Mechanisms Underlying Kcnq1channel Cell Volume Sensitivity

THE PHD SCHOOL OF SC IENCE FACULTY OF SCIENCE

UNIVERSITY OF COPENHAGEN

PhD thesis Sofia Hammami

Mechanisms underlying KCNQ1channel cell volume sensitivity

Submitted: 10/05/10

TABLE OF CONTENTS Preface ...... 5 Acknowledgements...... 5 Publications ...... 6 Summary ...... 7 Dansk resumé ...... 8 Abbreviations ...... 9 Table of figures ...... 10 BACKGROUND ...... 11 Ion Channels ...... 11 Mechanosensitive ion channels ...... 12 Cell volume sensitive ion channels ...... 17 The KCNQ1 channel ...... 18 1. Expression and role in epithelia and cardiac tissue ...... 19 2. Regulation ...... 20 a. Regulation by β subunits ...... 21 b. Regulation by cell volume ...... 23 Role of volume sensitive KCNQ1 in mammary epithelium ...... 23 Role of volume sensitive KCNQ1 in liver cells ...... 24 Role of volume sensitive KCNQ1 in cardiomyocytes ...... 24 Purinergic receptors and ATP signalling ...... 25 Receptors ...... 25 ATP release mechanisms ...... 26 THESIS OBJECTIVES ...... 28 METHODS ...... 29 Two-Electrode voltage clamp technique (TEVC) ...... 29 The patch clamp technique ...... 30 ATP bioluminescent assay ...... 30 Enzyme linked immunoassay for surface expression ...... 31 RESULTS AND DISCUSSION ...... 32 Cell swelling vs. membrane stretch ...... 33 ATP release and cell volume changes ...... 35 KCNQ1 association with KCNE1 and volume sensitivity ...... 36 The cytoskeleton ...... 38 Intracellular calcium ...... 38 Cytosolic pH ...... 38 Membrane PIP2 ...... 39 Kinases ...... 39 Specific residues for the volume sensitive potassium channels ...... 40 CONCLUSION ...... 42 REFERENCE LIST ...... 43 APPENDIX ...... 56 Manuscript I: Cell volume and membrane stretch independently control K + channel activity ...... 57 Manuscript II: KCNQ1 channel response to cell volume changes is not mediated by ATP release...... 64 Manuscript III: KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced plasma membrane expression ...... 86 Related paper: Cell swelling and membrane stretch – A common trigger of potassium channel activation? ...... 105

PhD thesis Sofia Hammami

Preface

This presented PhD thesis is the result of three years of work under supervision of Associate Prof. Niels J. Willumsen and Prof. Ivana Novak at the Department of Biology as well as Prof. Dan A. Klærke from the Department of Physiology and Biochemistry, IBHV, at LIFE.

This thesis starts with a general introduction to ion channels followed by an overview of mechanosensitive ion channels with emphasis on volume sensitive potassium channels. Subsequently, I give a description of the KCNQ1 channel and how it is regulated by KCNE1 and by cell volume. This is followed by a brief description of purinergic receptors and ATP signalling. The basics of the different techniques used are described in the method section. Attached to the thesis, are one published article and two other submitted manuscripts. In the discussion, the main findings from these studies are briefly discussed and other possible mechanisms will also be included. In addition, I have attempted to propose different perspectives related to the further identification of the so far unknown mechanisms behind channel activation upon small, fast changes in cell volume.

Acknowledgements

Many people are deserving acknowledgement at this time for their help in making this project possible. First and foremost, I would like to thank my supervisors, Niels and Dan, for excellent guidance and continuous support. It has been a great pleasure to be under their wings during my master and PhD for almost 5 years. I also owe a great thank to Prof. Ivana Novak for the kind supervision and support I received from her during the last 1½ year of my work. I would like to thank all people from the 3rd floor at August Krogh building for a great working environment as well all the members of Dan Klærkes lab, former as well new members. A special thank goes to Zaida Rasmussen for technical help and support. Last, but not least, I‟m thankful to my family, friends and especially Martin for giving me moral support and for always being there when I needed him.

PhD thesis Sofia Hammami

Publications

My work as a PhD student has resulted in 1 published manuscript, 3 published abstracts and 2 submitted manuscripts. Additionally I was invited to write a short review article related to my first manuscript to be published in Physiology News magazine.

1 published article, 2 manuscripts and the related review article are included in the thesis (attached in Appendix):

I. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA (2009). Cell volume and membrane stretch independently control K+ channel activity. J Physiol 587, 2225-2231.

II. Hammami S., Willumsen NJ, Klaerke DA, & Novak I. (2010). KCNQ1 channel response to cell volume changes is not mediated by ATP release. (To be submitted)

III. Hammami S, Klaerke DA & Willumsen NJ (2010). KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced plasma membrane expression (submitted)

Related paper: Cell swelling and membrane stretch – A common trigger of potassium channel activation?

PhD thesis Sofia Hammami

Summary

Cells are constantly exposed to changes in cell volume during cell metabolism, nutrient uptake, cell proliferation, cell migration and salt and water transport. In order to cope with these perturbations, potassium channels in line with chloride channels have been shown to be likely contributors to the process of cell volume adjustments. A great diversity of potassium channels being members of either the 6TM, 4 TM or 2 TM K+ channel gene family have been shown to be strictly regulated by small, fast changes in cell volume. However, the precise mechanism underlying the K+ channel sensitivity to cell volume alterations is not yet fully understood.

The KCNQ1 channel belonging to the voltage gated KCNQ family is considered a precise sensor of volume changes. The goal of this thesis was to elucidate the mechanism that induces cell volume sensitivity. Until now, a number of investigators have implicitly assumed that changes in cell volume are associated with parallel changes in membrane stretch, and, consequently, that regulation by cell volume and by membrane stretch constitute a common regulatory mechanism. This assumption was challenged in Manuscript I where we analyzed and compared the effects of (1) osmotic cell swelling and (2) local membrane stretch on the highly volume sensitive KCNQ1 channel and the highly stretch sensitive BK channel. In this study we present evidence against this assumption by showing that activation of BK channels by local membrane stretch is not mimicked by cell swelling, and activation of KCNQ1 channels by cell volume increase is not mimicked by stretch of the cell membrane. Thus, we conclude that stretch- and volume-sensitivity can be considered two independent regulatory mechanisms.

