ], where X is the NPS-ACTH concentration, and B is the Hill coefficient. EC 50 is the concentration that produces 1/2 of the maximum response. (D) Effect of PKA inhibitors on NPS-ACTH inhibition of bTREK-1. bTREK-1 current amplitudes are plotted against time. Pipette solution contained PKI (6–22)amide (4 µM) and H-89 (10 µM) .

Fig. 4.4. Effect of PKA inhibitors on bTREK-1 inhibition by forskolin . Whole cell K + currents were recorded from AZF cells in response to voltage steps applied from –80 mV at 30-s intervals with or without 10-s depolarizing prepulses to –20 mV. Patch pipettes contained standard solution or the same solution supplemented with PKI(6–22) amide (4 µM) alone or in combination with H-89 (5 or 10 µM). After bTREK-1 current reached a stable maximum, cells were superfused with forskolin (2.5 µM). (A and B) K + current traces recorded with (right traces) and without (left traces) prepulses, and corresponding plot of bTREK-1 amplitudes with (open circles) or without (closed circles) depolarizing pulses. Numbers on traces correspond to those on plots. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of bTREK-1 inhibition by forskolin (2.5 µM) with or without PKA inhibitors, as indicated.

Fig. 4.5. Effect of 8-pCPT-cAMP on bTREK-1 currents in perforated patch recordings . (A and B) Perforated patch recordings: whole cell K + currents were recorded in the nystatin perforated patch configuration in response to voltage steps applied at 30-s intervals from a holding potential of –80 to +20 mV with (right traces) or without (left traces) depolarizing prepulses. After bTREK-1 reached a stable amplitude, cells were superfused with 8-pCPT-2'-O-Me-cAMP (designated EA for Epac activator) (30 µM), ACTH (200 pM), or 8-pCPT-cAMP (30 or 300 µM), as indicated. Numbers on current traces correspond to those on plot at right. (C) Summary of experiments as in A and B. Bars indicate mean ± SEM of indicated number of determinations .

Fig. 4.6. Concentration-dependent inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP . K+ currents were recorded from AZF cells with standard pipette solution or the same solution supplemented with 8-pCPT-2'-O-Me-cAMP (EA) at concentrations from 1 to 30 µM. Currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of –80 mV with and without depolarizing prepulses. (A– C) Time-dependent increase in bTREK-1 and inhibition by 8-pCPT-2'-O-Me-cAMP (EA). Current traces recorded with (right) and without (left) depolarizing prepulses at indicated times. bTREK-1 amplitudes are plotted at right. Open circles indicate traces recorded with depolarizing prepulse. (D) Summary of experiments as in A–C. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (E) Effect of 8-pCPT-2'-O-Me-cAMP (EA) on K v1.4 current. Bars indicate K v1.4 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations in control saline and in the presence of 8-pCPT-2'-O- Me-cAMP (30 µM) (EA) . 89

Fig. 4.7. bTREK-1 inhibition by 8-pCPT-2'-O-Me-cAMP is independent of PKA. (A–C) Whole cell K + currents were recorded in response to voltage steps applied at 30-s intervals from –80 to +20 mV with or without depolarizing prepulses. Pipettes contained standard solution or the same solution supplemented with 8-pCPT-2'-O-Me-cAMP (EA), either alone or in combination with PKA inhibitors H-89 (10 µM), PKI(6–22) amide (4 µM), and Rp-cAMPS (500 µM), as indicated. When pipettes contained PKA inhibitors H-89 and PKI(6–22), cells were also pretreated for 30 min with H-89 (10 µM) and myristolated PKI(14–22) peptide (4 µM) before initiating recording. (A) Plots of bTREK-1 amplitude against time with pipettes containing indicated solutions. (B) Summary of experiments as in A. Bars indicate bTREK-1 current density in pA/pF expressed as the mean ± SEM of the indicated number of determinations. (C) Effect of 8-pCPT-2'-O-Me-cAMP (EA) and PKA inhibitors on PKA activity in AZF cell lysates: PKA activity was determined in cell lysates after incubating AZF cells either without (untreated) or with PKA inhibitors for 30 min. PKA activity in lysates from untreated cells was measured after 5 min with no further addition (control) or after addition of cAMP (5 µM) or 8-pCPT-2'-O-Me-cAMP (1– 30 µM) (EA). PKA activity in lysates from PKA inhibitor–treated cells was measured after 5 min incubation with cAMP (5 µM) or 8-pCPT-2'-O-Me-cAMP (1–30 µM) (EA) and the PKA inhibitors as indicated. PKA activity is expressed as % of that activatable by 5 µM cAMP in control lysates. (D) Effect of AMP-PNP on 8-pCPT-2'-O-Me-cAMP inhibition of bTREK-1 current. bTREK-1 current was measured at 30-s intervals using patch pipettes containing AMP-PNP (2 mM) in place of ATP, or this same solution plus 8-pCPT-2'-O- Me-cAMP (30 µM) (EA). Bars represent mean ± SEM of maximum current density for the indicated number of determinations . 90

Fig. 4.8. Inhibition of bTREK-1 by 8-pCPT-2'-O-Me-cAMP in twice-patched cells. Whole cell K + currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from –80 mV, with or without depolarizing prepulses. Cells were sequentially patched with two pipettes containing standard solution, or the same solution supplemented with PKI(6–22) amide (4 µM), H-89 (10 µM), or 8-pCPT-2'-O-Me-cAMP (EA) as indicated. When bTREK-1 reached a stable maximum, the first pipette was withdrawn and the cell patched again with the second pipette. (A–C) Current traces and corresponding plots of bTREK-1 amplitude against time for cells patch clamped with pipettes containing the additions indicated. Closed circles represent pipette #1, closed triangles, pipette #2. Numbers on traces at left correspond to those on plot at right. Break in graph denotes time required to change patch pipettes .

Fig. 4.9. Effect of suppression of Epac2 expression on bTREK-1 inhibition by 8- pCPT-2'-O-Me-cAMP and 8-br-cAMP. (A) Northern blot analysis of ACTH inhibition of Epac2 mRNA expression. AZF cells were cultured either without (control) or with ACTH (2nM). mRNA was isolated after 48 h and analyzed as described in Materials and methods. (B) 8-pCPT-2'-O-Me-cAMP and bTREK-1 expression in ACTH-treated cells. AZF cells were exposed to ACTH (10 nM) for 72–96 h before patch clamping with pipettes containing PKI amide (6–22) (4 µM) and H89 (10 µM) (control), this same solution supplemented with 8-pCPT-2'-O-Me-cAMP (5 µM) (EA), or a solution containing 8-br-cAMP (15 µM). bTREK-1 current amplitudes are plotted against time. Bar graphs: summary of bTREK-1 maximum current densities. Values are mean ± SEM of indicated number of determinations. (C and D) Effect of ACTH treatment on bTREK-1 inhibition by 8-pCPT-2'-O-Me-cAMP in twice-patched cells. AZF cells were cultured in serum-supplemented media with (C) or without (D) 10 nM ACTH for 72–96 h before sequentially recording K + currents with pipettes containing PKI amide (6–22) (4 µM) plus H89 (10 µM) and then these two agents plus 8-pCPT-2'-O-Me- cAMP (EA) (5 µM). K + current traces recorded with (right traces) and without (left traces) depolarizing prepulses. Numbers on traces correspond to those on plots of bTREK-1 amplitude at right . 92

Fig. 4.10. 6-Bnz-cAMP but not 8-pCPT-2'-O-Me-cAMP inhibits bTREK-1 channels expressed in HEK293 cells. Whole cell K + currents were recorded from HEK293 cells that had been transiently transfected with pCR3.1 uni-bTREK-1 cDNA. K + currents were activated by voltage steps to +20 mV, applied at 30-s intervals from a holding potential of –80 mV. Patch pipettes contained control saline or this same solution supplemented with 8-pCPT-2'-O- Me-cAMP (5 or 15 µM) (EA) or 6-Bnz-cAMP (15 µM). (A and B) Effect of 8-pCPT-2'-O- Me-cAMP (EA) and 6-Bnz-cAMP on bTREK-1 current. bTREK-1 current traces and associated time-dependent plots of current amplitudes. Numbers on traces correspond to those on plots at right. (C) Summary of experiments as shown in A and B. Bars indicate maximum bTREK-1 current density expressed as pA/pF. Values are mean ± SEM for indicated number of determinations. (D) Northern blot analysis of Epac2 in bovine AZF and HEK293 cells. Lanes contained 7 µg of poly(A) + RNA from the indicated cells. Hybridization with hEpac2 probe was performed as described in Materials and methods .

CHAPTER 5

POTENT INHIBITION OF NATIVE TREK-1 K + CHANNELS BY SELECTED

DIHYDROPYRIDINE Ca 2+ CHANNEL ANTAGONISTS

The discovery presented in this chapter has been published in J

Pharmacol Exp Ther. (Liu et al., 2007b).

5.1. Introduction

TREK-1 belongs to the mechanogated, thermosensitive phospholipid- and fatty acid-activated family of two pore/four transmembrane segment, leak-type K + channels (Lesage and Lazdunski, 2000). In humans, TREK-1 channels are widely expressed in CNS neurons, the small intestine, and ovaries (Lesage and

Lazdunski, 2000). In bovine adrenocortical cells, where TREK-1 was first identified, these channels set the resting membrane potential, and they function pivotally in the physiology of ACTH- and ANG II-stimulated cortisol and aldosterone secretion (Enyeart et al., 2005).

In neurons, TREK-1 channels may mediate pain and thermosensitivity, confer neuroprotection in global ischemia, and serve as a target for general anesthetics (Franks and Honoré, 2004). Recently, a role for TREK-1 K + channels

in clinical depression has emerged when it was observed that TREK-1 knockout mice displayed a remarkable depression-resistant phenotype (Heurteaux et al.,

2006). This finding suggests TREK-1 as a potential target for a new generation of antidepressants that would function by selective inhibition of these channels in

CNS neurons.

TREK-1 K + channels display a unique pharmacological profile.

Significantly, they are relatively insensitive to standard antagonists of voltage- gated K + channels, including TEA and 4-AP (Gomora and Enyeart, 1999b). In contrast, we discovered that several agents that potently and preferentially inhibit low-voltage-activated T-type Ca 2+ channels are also potent TREK-1 antagonists.