Alternatively, volume-activation of ion channels could be mediated by an autocrine mechanism in which ATP released from the cells in response to volume changes activates signaling pathways that subsequently lead to ion channel stimulation. Whether volume sensitivity of KCNQ1 is modulated by ATP release was investigated in Manuscript II. ATP release from KCNQ1 injected oocytes was monitored by a Luciferin/Luciferase assay during cell volume changes and the effect of exogenously added ATP and apyrase on the cell volume induced KCNQ1 current was studied. Based on our data to date, we postulate that KCNQ1 does not seem to be responsive to ATP during cell volume changes, which indicates another mechanism of regulation. Besides being regulated by cell volume, KCNQ1 is also modulated by the interaction of the β subunit KCNE1 giving rise to the cardiac IKs delayed rectifier potassium current. Apart from altering the kinetic characteristics of the KCNQ1 channel current, KCNE1 also augments the KCNQ1 current. It is debated whether this increase in macroscopic current upon expression of KCNQ1 with KCNE1 is due to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N). The latter was quantified by measuring the level of KCNQ1 surface expression by using an enzyme-linked immunoassay (Manuscript III). To do this, a HA- tagged version of the KCNQ1 channel was expressed with and without KCNE1 in Xenopus oocytes. The results show that the KCNQ1 surface expression was significantly lower when KCNE1 is coexpressed compared to KCNQ1 alone despite the higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall increase of the KCNQ1 current, when KCNE1 is coexpressed, is not due to an increase in ion channel surface density but rather to an increase in single-channel conductance or in open state probability.

PhD thesis Sofia Hammami

Dansk resumé

Celler er konstant udsatte for volume ændringer ved celle migration, metabolisme, proliferation og ved optagelse af næringsstoffer samt salt og vand transport. For at klare disse forstyrrelser, har kalium kanaler, på samme vis som kloridkanaler, vist sig at være væsentlige i tilbage justering af celle-volumen og dermed bidrage til den regulatorisk volume proces. En stor del af kalium kanaler, som er medlem af enten 6TM, 4 TM eller 2 TM gen familierne har vist sig at være nøje reguleret af små, hurtige ændringer i cellevolumen. Imidlertid er den mekanisme der ligger til grund for kalium kanalers følsomhed over for celle-volume ændringer, endnu ikke helt forstået. KCNQ1 kanalen, der tilhører den spændingsafhængig KCNQ familie betragtes, som en præcis sensor af volume-ændringer. Målet med denne afhandling er at klarlægge den mekanisme, der inducerer kanalens celle-volume følsomhed. Indtil nu har mange studier implicit antaget, at ændringer i cellevolumen er forbundet med parallelle ændringer i membran stræk, og derfor, at de begge reelt udgør en fælles reguleringsmekanisme. Denne antagelse bliver udfordret i manuskript I, gennem en analyse og sammenligning af virkningerne af (1) osmotisk celle svulmning og (2) lokalt membran stræk på den volumen følsomme KCNQ1 kanal samt den stræk-følsomme BK kanal. I denne undersøgelse præsenteres beviser mod denne antagelse. Der vises, at aktivering af BK kanaler ved membran stræk ikke er efterlignet af membranspænding fremkaldt af celle svulmning, samt at aktivering af KCNQ1 kanaler ved celle svulmning ikke er medieret af de lokale spændinger i celle membranen. Således konkluderes, at stræk og volumen-følsomheden skal betragtes som to uafhængigt af hinanden regulerende mekanismer. Alternativt, kunne aktivering af ion kanaler ved volumen ændringer, være medieret af en autocrine mekanisme, hvor ATP frigives fra cellerne og aktiverer signalveje, som derefter fører til stimulering af ion kanaler. Hvorvidt den volumen-følsomme KCNQ1 moduleres af ATP frigivelse undersøges i Manuskript II. Ved denne undersøgelse blev ATP frigivelse fra oocyter, der udtrykker KCNQ1, målt ved hjælp af Luciferin/luciferase assay ved forskellige volume ændringer. Endvidere, blev virkningen af eksogent tilsat ATP og apyrase undersøgt på KCNQ1 kanal strømmen. Baseret på disse data, er det vores opfattelse, at KCNQ1 ikke er reguleret af ATP under ændringer i cellevolumen. Ud over at være reguleret af celle volume ændringer, er KCNQ1 også moduleret af β- subunit KCNE1. Når disse udtrykkes sammen, giver det ændringer i de kinetiske egenskaber af KCNQ1 kanalen herunder en væsentlig forøgelse af KCNQ1 strømmen. En del studier har diskuteret om denne stigning i makroskopisk strøm skyldes en stigning i ion kanal konduktansen (γ), åbnings sandsynligheden (Po) eller en stigning i antallet af kanaler i plasmamembranen (N). Sidstnævnte blev i dette studie kvantificeret ved, at måle niveauet af udtrykte KCNQ1 kanaler på membranoverfladen ved hjælp af enzym-linked immunoassay (Manuskript III). For at gøre dette, er en HA-mærket KCNQ1 kanal blev udtrykt med og uden KCNE1 i Xenopus oocyter. Resultaterne viser, at KCNQ1 overflade ekspressionen blev markant lavere, når KCNE1 er co-udtrykt sammenlignet med KCNQ1 alene. Dette på trods af, at der måles en højere strøm når KCNQ1 og KCNE1 er udtrykt sammen. Dette viser, at den samlede stigning i KCNQ1 strømmen, ved co-udtrykt KCNE1 ikke skyldes en stigning i ion kanalens overflade ekspression, men snarere skyldes en stigning i enkelt-kanal konduktansen eller i åbnings-sandsynligheden.

PhD thesis Sofia Hammami

Abbreviations AA Amino Acids Kv Voltage activated potassium AC Adenylate cyclase LQT Long QT AMP Adenosine monophosphate MAPK Mitogen Activated Protein ADP Adenosine Diphosphate Kinase AP Action Potential mOsm milli osmolarity AQP Aquaporin MS Mechanosensitive ATP Adenosine Tri-Phosphate NTPDase nucleoside triphosphate BK Big Conductance calcium- diphosphohydrolases

activated Potassium channel Po Open state probability BNFC Benign Neonatal Familial PIP2 Phophatidylinositol 4,5 Convulsions biphosphate cAMP Cyclic AMP PKA protein kinase A CFTR Cystic Fibrosis PKC protein kinase C Transmembrane Regulator PLC Phospholipase C CHIF Corticosteroid hormone RVD Regulatory Volume induced factor Decrease ClC Chloride channel RVI Regulatory Volume Increase DAG Diacylglycerol RW Romano-Ward DFNA Deafness autosomal SAC Stretch Activated Channel dominant nonsyndromic SK Small conductance sensorineural Potassium ENaC Endothelial Sodium channel TASK Twik-related Acid-Sensitive HCN Hyperpolarization-activated K+ channel nucleotide-gated channel TEVC two electrodes Voltage HERG Human Ether-a-go-go Clamp related Gene TM Transmembrane IK Intermediate conductance TRAAK Twik related arachidonic calcium-activated Potassium acid K+ channel channel TREK Twik related K+ channel IP3 Inositol Triphosphate TRP Transient receptor potential JLNS Jervall-Lange-Nielsen channel Syndrome TRPP TRP polycystin 2+ KCa Calcium activated potassium TRPV TRP vanilloid channel TWIK Tandem, weak inward KCNE K+ channel Nomenclature rectifier K+ channel family E, a family of β- UDP Uridine Diphosphate subunits UTP Uridine-5'-triphosphate KCNQ K+ channel Nomenclature VRAC Volume regulated anion family Q channel Kir Inward Rectifier Potassium VSOR Volume sensitive outwardly channel rectifying anion channel