Specifically, in studies on bovine AZF cells, diphenylbutylpiperidine (DPBP) antipsychotics, including penfluridol, fluspirilene, and pimozide, inhibit native bTREK-1 channels with IC 50 values ranging from 187 to 354 nM (Gomora and

Enyeart, 1999b). Penfluridol inhibited cloned bTREK-1 channels expressed in human embryonic kidney 293 cells with similar potency (Enyeart et al., 2002a).

Mibefradil, a novel Ca 2+ antagonist that preferentially inhibits T- over L-type Ca 2+ channels, blocked native bTREK-1 channels with an IC 50 value of 0.5 µM (Alloui et al., 2006; Gomora and Enyeart, 1999a).

The potent inhibition of bTREK-1 channels by these Ca 2+ antagonists was unexpected, and it suggested that perhaps other organic Ca 2+ antagonists might also inhibit bTREK-1. In this regard, DHP Ca 2+ antagonists were of particular interest. DHPs are widely prescribed for the treatment of hypertension and angina (Hoffman, 2005; Michel, 2005). Although the DHPs uniformly and potently 95

block L-type Ca 2+ channels, it has been reported that several of these agents also inhibit T-type Ca 2+ channels (Heady et al., 2001).

In patch-clamp recordings from bovine AZF cells, we have explored the inhibition of native bTREK-1 channels by several DHP Ca 2+ antagonists, including one antagonist that potently inhibits T-type Ca 2+ channels, and by two additional T-channel blockers (Fig. 5.1). We discovered that two DHP Ca 2+ antagonists, amlodipine and niguldipine, inhibited bTREK-1 K + channels at submicromolar concentrations. Amlodipine, the most potent bTREK-1 antagonist, did not inhibit T-type Ca 2+ channels in these cells.

5.2. Results

+ Bovine AZF cells express voltage-gated, rapidly inactivating K v1.4 K channels and bTREK-1 background K + channels (Mlinar et al., 1993a; Mlinar and

Enyeart, 1993; Enyeart et al., 2002a). In whole-cell patch-clamp recordings, bTREK-1 amplitude increases over a period of minutes before reaching a stable maximum. Expression of bTREK-1 is enhanced when pipette solutions contain nucleotide triphosphates at millimolar concentrations (Enyeart et al., 2002a). The absence of time- and voltage-dependent bTREK-1 inactivation allows this current to be isolated in whole-cell recording using either of two voltage-clamp protocols.

When voltage steps of 300-ms duration are applied from a holding potential of –

80 mV, bTREK-1 can be measured near the end of a voltage step when the transient K v1.4 current has inactivated (Fig. 5.2A, left traces). Alternatively, bTREK-1 can be selectively activated by an identical voltage step after a 10-s 96

prepulse to –20 mV has completely inactivated K v1.4 channels (Fig. 5.2A, right traces). Measurement of bTREK-1 by either method provided nearly identical results.

5.2.1. Inhibition of bTREK-1 by DHP Ca 2+ Channel Antagonists .

Three DHP Ca 2+ antagonists, amlodipine, niguldipine, and nifedipine, were compared with respect to their potency as inhibitors of bTREK-1 K + channels in bovine AZF cells. Of these three antagonists, amlodipine and niguldipine inhibited bTREK-1 at submicromolar concentrations, whereas nifedipine was significantly less potent. In the experiments illustrated in Fig. 5.2, A and B, bTREK-1 grew to a stable maximal amplitude over a 15-min period before the cell was superfused with amlodipine at concentrations between 0.2 and 10 µM.

Amlodipine specifically inhibited bTREK-1 in a concentration-dependent manner with an IC 50 of 0.43 µM (Fig. 5.2C). Amlodipine is charged, but highly lipid soluble at physiological pH (Mason et al., 1989). Inhibition of bTREK-1 by amlodipine was slowly reversible with washing (Fig. 5.2B, right).

Although it is a background or leak-type K + channel, bTREK-1 activation is weakly voltage-dependent with open probability enhanced at more positive potentials (Enyeart et al., 1997). Amlodipine inhibited bTREK-1 with equal effectiveness over a wide range of test potentials. The experiment illustrated in

Fig. 5.3A shows current-voltage relationships recorded before and after superfusing a cell with 10 µM amlodipine. In the presence of amlodipine, bTREK-

1 was completely inhibited at all test potentials between –60 and +40 mV. In 97

contrast, the rapidly inactivated K v1.4 current was prominently expressed in the presence of amlodipine (Fig. 5.3A, right traces).

The selectivity of amlodipine as an inhibitor of bTREK-1 compared with

Kv1.4 was quantitated by comparing K v1.4 current amplitudes before and after inhibition of TREK-1 currents by amlodipine (Fig. 5.3B). At concentrations of 2

+ and 10 µM, amlodipine inhibited K v1.4 K currents by only 4.3 ± 1.1% (n = 6) and

12.7 ± 6.9% (n = 4), respectively. By comparison, in the same experiments, bTREK-1 was inhibited by 76.4 ± 3.8% (n = 8) and 96.1 ± 1.9% (n = 5), respectively (Fig. 5.3C).

5.2.2. Amlodipine Reduces bTREK-1 Open Probability but Not Unitary

Conductance .