PhD thesis Sofia Hammami

Table of figures Figure 1 Studying mechanosensitivity of ion channels...... 14 Table 1 Overview of mechanosensitive ion channels...... 16 Figure 2 Overview of volume sensitive potassium channels across the K+ family tree...... 18 Figure 3 Topology and channel architecture of KCNE and KCNQ1 proteins...... 19 Figure 4 Regulation of KCNQ1 by KCNE1 and cell volume...... 21 Figure 5 Overview of purinergic receptors...... 26 Figure 6 Measurement of surface expressed proteins through enzyme immunoassay...... 31 Figure 7 Possible mechanisms for KCNQ1 cell volume sensitivity ...... 41

PhD thesis Sofia Hammami

BACKGROUND

Ion Channels

All living cells are delimited by a plasma membrane separating the intracellular content from their extracellular surrounding. The plasma membrane is composed of amphipathic phospholipids where small uncharged molecules such as O2 and CO2 can easily cross the membrane whereas other such as amino acids, ions, nucleic acids and carbohydrates, can pass only with the aid of membrane proteins. Among these proteins are ion channels which are integral membrane proteins forming pores that allow passage of inorganic ions mainly Na+, K+, Cl- and Ca2+ across the membrane down their electrochemical gradients.

Ion channels are of pivotal physiological importance for many cellular functions such as the regulation of the membrane potential, control of cardiac excitability, hormone secretion, cell volume regulation, cell proliferation, intracellular signalling and many other biological processes. Because of their great relevance and their specific expression in various tissue cells and organs, ion channels are also involved in many pathophysiological conditions. Diseases involving ion channel dysfunction due to mutations in the genes encoding for ion channels are termed „Channelopathies‟ (Ashcroft, 2000). Today a multitude of human disorders including epilepsy, cystic fibrosis, arrhythmias and many others have been linked to ion channel dysfunction. Therefore ion channels have become major targets for a number of therapeutic agents developed for the treatment of various diseases.

Three major methodological advances have been gathered around the study of ion channel structure and function and have facilitated ion channel research during the past 35-40 years. First the patch clamp technique invented by Bert Sakmann and Erwin Neher in 1976 allowed ionic currents flowing through single channel proteins to be measured with unique precision thereby deducting the single-channel conductance and channel kinetics. The technique is based on making a high resistance seal, a so called giga seal between the glass electrode tip and the cell plasma membrane by applying a negative pressure in the pipette. This high resistance seal enables us to record small amplitudes of single channels in picoAmpere range with very low level of background current noise. Second, advances in molecular genetics have made it possible to clone individual channels and thus relate ion channel function to the protein sequence from which they are constructed. Finally, another major breakthrough

PhD thesis Sofia Hammami came in 1998 when Roderick MacKinnon‟s group used X-ray crystallography to resolve the first three dimensional structure of an ion channel - the bacterial KcsA potassium-channel from Streptomyces lividans (Doyle et al., 1998). This discovery provided an excellent model of eukaryotic K+ channel architecture, an insight into how the ion specificity of channel proteins is achieved and how the voltage sensor of voltage gated ion channels functions.

Three main features distinguish ion channels from other membrane proteins: 1. They conduct ions rapidly nearly as quickly as the ions move through free fluid; 2. They show ion selectivity permitting some ions to pass but not others. This depends on the diameter and shape of the ion channel and on the distribution of charged amino acid (AA) in its pore lining 3. They are gated i.e. they fluctuate randomly between two or more functional states, usually an open and closed state by a change in confirmation. The transition between the states is governed by the rate constants affecting the time spent by the channel in the open or closed state. This open state probability is regulated by an external stimulus such as ligand binding, membrane voltage, temperature and mechanical stimulus such as stretch, shear stress or volume change depending on the type of channel.

This thesis is focused on ion channels regulated by mechanical stimuli more precisely ion channels sensitive to cell volume changes. A brief general introduction to mechanosensitive ion channels is presented in the next section with focus on volume sensitive potassium channels.

Mechanosensitive ion channels

Cells are constantly subjected to mechanical stimuli, such as changes in cell volume, stretch and shear stress. For instance, changes in cell volume take place during physiological processes such as secretion and salt and water transport in the intestine, kidneys and exocrine glands, but may also occur during pathological conditions as in brain and heart ischemia, diabetes and dehydration. Rhythmical stretch and relaxation of the lung epithelium occur during breathing. However, this can be exaggerated during asthma and lung diseases. Shear stress is likely to be seen in all kinds of tubular structures, such as kidney tubules as well as in blood vessels due to the pulsatile nature of blood pressure and flow.

PhD thesis Sofia Hammami

Studies have revealed the presence of many intracellular and membrane bound components that perceive and react upon such mechanical perturbations (Kalapesi et al., 2005). Today, a great knowledge has been gathered about mechanosensitive (MS) ion channels and their association to several major human diseases (Ingber, 2003), such as neuronal and muscular degeneration, cardiac arrhythmias (Kohl et al., 2006), hypertension, polycystic kidney diseases, atherosclerosis (Gautam et al., 2006), muscular dystrophy (Hamill, 2006), brain and cardiac ischemia and much more (Ingber, 2003).

MS ion channels are of various ionic selectivities (Table 1), existing in more than 30 types of cells, from animals to plants to fungi and even bacteria (Hu & Sachs, 1997;Sachs, 1988;Morris, 1990). One common characteristic for MS ion channels is that their gating is altered in response to mechanical stimuli (membrane stretch, cell volume changes or shear stress) generating an ionic current that is subsequently transformed into an electrical response.

Early after the development of the patch clamp technique, the first recordings of cell swelling and stretch-activated channel currents were obtained (Guharay & Sachs, 1984;Hamill, 1983). With the patch clamp technique the mechano-sensitivity of ion channels can be studied while applying or subjecting the cells to different mechanical stimuli (see figure 1).

The most studied class of MS channels is the stretch-activated channels (SAC) (Sachs & Morris, 1998)(Table 1). SACs were first detected in chick skeletal muscles (Guharay & Sachs, 1984). Their open state probability increases with increasing pressure applied at the patch pipette. This mechanical stretch affect channel gating but without significant alteration in current amplitude or conductance.

PhD thesis Sofia Hammami

Cell volume changes Suction Shear stress Δ Osmolarity Negative pressure Fluid flow

Figure 1 Studying mechanosensitivity of ion channels. Mechanosensitivity of ion channels can be studied by 1) exposing the cell to different extracellular osmolarities thus provoking cell swelling or shrinkage; 2) Applying local pressure (stretch) in membrane patches which are in contact with the pipette tip; and 3) inducing shear stress by exposing the cell to fluid flow via the perfusion system (courtesy of N. Willumsen)

The mechanisms linking mechanical stimuli to the subsequent modulation of ion channels are still not clearly understood and many possibilities are under discussion. Multiple studies have provided evidence for the involvement of the underlying cytoskeleton, which exerts forces on the channel leading to channel gating (Kalapesi et al., 2005;Jorgensen et al., 2003;Grunnet et al., 2003;Grunnet et al., 2002b). Others postulate that solely membrane tension mediates channel activation: reconstitution of completely functional stretch activated bacterial channel into liposomes shows that membrane tension can directly be transferred to the channel via the lipid bilayer independently of a underlying cytoskeletal network (Sukharev et al., 1994;Sukharev et al., 1993;Markin & Martinac, 1991). Activation of membrane-bound phospholipases during mechanical deformation was suggested to release fatty acids from the membrane, which subsequently modulates ion channels (Kirber et al., 1992;Ordway et al., 1995). Channel activation may also be a result of mechanical-induced stimulation of a response that has no physical connection with the channel, for example Ca2+ release, ATP release, phosphorylation or alteration of other signalling molecules (e.g. mitogen activated protein kinase (MAPK), Protein kinase A and C) (Aikawa et al., 2002;Chen et al., 1999;Giancotti & Ruoslahti, 1999). Other studies have revealed that shear stress provokes bending of primary cilia which are non-motile structures projecting from the centriole e.g. in

PhD thesis Sofia Hammami renal tubular cells, resulting in Ca2+ influx through mechanosensitive channels residing in the cilium (Schwartz et al., 1997;Praetorius & Spring, 2001).

Table 1 on the next page lists some examples of ion channels that are either regulated by cell volume changes, stretch or shear stress. This table does not include all known mechanosensitive channels though the most important ones are mentioned.

PhD thesis Sofia Hammami

Table 1 Overview of mechanosensitive ion channels. Type of ion channels Ion channel References shear stress sensitive Na+ channels ENaC (Satlin et al., 2001)

K+ channels Kir2.1 (Olesen et al., 1988;Hoger et al., 2002) IK (Brakemeier et al., 2003)

Cl- channels Endothelial chloride channel (Gautam et al., 2006) Outward rectifying CLC

Cation channels TRPV2 and TRPV4 , (O'Neil & Heller, 2005) TRPpolycystin 1 and 2 Stretch sensitive K+ channels (BK) (Kirber et al., 1992;Gasull et al., 2003) Cl- channels CLC5 (Wang et al., 2010) Cation channels Stretch activated cation channels (Hu & Sachs, 1997;Guharay & (SACs) Sachs, 1984)

Volume sensitive K+ channels SK and IK (Grunnet et al., 2002b;Jorgensen et al., 2003);

KCNQ1, KCNQ4, and KCNQ5 (Grunnet et al., 2003;Jensen et al., 2005;Hougaard et al., 2004)

TASK-2, TREK-1 and TRAAK (Kalapesi et al., 2005;Maingret et al., 2002;Lesage et al., 2000;Kelly et al., 2006;Niemeyer et al., 2001)

Slick (Slo2.1) (Personal communication-Stolpe K. and Tejada. M)

Kir4.1 and Kir4.1-Kir5.1 (Soe et al., 2009)

Kv1.3 and Kv1.5 (Deutsch & Chen, 1993;Felipe et al., 1993)

Cl- channels Volume-regulated anion channels (Christensen & Hoffmann, (VRAC) or volume sensitive 1992;Hoffmann & Pedersen, outwardly rectifying anion 2006;Pasantes-Morales et al., channels (VSOR) 2006)

Calcium activated chloride (Almaca et al., 2009) channel TMEM16 Cation channels Hyperpolarization-activated cyclic (Calloe et al., 2005) nucleotide-gated channel 2 (HCN2)

PhD thesis Sofia Hammami

Cell volume sensitive ion channels

During physiological processes such as secretion, cell migration, growth, proliferation, cell metabolism and salt and water transport, animal cells are constantly exposed to variations in intracellular or extracellular osmolorarities resulting in smaller or larger changes in cell volume. In the absence of any kind of mechanism for volume regulation, cells would swell up to the point of lysis or shrink and loose normal functionality, therefore the maintenance of a constant volume is very crucial for cell survival and proper cell function.

In order to counteract the changes in cell volume, cells respond to swelling and shrinkage by processes called regulatory volume decrease (RVD) and increase (RVI), respectively. During cell volume increase, potassium and negatively charged ions (KCl) exit the cell, thereby decreasing the osmolarity of the cytosol. Subsequently, water flux out of the cell to drive volume recovery. Osmotically shrunken cells, in contrast, initiate a gain of KCl and water thereby increasing cell volume to the initial value (Hoffmann et al., 2009). In the last decade, besides chloride channels, studies have been focused on potassium channels as having an important role in sensing the changes in cell volume and triggering regulatory volume mechanisms. Activation of potassium current during volume changes has indeed been reported in a great variety of cell types. These currents are transported by channels belonging to distinct classes of K+ channels, the 6TM, 4TM or 2TM K+ channel family (See figure 2). Interestingly, channels that are homologous have distinct behaviour with respect to cell volume. Some are sensitive to volume and some are not (KCNQ1 vs. KCNQ2 or Slick vs. Slack), probably indicating different cellular expression and functions.