In single-channel recordings from excised outside-out patches, amlodipine reduced the activity of bTREK-1 channels without changing the amplitude of the unitary currents. Figure 5.4 shows unitary bTREK-1 currents, recorded from an excised outside-out patch in response to voltage steps to +30 mV. Under these conditions, a single type of K + channel was typically active in the membrane patch. Histogram analysis of unitary current amplitudes showed a major peak with a mean of 4.10 ± 0.55 pA (Fig. 5.4A).

As in whole-cell recordings, bTREK-1 channel activity increased spontaneously with time in outside-out patches. As channel activity increased, histogram analysis showed several peaks, each with a mean amplitude a multiple of the first (Fig. 5.4B). However, in contrast to whole-cell recordings, 98

bTREK-1 activity did not reach a stable maximal value in excised patches. In spite of continuous channel run-up, bTREK-1 channel activity was markedly inhibited upon superfusing 4 µM amlodipine (Fig. 5.4C). Although channel open probability was reduced by amlodipine, the unitary current amplitude remained essentially constant at 4.04 ± 0.81 pA. The inhibition of bTREK-1 activity was reversible with washing, revealing the continuous time-dependent increase in bTREK-1 activity (Fig. 5.4D). Overall, these results clearly demonstrate that amlodipine affects bTREK-1 gating, rather than permeation.

Dwell time analysis of unitary bTREK-1 currents showed that under control

conditions, channel kinetics could be described by single open ( o) and closed ( c)

time constants. Amlodipine increased c with little effect on the mean open time.

In the experiment illustrated in Fig. 5.4, amlodipine increased c from the control

value of 10.9 ms to 18.4 ms. By comparison, o in control saline (0.773) did not differ significantly from that determined in the presence of amlodipine (0.680 ms).

The increase in c observed in the presence of amlodipine occurred despite the spontaneous increase in single-channel activity. Accordingly, in the experiment

shown, c decreased markedly upon washing from 18.4 to 2.62 ms.

5.2.3. Effects of Other Ca 2+ Channel Antagonists on bTREK-1.

Two other DHP Ca 2+ antagonists, niguldipine and nifedipine, were compared with respect to their potency as inhibitors of bTREK-1. Although both of these agents inhibit L-type Ca 2+ channels, only niguldipine has been reported to also block T-type channels with similar potency (Romanin et al., 1992). We found 99

+ that niguldipine also potently inhibited bTREK-1 K channels with an IC 50 value of

0.75 µM (Fig. 5.5, A and C). In contrast, nifedipine inhibited bTREK-1 only at

much higher concentrations (IC 50 value of 8.18 µM) (Fig. 5.6B). The inhibition of bTREK-1 by niguldipine was voltage-independent, specific, and not easily reversed with washing. As illustrated in Fig. 5.5B, 10 µM niguldipine inhibited bTREK-1 with equal effectiveness at test potentials between –60 and +40 mV.

With regard to selectivity, niguldipine at concentrations of 2 and 10 µM

+ inhibited K v1.4 K currents by only 0.61 ± 0.39% ( n = 5) and 3.79 ± 2.69% ( n = 4), respectively. In contrast, in the same experiments, bTREK-1 was inhibited by

79.2 ± 2.92% ( n = 7) and 94.7 ± 1.58% ( n = 5), respectively (Fig. 5.5D).

Flunarizine is a diphenyldiperazine that is used clinically to treat migraine and epilepsy. Flunarizine blocks L-, T-, and N-type Ca 2+ channels (Heady et al.,

2001; Santi et al., 2002). Two of the three subtypes of T-type Ca 2+ channels are blocked by flunarizine at submicromolar concentrations (Santi et al., 2002).

+ Flunarizine also inhibited bTREK-1 K channels with an IC 50 value of 2.48 µM

(Fig. 5.6, A and B).

Anandamide is an endogenous cannabinoid found in the brain that directly

2+ blocks two T-type Ca channel subtypes, with IC 50 values of approximately 1 µM

(Chemin et al., 2001). Anandamide also inhibited bTREK-1 channels but less

potently (IC 50 value of 5.10 µM) (Fig. 5.6, A and B).

100

5.2.4. Amlodipine Inhibition of T-type Ca 2+ Current .

2+ Bovine adrenocortical cells express α1H (Ca v3.2) T-type Ca channels that are inhibited by submicromolar concentrations of DPBPs and mibefradil

(Enyeart et al., 1993;Gomora et al., 2000; Schrier et al., 2001). Previous studies on native and cloned bTREK-1 demonstrated that these K+ channels were potently inhibited by the same organic Ca 2+ channel antagonists that potently block T-type Ca 2+ channels in bovine AZF cells (Enyeart et al., 2002a; Gomora and Enyeart, 1999b; Gomora and Enyeart, 1999a).

Experiments were done to determine whether amlodipine would also inhibit T-type Ca 2+ current in AZF cells. The rapidly inactivating and slowly deactivating T-type Ca 2+ current was recorded in response to either long or short depolarizing steps (Fig. 5.7A). At concentrations that produced near complete inhibition of bTREK-1, amlodipine was ineffective at blocking T-type current in these cells. Even at higher concentrations of 10 and 20 µM, amlodipine inhibited

T currents by only 15.6 ± 2.0% ( n = 5) and 28.8 ± 4.5% ( n = 4), respectively (Fig.

5.7, B and C).