Most of these potassium channels are regulated by instantaneous small changes in cell volume as shown by Grunnet (Grunnet et al., 2003;Grunnet et al., 2002b). In these experiments channels were expressed together with Aquaporin 1 (AQP1) in Xenopus oocytes. Since oocytes are devoid of endogenous water channels, AQP1 was used as a “tool” to make the oocytes swell or shrink. Oocytes exposed to osmotic challenges corresponding to a 27% decrease in bath osmolarity showed an increase of approximately +8% in cell volume. A reversible change in cell volume (-8%) was monitored upon a 27% increase in bath osmolarity (Figure 4 B). These small changes in cell volume evoked dramatic current responses. For instance KCNQ4 currents increased to 258% of control upon cell swelling and decreased 30% of control upon cell shrinkage (Grunnet et al., 2003).

PhD thesis Sofia Hammami

2TM

6TM

Kir1 Kir2 Kir3 Kir4 Kir5 Kir6 Kir7 4TM

Kir4.1 Kir4.1-5.1

Eag KCa2+ Kv KCNQ

Kv1 Kv2 Kv3 Kv4 Kv5 Kv6 Kv8 Kv9

eag erg elk Kv1.3 Kv1.5 KCNQ1 KCNQ2 KCNQ3 KCNQ4 KCNQ5

BK IK SK

TWIK TREK TASK TRAAK THIK TALK KCNK

Slo1 Slo2 Slo3 TASK1 TASK2

Slick Slack

Figure 2 Overview of volume sensitive potassium channels across the K+ family tree. Blue: Volume sensitive K+ channels. Red: Homologeous K+ channels that are not volume sensitive. Modified from (Coetzee et al., 1999;Goldstein et al., 2001)

The KCNQ1 channel

The experimental work in my thesis has been focused on KCNQ1 channel regulation by the β subunit KCNE1 and by cell volume. The following paragraph will therefore mainly concentrate on KCNQ1 and its regulation. KCNQ1, previously named KvLQT1, was the first member of the KCNQ family (KCNQ1-5) to be cloned (Wang et al., 1996a). KCNQ1 channels belong to the 6 TMD family of K+ channels and have 4 positively charged amino acids in the 4th TMD making them voltage gated. Four subunits assemble to make a functional channel (Figure 3).

PhD thesis Sofia Hammami

Figure 3 Topology and channel architecture of KCNE and KCNQ1 proteins. Left: Topology of the KCNE and KCNQ1 proteins with indications of some of the domains important for regulation of the channel. Right: KCNQ1/KCNE channel architecture. Four KCNQ1 -subunits assemble to form the basic channel. KCNQ1 and KCNE coassemble in a 4:2 stoichiometry. (figure from (Jespersen et al., 2005).

1. Expression and role in epithelia and cardiac tissue

KCNQ1 channels have been found in a number of epithelial tissues and have been demonstrated to be essential for transepithelial transport and for participating in potassium absorption and secretion. In the inner ear, these channels play a role in maintaining the proper ion balance needed for normal hearing. KCNQ1 and the auxiliary subunit KCNE1 are expressed in the marginal cells at the apical membrane of the stria vascularis in the cochlea. These cells secrete the endolymph, which is a K+ rich fluid that bathes the stereocilia of the sensory hairs cells and is a prerequisite for the sense of sound and balance. Any mutations in KCNQ1 or KCNE1 leads to a low K+ concentration in the endolymph which results in the degeneration of the sensory hairs cells in the auditory pathway and consequently hearing and balance defects (Ashcroft, 2000;Bleich & Warth, 2000).

In other epithelia, such as pancreas, kidney and airway, KCNQ1 and KCNE1 are crucial for providing a basolateral K+ conductance essential for driving apical Cl- secretion. This

PhD thesis Sofia Hammami important role was demonstrated in experiments where chromanol 293B specifically inhibited KCNQ1 and further abolished Cl- secretion (Bleich & Warth, 2000). KCNQ1 together with KCNE2 have also been localized in the luminal membrane of gastric parietal cells having a crucial role in gastric acid secretion (Heitzmann et al., 2004)

In cardiac tissue, KCNQ1 channels contribute to the repolarisation of the cardiac action potential thereby recharging the muscle after each contraction to maintain a regular heartbeat. Mutations in KCNQ1 gene give rise to long QT syndrome which is a cardiac disorder that may cause arrhythmias, loss of consciousness and sudden death. It is characterized by an abnormally long QT interval in the electrocardiogram (ECG) which reflects the delayed repolarisation of the ventricular action potential. This prolonged action potential can provoke a life-threatening arrhythmia called “torsade de pointes” where the QRS wave changes continuously and swing up and down around the baseline in a chaotic fashion.

Two forms of LQT have been described: an autosomal dominant form known as Romano- Ward syndrome and a much rarer recessive form known as Jervall-Lange-Nielsen (JLN) syndrome. Besides having cardiac abnormalities, patients with JLN syndrome also suffer from deafness. This disease is also linked to mutations in KCNE1 β-subunit.

2. Regulation

KCNQ1 channel activity is regulated by many factors such as calcium, pH, protein kinases, auxiliary β subunits and cell volume. In the following section I will only consider the two last mentioned factors. However, the remaining will be mentioned along with the discussion.

PhD thesis Sofia Hammami

A) Regulation by KCNE1

B) Regulation by cell volume

Figure 4 Regulation of KCNQ1 by KCNE1 and cell volume. A) When expressed alone, the KCNQ1 gives rise to a voltage dependent outward current that reaches a steady state within 1s. Co-assembly of the regulatory β-subunit KCNE1 with KCNQ1 results in a significant change of the electrophysiological properties of the channel. This induces a slowly activating delayed rectifier current called IKs. The voltage activation threshold is shifted to a more positive potential and the inactivation is almost completely absent. The KCNQ1/KCNE1 complex contribute to the repolarisation of the cardiac action potential at the plateau phase (From Jespersen et al., 2005) B) KCNQ1 channels expressed with AQP1 in Xenopus laevis oocytes challenged with a hyposmolar or hyperosmolar extracellular solution . Oocyte cell volume (upper traces) and currents (lower traces) were simultaneously measured. KCNQ1 currents increased to 172% of control upon 8% increase in cell volume and decreased 55% of control upon 8% decrease in cell volume. Co- expression of KCNE1 significantly attenuated the swelling-induced increase in KCNQ1 current, whereas the response to cell shrinking was unchanged. (From Grunnet et al., 2003).

a. Regulation by β subunits

KCNQ1 associates with accessory proteins encoded by the gene family of KCNE which have overlapping tissue distribution with KCNQ1. These proteins, β subunits, are small with

PhD thesis Sofia Hammami a single transmembrane domain (Figure 3). They do not produce current by themselves, however, they change the actual function of the KCNQ1 channel and give it new electrophysiological properties when they associate with it. Until now five different β subunits for the KCNQ1 channel have been identified (KCNE1-KCNE5). These subunits alter cell surface expression and modulate gating properties (Tinel et al., 2000;Angelo et al., 2002;Grunnet et al., 2002a;Jespersen et al., 2005).

The association of KCNQ1 with KCNE1 is the most studied example as they form the + slowly activated delayed rectifier K current, IKs, which contributes to the repolarisation of the cardiac action potential and any mutations in KCNQ1 or KCNE1 has been related to LQT syndrome (Ashcroft, 2000).

KCNE1 considerably increase the KCNQ1 current amplitude, delay its activation, inactivation and shifts the voltage dependence of activation (Figure 4 A). It is now well established that KCNE1 lies in close proximity to the KCNQ1 pore and thereby influencing KCNQ1 conducting properties and pharmacology. Studies have shown that KCNE1 directly interacts with the S5-P-S6 pore domain and sits in a cleft between this pore domain and adjacent voltage sensor (Kang et al., 2008;Panaghie et al., 2006;Melman et al., 2004). Moreover, some residues from the KCNE1 transmembrane domain modulates channel activation (Chen & Goldstein, 2007;Melman et al., 2002) whereas the juxtamembrane C- terminal domain of KCNE1 is important in preventing channel inactivation (Chen et al., 2009;Tapper & George, Jr., 2000).

Studies have shown that KCNE1 requires KCNQ1 co-assembly prior to reaching the cell surface (Chandrasekhar et al., 2006;Vanoye et al., 2010) and newer studies reveal, that once the subunits have been delivered to the membrane they can dissociate from each other (Poulsen & Klaerke, 2007;Jiang et al., 2009) and that KCNQ1 can alternate between being associated with KCNE1 and KCNE2 (Jiang et al., 2009).

Recently, another member of the one transmembrane segment protein family, the corticosteroid hormone induced factor (CHIF) have been shown to be capable of modulating KCNQ1 channel by making the channel constitutively open at all potentials but so far evidence for an actual co-localization of CHIF and KCNQ1 channels in native tissue is lacking (Jespersen et al., 2006).

PhD thesis Sofia Hammami

b. Regulation by cell volume

Sasaki et al (Sasaki et al., 1994) were the first to report an increase in the slowly activating current IKs, during exposure to cell swelling in guinea pig ventricular myocytes. Subsequently, newer studies supported this finding and suggested the channel to contribute to the regulatory volume response in similar and different cell types such as in canine ventricular myocytes (Zhou et al., 1997), primary neonatal rat cardiomyocytes (Vandenberg et al. 1996; Calloe et al. 2007), airway epithelial cells (Lock & Valverde, 2000), rat hepatocyte (Lan et al., 2005), guinea-pig ventricular myocytes (Missan et al., 2006) and mammary epithelial cells (vanTol et al., 2007). Volume-sensitivity of homomeric expressed KCNQ1 channels was also demonstrated in Xenopus oocytes and in COS cells (Kubota et al., 2002;Grunnet et al., 2003). This indicates that KCNQ1 activity upon volume changes is independent of KCNE1 and of the expression system.

Grunnet et al., (2003) have demonstrated that the channel activity augments with increased cell volume and decreased when the cell volume diminished. These cell volume changes where within physiological ranges (8-10% volume increase or decrease) (Figure 4 B). Since it is well documented that epithelial cells change volume during transport of salt and water, the property of being a precise sensor of even small changes in cell volume may explain how the activity of this otherwise “voltage regulated” K+ channel can be modulated in epithelia and play a significant physiological role.

Here are some examples of the role of the volume sensitive KCNQ1 channels in physiological and ischemic conditions:

Role of volume sensitive KCNQ1 in mammary epithelium

Mammary epithelial cells experience changes in volume as a result of variations in milk metabolism and to the presence of the higher content of impermeable solutes such as lactose in milk. Furthermore, the K+ concentration of milk is actually several fold higher than that of plasma suggesting that some mechanisms for K+ secretion and volume regulation must be present in mammary epithelial cells (Shennan & Gow, 2000). In 2007, KCNQ1 expression has been reported for the first time in mammary epithelial cell line. KCNQ1 channels have been exclusively localized at the apical membrane. By using both pharmacological (293B and XE991) and molecular (heterologous expression of dominant negative ΔN-KCNQ1

PhD thesis Sofia Hammami construct) means, vanTol et al have demonstrated that inhibition of KCNQ1 activity abolished the ability of MCF-7 cells to undergo RVD suggesting a physiological role for KCNQ1 in regulating mammary epithelial cell volume (vanTol et al., 2007).

Role of volume sensitive KCNQ1 in liver cells

Hepatocellular nutrient uptake and bile formation in the liver is accompanied by cell swelling, activation of K+ currents and subsequent RVD. Lan W-Z et al, have demonstrated that KCNQ1 channels are indeed participating in the RVD-induced K+ efflux in intact liver. Furthermore, PIP2 indirectly regulates swelling activated potassium current through a PLC- dependent process involving PKC activation and cytoskeletal rearrangement (Lan et al., 2006).

Role of volume sensitive KCNQ1 in cardiomyocytes

During ischemia and reperfusion, cardiomyocytes may experience significant cell swelling due to the breakdown of high energy phosphates and macromolecules (e.g., glycogen, free fatty acids) and the accumulation of lactate within the cell causing swelling. Cell swelling induces activation of IKs current which promotes repolarisation and shortening of the cardiac AP, which in turn may restrict Ca2+ influx and protect myocytes against deleterious Ca2+ overload. Calloe et al, have demonstrated that a slowly activating current mediated by the KCNQ1 channels in complex with the beta subunit KCNE1 is activated in neonatal rat cardiomyocytes upon cell swelling. This current was shown to contribute to the RVD response after ischemia by the help of an intact F-actin cytoskeleton (Calloe K et al 2007). 2+ The swelling induced IKs current may protect the cells from Ca overload however the shortening of the cardiac AP can lead to cardiac arrhythmias.

PhD thesis Sofia Hammami

The following section is written as an introduction to purinergic receptors related to manuscript II.