5.3. Discussion

In this study, it was discovered that selected DHP Ca 2+ channel antagonists potently inhibit native bTREK-1 K + channels. The most potent of these antagonists, amlodipine, inhibits bTREK-1 half-maximally at a concentration of 0.43 µM, ranking it among the most potent TREK-1 antagonists yet identified (Table 5.1). In this regard, it is interesting to note that among the 29 101

drugs listed in Table 5.1, the six most potent drugs are organic Ca 2+ antagonists.

Each of these antagonists inhibits TREK-1, with an IC 50 value less than 1 µM.

These Ca 2+ antagonists inhibit TREK-1 at concentrations more than 10,000-fold lower than traditional antagonists of voltage-gated K + channels, including 4-AP and TEA. The potent inhibition of bTREK-1 by amlodipine is of particular significance, because amlodipine is used clinically in the treatment of hypertension, and it is among the most frequently prescribed drugs in cardiovascular pharmacology.

5.3.1. Comparison of T-Type Ca 2+ Channel and bTREK-1 K + Channel Inhibition .

Previous studies suggested that TREK-1 was inhibited by Ca 2+ antagonists that potently inhibit T-type Ca 2+ channels (Gomora and Enyeart,

1999b; Gomora and Enyeart, 1999a; Enyeart et al., 2002a). The findings of this present study indicate that the previously noted correlation was coincidental.

Specifically, amlodipine is at least 100-fold less potent than mibefradil and

DPBPs as an inhibitor of T-type Ca 2+ channels in AZF and other cells (Gomora et al., 2000; Enyeart et al., 1993; Santi et al., 2002; Heady et al., 2001). In contrast, amlodipine inhibits bTREK-1 with an IC 50 value comparable with that of the

DPBPs and mibefradil (Gomora and Enyeart, 1999b; Gomora and Enyeart,

1999a; Enyeart et al., 2002a). Although niguldipine is 50 to 100 times more potent than amlodipine at inhibiting T-type Ca 2+ channels in various cells, it was slightly less potent than amlodipine as an inhibitor of bTREK-1 (Heady et al.,

2001; Stengel et al., 1998; Romanin et al., 1992). In bovine AZF cells, 20 µM 102

amlodipine inhibited T-type Ca 2+ channels by only 29.0%, indicating that it was approximately 100-fold more potent as an inhibitor of bTREK-1.

Flunarizine is a relatively potent T-channel antagonist, and it inhibits α1G and α1I channels, with IC 50 values less than one micromolar (Santi et al., 2002).

However, flunarizine was 5-fold less potent than amlodipine as an inhibitor of native bTREK-1 channels. Likewise, anandamide, which inhibits α1H and α1I T- type currents, with IC 50 values of approximately 1 µM, was 10-fold less potent than amlodipine as a bTREK-1 inhibitor (Chemin et al., 2001). Previously, 3 µM anandamide was reported not to significantly inhibit rat TREK-1 K + channels expressed in COS cells (Maingret et al., 2001). In our experience, continuous run-up of TREK-1 activity in transfected cells makes it difficult to measure block at low antagonist concentrations.

5.3.2. Mechanism of bTREK-1 Inhibition by Amlodipine .

cAMP inhibits hippocampal TREK-1 K + channels by a mechanism that reportedly involves the interconversion between leak-type and voltage-dependent phenotypes (Bockenhauer et al., 2001). Consequently, cAMP was much less effective at inhibiting these neuronal K + channels at increasingly positive test potentials. In contrast, amlodipine inhibited bTREK-1 with equal effectiveness over a wide range of test voltages. Although amlodipine did not alter the voltage- dependent activation of bTREK-1, single-channel recording suggested that inhibition occurred through an effect on channel gating rather than permeation.

Specifically, although TREK-1 channel open probability was markedly reduced, 103

the unitary conductance remained unchanged. Nevertheless, our results do not exclude the possibility that this charged DHP inhibits bTREK-1 by pore occlusion.

5.3.3. Amlodipine and TREK-1 in the Cardiovascular System .

Amlodipine is one of the most effective Ca 2+ antagonists prescribed in the treatment of hypertension (Fleckenstein et al., 1989). It is not known whether any of the actions of amlodipine on the cardiovascular system could be due to inhibition of TREK-1 channels. In humans, TREK-1 channels are primarily expressed in the brain, ovaries, and the small intestine (Lesage and Lazdunski,

2000). But TREK-1-like K + channels have also been identified in the mammalian heart, including atria and ventricles, where they are modulated by beta adrenergic agonists and ATP (Tan et al., 2002; Xian et al., 2006; Terrenoire et al.,

2001; Aimond et al., 2000). The role of TREK-1 channels in cardiovascular function has not been determined. At therapeutic concentrations, amlodipine and other Ca 2+ antagonists may interact with TREK-1 K + channels and L-type Ca 2+ channels in the cardiovascular system.

5.3.4. TREK-1 Antagonists in the CNS and Depression .

TREK-1 K + channels are also widely distributed throughout the brain and spinal cord, with particularly high expression in the basal ganglia, hippocampus, and parts of the cerebral cortex (Talley et al., 2001; Hervieu et al., 2001). At the cellular level, TREK-1 channels are distributed over the entire neuronal membrane, including the cell body and processes. Although amlodipine is 104

charged at physiological pH, it has an extremely high lipid-to-water partition coefficient (Mason et al., 1989). The possibility that amlodipine could produce

CNS effects through interaction with neuronal TREK-1 channels cannot be excluded.

In this regard, the recent finding that deletion of TREK-1 in knockout mice produced a depression-resistant phenotype raises the possibility that drugs that potently block TREK-1 would have therapeutic value as antidepressants

(Heurteaux et al., 2006). Interestingly, fluoxetine, a widely used antidepressant that acts primarily as a serotonin uptake inhibitor, also inhibits TREK-1 K + channels (Kennard et al., 2005) (Table 5.1).

Several DHP Ca 2+ channel antagonists, including nifedipine have been reported to possess antidepressant activity in tests on mice and rats (Czyrak et al., 1989; Cohen et al., 1997). However, amlodipine lacked activity in these animal models of depression (Cohen et al., 1997). Since amlodipine is more than

10 times more potent than nifedipine as a TREK-1 antagonist, its lack of antidepressant activity argues against a role of this or other TREK-1 blockers as antidepressants. Accordingly, other drugs that potently inhibit bTREK-1, including the DPBPs niguldipine and mibefradil, have not been reported to possess antidepressant properties.

Thus, although some overlap has been reported between inhibition of

TREK-1 K + channels and antidepressant activity for several drugs, including fluoxetine and selected DHPs, the correlation seems to be coincidental. It remains to be seen whether TREK-1 channel antagonists like amlodipine will 105

modulate other TREK-1-related functions including nociception, neuroprotection, and anesthesia.

106

Fig. 5.1. Chemical structure of Ca 2+ channel and bTREK-1 K + channel antagonists .

107

Fig. 5.2. Concentration-dependent inhibition of bTREK-1 by amlodipine . Whole-cell K + currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of –80 mV with or without 10-s prepulses to –20 mV. After bTREK-1 reached a maximal amplitude, cells were superfused with amlodipine at concentrations ranging from 0.2 to 10 µM. A, K + current records with (right traces) or without (left traces) 10-s prepulses to –20 mV. Numbers on traces correspond to currents recorded at times indicated in B. B, bTREK-1 amplitudes recorded with ( ) or without ( ) depolarizing prepulses are plotted against time. Amlodipine was superfused as shown at the concentrations indicated. Numbers on plot at left correspond to currents in A. C, inhibition curve: fraction of unblocked bTREK-1 current is plotted against amlodipine concentration. Data were fit with an equation of the X form I/IMAX = 1/[1 + ( B/IC 50 ) ], where B is the amlodipine concentration. IC 50 is the concentration that reduces bTREK-1 by 50%, and X is the Hill coefficient. Values are mean ± S.E.M. of indicated number of determinations .

108

Fig. 5.3. Amlodipine inhibition of bTREK-1 is voltage-independent and specific . A, voltage-independent inhibition. K + currents were activated at 30-s intervals by voltage steps of varying size from a holding potential of –80 mV, before and after superfusing 10 µM amlodipine. bTREK-1 current amplitudes in the absence and presence of amlodipine are plotted against test potential. B, specificity: K + currents were recorded in response to voltage steps to +20 mV applied at 30-s intervals from a holding potential of –80 mV. After bTREK-1 reached a maximal amplitude, cell was superfused with 10 µM amlodipine. Current traces are recorded at indicated times in the absence and presence of amlodipine. C, summary of experiments as described in B. Bars indicate percentage + of K v1.4 or bTREK-1 K current inhibited by 2 or 10 µM amlodipine as indicated. Values are mean ± S.E.M. of indicated number of separate determinations .

109

Fig. 5.4. Effect of amlodipine on unitary bTREK-1 channel activity . Unitary bTREK-1 currents were recorded from excised patches in the outside-out configuration in response to voltage steps to +30 mV, applied from a holding potential of –40 mV. Each amplitude histogram was constructed from idealized channel opening obtained from unitary currents recorded during 80 to 96 separate voltage steps of 300- ms duration. Unitary current amplitudes were distributed into bins of 0.18-pA width. Currents were filtered at cut-off frequency of 2 kHz and sampled at 5 kHz. Similar results were obtained in each of four outside-out patches. Traces and corresponding amplitude histograms in control saline (A), in control saline after 9 min of recording to show run-up of current (B), in the presence of 4 µM amlodipine (C), and after washing with control saline (D). The continuous line in the histograms represents the first-order (A), second- order (B and C), or third-order Gaussian fit (D) of the data. Similar results were obtained in each of four outside-out patches . 110

Fig. 5.5. Niguldipine inhibition of bTREK-1. A and C, concentration-dependent inhibition of bTREK-1. Whole-cell K + currents were recorded at 30-s intervals in response to voltage steps to +20 mV, applied from –80 mV with or without 10-s prepulses to –20 mV. After bTREK-1 reached a maximal amplitude, cells were superfused with niguldipine at concentrations from 0.2 to 10 µM. A, K + current records with (right traces) or without (left traces) 10-s prepulses to –20 mV. Numbers on traces correspond to those on plot of bTREK-1 amplitudes at right. B, voltage-independent inhibition. K + currents were activated by voltage steps of varying size from –80 mV before and after superfusing 10 µM niguldipine. bTREK-1 current amplitudes in the absence and presence of niguldipine are plotted against test potential. C, inhibition curve: fraction of unblocked bTREK-1 current is plotted against niguldipine concentration. Data X were fit with an equation of the form I/IMAX = 1/[1 + ( B/IC 50 ) ], where B is niguldipine concentration, IC 50 is the concentration that reduces bTREK-1 by 50%, and X is the Hill coefficient. Values are mean ± S.E.M. of indicated number of determinations. D, specificity: K + currents were recorded in response to voltage steps to +20 mV from a holding potential of –80 mV. After bTREK-1 reached a maximal amplitude, cells were superfused with 2 or 10 µM niguldipine. Bars indicate percentage of K v1.4 or bTREK-1 K+ current inhibited by 2 or 10 µM niguldipine as indicated. Values are mean ± S.E.M. of indicated number of separate determinations . 111