Purinergic receptors and ATP signalling

Extracellular ATP release is reported in various cell types, e.g. in neuronal cells acting as a neurotransmitter, as well as in epithelial cells acting as an autocrine/paracrine messenger modulating many cellular functions. This ATP release can be triggered by neuronal and hormonal agonists (Abbracchio et al., 2009;Joseph et al., 2003;Novak, 2003) and by mechanical stimuli such as shear stress (Woo et al., 2008;Grierson & Meldolesi, 1995;Maroto & Hamill, 2001), compression (Sauer et al., 2000), stretch (Grygorczyk & Hanrahan, 1997) and cell volume changes (Boudreault & Grygorczyk, 2004;Grygorczyk & Guyot, 2001;Aleu et al., 2003). Once outside it interacts with purinergic receptors located at the cell membrane and subsequently triggers distinct intracellular signalling pathways dependent on the receptor type. Released ATP during cell swelling has been reported to activate ion channels such as chloride and potassium channels and to contribute to the regulatory volume decrease (Wang et al., 1996b;Roman et al., 1997;Feranchak et al., 2000;Perez-Samartin et al., 2000;Hafting et al., 2006;Almaca et al., 2009).

Receptors

Purinergic receptors are divided into two families: 1) P1 receptors that recognize adenosine and which are divided into 4 subtypes A1, A2A, A2B and A3. They are coupled to G proteins and 2) P2 receptors that recognize ATP, ADP, UTP and UDP and which are divided into two subfamilies: ionotropic P2X receptors, which are ligand gated ion channels, and metabotropic P2Y receptors which are G-protein coupled (Figure 5).

Binding of an agonist to the extracellular loop of P2X receptors will mediate receptor conformational change resulting in a rapid non selective transport of cations (Na+, K+, Ca2+) across the cell membrane. A subsequent increase in intracellular calcium elicits membrane depolarization and activation of calcium dependent processes.

Adenosine and P2Y receptors are G-protein coupled receptors that couple to 1) Gi/Go proteins which inhibit adenylate cyclase (AC) and subsequently decrease cAMP, 2) Gs proteins which activate AC and increase cAMP or 3) Gq proteins which activates membrane bound phospholipase C (PLC) that hydrolyses Phophatidylinositol 4,5 biphosphate (PIP2)

PhD thesis Sofia Hammami into inositol triphosphate (IP3) and diacyglycerol (DAG). IP3 will help calcium release from calcium channels at the ER membrane and DAG will lead to activation of protein kinase C which phosphorylates many other proteins.

Figure 5 Overview of purinergic receptors. ATP is released to the extracellular space either by exocytosis or with the aid of membrane transporters. Released ATP acts on P2X and P2Y receptors. P2X receptors which are ligand gated ion channels consists of three subunits that allow the passage of cations. P2X receptors are encoded by seven distinct genes (P2X1 to P2X7). P2Y receptors are proteins consisting of 7 transmembrane domains where the C-terminal is coupled to an intracellular G-protein. Two distinct subgroups of P2Y receptors are recognized dependent on the G-protein they couple to. The P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11 subgroup use Gq and the P2Y12, P2Y13 and P2Y14 subgroup couple to Gi/o protein. ATP is hydrolyzed by membrane bound ecto-nucleotideases (NTPDase: ecto-nucleoside triphosphate diphosphohydrolases) to ADP and AMP. ADP acts also on P2Y receptors. 5- nucleotidase (5'-NT) catalyses the hydrolysis of AMP to adenosine which activates P1 receptors which are also G-protein coupled receptors. (Figure taken from http://www.uni- leipzig.de/~straeter/research/ntpdase.html).

ATP release mechanisms

Under basal conditions, intracellular concentration of ATP lies between 1-5 mM. Extracellularly, ATP concentrations are regulated due to the action of membrane bound ecto-nucleotideases, enzymes that degrades ATP. This concentration gradient favours the

PhD thesis Sofia Hammami movement of ATP out of the cell. Several mechanisms have been suggested regarding how ATP is released under basal and stimulated conditions. It can be released by a non-ionic process through mechanical induced constitutive release of vesicles (Maroto & Hamill, 2001) and through hormonal/neuronal regulated exocytosis (Lazarowski et al., 2003). Moreover release can take place by a conductive ionic process through membrane channels, such as mechanically gated ion channels (Aleu et al., 2003), hemichannels such as connexins (Bahima et al., 2006) and pannexins (Huang et al., 2007), maxi anion channels (Liu et al., 2008), volume regulated anion channels (Hisadome et al., 2002;Fitz, 2007), CFTR (Schwiebert et al., 1995) and P2X7 receptors (Suadicani et al., 2006).

When studying the role of ATP in regulatory volume decrease or increase, some classical tests are used such as looking upon the effect of exogenously added ATP, purinergic receptor blockers or agents that degrade released ATP (such as apyrase) on the cell volume induced currents. Some of these methods were also used in one of the studies in this thesis (manuscript II).

PhD thesis Sofia Hammami

THESIS OBJECTIVES

The overall objective of the whole study was to identify the different mechanisms underlying KCNQ1 sensitivity upon changes in cell volume.

The detailed aims of this thesis were:

1) To test whether potassium channel sensitivity upon changes in cell volume is mediated through sensitivity to changes in membrane stretch (Manuscript I).

We intended to differentiate between membrane stretch and cell volume and examined whether these phenomena share a common mechanism or they are different on their way affecting ion channel regulation. This was examined by the use of the patch clamp technique on different ion channels that have been reported to be activated by one of the two mechanisms (the volume sensitive KCNQ1 channel and the stretch-sensitive BK channel).

2) To test whether volume-activation of KCNQ1 channels could be mediated by an autocrine mechanism in which ATP released in response to volume changes activates signalling pathways that subsequently lead to ion channel stimulation (Manuscript II).

Several studies have shown that ATP released during mechanical stimuli for example upon changes in cell volume has an important role in cell volume regulation and modulating ion channel activity. The goal of the study was first, to monitor ATP release under basal conditions and during cell volume changes for the KCNQ1 injected oocytes and second, to investigate whether ATP release modulates KCNQ1 cell volume sensitivity and to examine the effect of addition or removal of ATP from the extracellular side.

3) To determine whether expression of KCNQ1 with the KCNE1 alters the number of ion channels translocated to the membrane (Manuscript III)

It is debated whether the increase in macroscopic current upon expression of KCNQ1 with KCNE1 is due to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N). The latter was quantified by measuring the level of KCNQ1 surface expression by using an enzyme-linked immunoassay.

PhD thesis Sofia Hammami

METHODS

In the following section, the techniques used during the thesis will be briefly presented. More details related to the specific experiments are described in the different manuscripts.

In this project, oocytes from the aquatic adult female frog Xenopus laevis are used for the heterologous expression of ion channels. The frogs are supplied from the animal facility at the August Krogh Building or the Panum Institute where they are bred under ideal temperature and light conditions. Collagenase treated and defolliculated oocytes are also purchased from the German company Ecocyte. Xenopus oocytes have a developed apparatus for synthesis of foreign injected protein meanwhile lowering the production of their own endogenous proteins (Dascal, 1987). Many electrophysiological techniques can be applied on oocytes for example two-electrode voltage clamp (TEVC), cut open, patch clamp and macropatch or giant patch technique.