Fig. 5.6. bTREK-1 inhibition by flunarizine, anandamide, and nifedipine . Whole-cell K + currents were recorded from AZF cells at 30-s intervals in response to voltage steps to +20 mV applied from a holding potential of –80 mV with or without 10-s prepulses to –20 mV. After bTREK-1 reached maximal amplitude, cells were superfused with one of the three Ca 2+ antagonists at concentrations from 0.5 to 10 µM. A, flunarizine and anandamide. K + current records with (right traces) and without (left traces) 10-s prepulses to –20 mV in the presence of flunarizine (top) or anandamide (bottom). bTREK-1 amplitudes recorded with ( ) or without ( ) depolarizing prepulses are plotted against time at right. Numbers on plot correspond to those on current traces. B, inhibition curves. fraction of unblocked bTREK-1 current is plotted against niguldipine, flunarizine, or nifedipine concentration as indicated. Data were fit with an equation of the form I/IMAX X = 1/[1 + ( B/IC 50 ) ], where B is the drug concentration, IC 50 is the concentration that reduces bTREK-1 by 50%, and X is the Hill coefficient. Values are mean ± S.E.M. of from three to nine separate determinations . 112

Fig. 5.7. Inhibition of T-type Ca 2+ current by amlodipine . T-type Ca 2+ currents were activated by short (10-ms) or long (300-ms) voltage steps to 0 or –5 mV, applied at 30-s intervals from a holding potential of –80 mV. After recording Ca 2+ currents in standard saline, cells were superfused sequentially with amlodipine at 10 and 20 µM. A, T-type Ca 2+ current records in response to 300-ms (left) or 10-ms (right) depolarizing steps, showing steady-state block of T-type current by amlodipine. Ca 2+ current amplitudes are plotted against time in the presence of 10 and 20 µM amlodipine. C, bars indicate percentage of I T-Ca inhibited by 10 and 20 µM amlodipine. Values are mean ± S.E.M. of indicated number of separate determinations .

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TABLE 1 - PHARMACOLOGY OF TREK-1 K + CHANNEL INHIBITION

DRUG IC 50 (µM) SOURCE

Penfluridol 0.187, (Gomora and Enyeart, 1999b; Enyeart et al., 0.350 2002a) Fluspirilene 0.232 (Gomora and Enyeart, 1999b) Pimozide 0.354 (Gomora and Enyeart, 1999b) Amlodipine 0.43 present study Mibefradil 0.500 (Gomora and Enyeart, 1999a) Niguldipine 0.75 present study Flupenthixol 2.0 (Thümmler et al., 2007) Flunarizine 2.48 present study Chlorpromazine 2.70 (Meadows et al., 2001) Sipatrigine 4.0 (Meadows et al., 2001) Fluphenazine 4.7 (Thümmler et al., 2007) Anandamide 5.10 present study Haloperidol 5.5 (Meadows et al., 2001) Nifedipine 8.18 present study Fluoxetine 19.0 (Kennard et al., 2005) Loxapine 19.7 (Meadows et al., 2001) Quinidine 24.1 (Gomora and Enyeart, 1999b) l-cis -Diltiazem 24.8 (Gomora and Enyeart, 1999b) Yb 3+ 50 (Enyeart et al., 2002b) La 3+ 52 (Enyeart et al., 2002b) Glibenclamide 64 (Gomora and Enyeart, 1999b) Bupivacaine 113 (Gomora and Enyeart, 1999b) Caffeine 377 (Harinath and Sikdar, 2005) Theophylline 486 (Harinath and Sikdar, 2005) Zinc 659 (Gruss et al., 2004b) Tolbutamide 784 (Gomora and Enyeart, 1999b) Ba 2+ 1027 (Gomora and Enyeart, 1999b) 4-AP 2750 (Gomora and Enyeart, 1999b) TEA 24,270 (Gomora and Enyeart, 1999b)

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CHAPTER 6

SUMMARY AND CONCLUSION

The overall aim of this dissertation was to identify and characterize the molecular signaling pathways by which ANG II and ACTH inhibit bTREK-1 K+ channels in bovine AZF cells and the pharmacological profile of this channel.

TREK-1 channels set the RMP and function in regulating cortisol secretion from

AZF cells by linking receptor activation to membrane depolarization and Ca 2+ entry. Because of its pharmacological properties, TREK-1 provides a potential target for new drug development.

6.1. The ATP hydrolysis-dependent mechanism by which ANG II inhibits bTREK-

1 K+ channels in AZF cells .

Previous studies have demonstrated that ANG II inhibits bTREK-1 channels through activation of separate Ca 2+ and ATP hydrolysis-dependent signaling pathways in bovine AZF cells (Enyeart et al., 2005). In this thesis, I further investigated the ATP hydrolysis-dependent mechanism in whole cell patch clamp recordings from these cells. A novel mechanism that mediates the

ATP-dependent inhibition of bTREK-1by ANG II had been discovered.,

115

Specifically, this pathway is independent of PLC and its downstream effectors.

Application of PLC antagonists or PKC antagonists failed to reduce the ATP- dependent inhibition of bTREK-1 by ANG II. However, PDBu, a PKC activator, partially inhibits bTREK-1 through the ATP-dependent mechanism that can be blocked by PKC antagonist, BIM. Moreover, the inhibition of bTREK-1 by ANG II was unaffected by DiC 8PI (4, 5)P 2, suggesting that the ATP-dependent inhibition by ANG II is independent of PIP 2 depletion.

Furthermore, the ATP-dependent inhibition of bTREK-1 by ANG II is insensitive to antagonists of various kinases that are activated by ANG II in AZF cells. In addition, DiC 8PI (4, 5)P2 is a weak activator of bTREK-1 compared with the high-affinity ATP analog 6-PhEt-ATP. Thus, the modulation of bTREK-1 in

AZF cells is distinctive with respect to activation by phosphoinositides and nucleotides and inhibition by Gq-coupled receptors.

Together, these results indicate that under physiological conditions ANG II inhibits bTREK-1 channel through an unusual ATP-dependent mechanism that is independent of PLC activation, PIP 2 depletion and PKC activation.

6.2. The mechanism by which ACTH and cAMP inhibit bTREK-1 K+ channels in

AZF cells.

In AZF cells, inhibition of bTREK-1 channels by ACTH and cAMP is coupled to membrane depolarization-dependent Ca 2+ entry which triggers cortisol secretion. In this thesis, I explored the mechanism by which ACTH and cAMP inhibit bTREK-1 channels in AZF cells using whole cell patch clamp technique. 116

Several lines of evidence showed that ACTH, NPS-ACTH and cAMP inhibit bTREK-1 by multiple cAMP-dependent signaling pathways that involve the activation of both PKA and Epac2.

First, ACTH, NPS-ACTH and forskolin inhibited bTREK-1 in AZF cells.

The inhibitory effect was not affected in the presence of PKA antagonists at concentrations that completely blocked activation of PKA in these cells, indicating that inhibition by these agents is mediated through a PKA-independent pathway.

Second, Epac2 is highly expressed in bovine AZF cells. The selective

Epac activator, 8-pCPT-2'-O-Me-cAMP, potently inhibited bTREK-1 with an IC 50 of 0.63 µM. Inhibition by this agent was unaffected by PKA inhibitors but was blocked by replacing ATP with AMP-PNP. Therefore, Epac2 can inhibit bTREK-1 channels through a PKA-independent mechanism which requires ATP hydrolysis.

Third, culturing AZF cells in the presence of ACTH markedly reduced

Epac2 mRNA level. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 currents in

AZF cells that had been treated with ACTH whereas inhibition by 8-br-cAMP was intact. In addition, 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 channel expressed in HEK293 cells which express little or no Epac2.

In summary, ACTH, NPS-ACTH, and cAMP inhibit bTREK-1 in AZF cells through parallel PKA- and Epac-dependent mechanisms which may provide for failsafe membrane depolarization by ACTH.

117

6.3. Inhibition of bTREK-1 K+ channels by selected DHP Ca 2+ channel antagonists

TREK-1 channels have unique pharmacological properties. Previous studies have shown that bTREK-1 channels can be potently inhibited by several

Ca 2+ channel antagonists (Gomora and Enyeart, 1999b). In this thesis, I further demonstrated that five Ca 2+ channel antagonists including DHP Ca 2+ channel antagonists amlodipine and niguldipine, nifedipine, the diphenyldiperazine flunarizine, and the cannabinoid anandamide also inhibited bTREK-1 channels.

I found that amlodipine, a highly prescribed drug, is the most potent inhibitor of bTREK-1 channels among these five antagonists with an IC 50 of

0.43µM. Inhibition by this agent is voltage-independent and specific. At the single-channel level, amlodipine reduced bTREK-1 open probability without altering the unitary conductance. The potency of other four antagonists as inhibitors of bTREK-1 is niguldipine> flunarizine> anandamide> nifedipine with

IC 50 s of 0.75, 2.48, 5.07, and 8.18µM, respectively.

Among all TREK-1 inhibitors yet described, organic Ca 2+ channel antagonists rank as the most potent ones. Since TREK-1 is widely expressed in the CNS and cardiovascular systems, it is possible that some of the therapeutic effects of frequently prescribed drugs may be due to their interaction with TREK-

1 rather than L-type Ca 2+ channels.

In conclusion, the present study characterizes the mechanisms by which hormones inhibit native bTREK-1 channels in bovine AZF cells. Novel signaling 118

pathways that mediate bTREK-1 inhibition by ANG II and ACTH have been discovered in these cells. These results provide important evidence for future studies to understand the physiological roles of bTREK-1 in hormonal control of cortisol and aldosterone secretion and may serve as guidance for future drug development.

119

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