Two-Electrode voltage clamp technique (TEVC)

Because of its large size compared to other cells (e.g. CHO, HEK cells), Xenopus Oocyte whole cell currents can not be measured with the conventional patch clamp technique at the whole-cell configuration. The surface area of an oocyte is large with invaginations; therefore there is an enormous amount of membrane that must be charged in order to clamp the oocyte. Therefore the TEVC is a straightforward technique to measure whole cell currents on Xenopus oocytes without any effect or change on the intra-oocyte concentration. Recordings can be made directly on oocytes still coated with the vitelline membrane and experiments can be repeated up to a week after if the oocytes survive that long.

As the name refers to, the technique consists of two electrodes of very thin tips which are inserted into the oocyte. One intracellular electrode is used to record the actual intracellular potential (the voltage electrode) and the second electrode is used to pass current in such way as to maintain the desired potential (the current electrode). This is achieved using a feedback circuit. The principle of the technique is to inject a current which is equal in amplitude but opposite in sign to that which flows across the cell membrane. This technique measure the current that flows through the whole cell membrane. This whole cell current is the sum of the currents flowing through several different kinds of ion channel in the oocyte

PhD thesis Sofia Hammami

The recording chamber is connected to a perfusion system, which make it easy to apply different solutions for example employing solutions with different osmolarities in order to make cell volume experiments meanwhile measuring current alterations.

The patch clamp technique

The patch clamp technique is a method that allows direct observation of single-channel activity thereby deducing the single-channel conductance and channel kinetics. Moreover whole-cell currents from small cells are also recorded. The technique is based on making a high resistance seal, a so called giga seal between the glass pipette tip and the cell plasma membrane by applying a negative pressure in the pipette. This high resistance seal enable us to record small amplitude of single-channel in the order of pA at very low level of background current noise. This technique uses a single electrode both to control the membrane potential and to measure currents.

An advanced method in patch clamping which is commonly used for bigger cells such as the Xenopus laevis oocytes or muscle cells is the so called macropatch or giant patch technique (depending on how big a pipette tip is used). This method has been developed by Hilgemann D. W in 1989 on cardiac myocytes (Hilgemann, 1989). It is basically a cell-attached configuration that allows measuring macroscopic currents from much larger membrane areas containing hundreds of ion channels than in conventional single-channel recording. This requires large diameter patch pipettes of approx. 5-30 μm in tip diameter and a resistance of a few hundred kiloohms. The advantage of this method is that it allows macroscopic current measurements with high signal to noise ratio and because of the low access resistance, microsecond time resolution can be achieved.

ATP bioluminescent assay

In Manuscript II, Basal ATP release and release during cell volume changes is measured by ATP bioluminescent assay based on the firefly Luciferase. This is a widely used tool for conducting quantitative ATP measurements. The assay is based on the following reaction: ATP is consumed and light is emitted when firefly luciferase catalyzes the oxidation of D- luciferin: Firefly luciferase ATP + Luciferin Adenyl-luciferin + PPi Mg++

Adenyl-luciferin + O2 Oxyluciferin + AMP+ CO2 + Light

PhD thesis Sofia Hammami

The first reaction is reversible and the equilibrium lies far to the right. The second reaction is essentially irreversible. When ATP is the limiting reagent, the light emitted is proportional to the ATP present.

Enzyme linked immunoassay for surface expression

In Manuscript III, the surface expression of KCNQ1 channel was studied by means of enzyme linked immunoassay. This was conducted on a double Hemaglutunin (HA) tagged KCNQ1 channel. The two HA-epitopes, with the amino acid sequence YPYDVPDYA, are placed in the extracellular site between the S3 and S4 segment of the KCNQ1 protein. This allows us to measure the amount of surface expressed protein through enzyme immunoassay (Figure 6).

HA-tagged protein complex OPD Absorbance at 450 nm substrate Anti HA primary antibody

HRP- conjugated secondary antibody Color

Extracellular

S1 S2 S3 S4 S5 S6 Intracellular N C

Figure 6 Measurement of surface expressed proteins through enzyme immunoassay. The extracellular segment between S3 and S4 of KCNQ1 is tagged with two HA epitopes. If KCNQ1 is expressed on the cell surface, the HA epitope is accessible to the primary anti-HA antibody. In the second step, bound primary antibody is recognized by a HRP-conjugated secondary antibody. Bound HRP reacts with the OPD solution and gives a yellowish color which can be quantified measuring the absorbance at 450 nm.

PhD thesis Sofia Hammami

RESULTS AND DISCUSSION

Cells have several ways to accommodate for volume expansion and for preventing cell lysis when exposed to hypoosmotic conditions. Both physical changes related to the membrane and the cytoskeleton and intracellular changes that restore optimal intracellular concentrations of enzymes, osmolytes and metabolites occur in order to bring back normal cell volume. Potassium channels play a critical role in regulating cell volume as they are able to “sense” the changes in cell volume and subsequently having a regulatory effect on it. Several mechanisms for coupling of cell volume changes and K+ channel activation have been proposed and are still under discussion.

This thesis has focused on identifying the mechanisms underlying the volume sensitivity of KCNQ1 channel. KCNQ1 surface expression upon interaction with the regulatory subunit KCNE1 was also studied. The main findings are:

1. Cell swelling and membrane stretch are two independent regulatory mechanisms and i.e. KCNQ1 cell volume sensitivity is not mediated by membrane stretch (Figure 4 in manuscript I) 2. KCNQ1 channel response to cell volume changes is not mediated by ATP release (Figure 3 and 4 in manuscript II) 3. KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced plasma membrane expression (Figure 3 in manuscript III)

In the following discussion, these results will be discussed in line with other possible mechanisms. Where it is appropriate, I have attempted to propose different perspectives related to the further identification of the mechanisms underlying channel activation upon changes in cell volume.

PhD thesis Sofia Hammami

Cell swelling vs. membrane stretch

Activation of ion channels by membrane stretch and by cell volume changes have been considered until recently as one common mechanism. The study in Manuscript I, discloses that volume changes and membrane stretch are two distinct mechanisms. Ion channel activation during cell swelling seems not be a result from membrane stretch and even though a channel is activated by stretch, it does not seem to react upon cell swelling. This suggests that stretch and swelling may activate K+ channels by distinct mechanisms and that they do not depend on each other. From this finding we can conclude that KCNQ1 cell volume sensitivity is not mediated by membrane stretch.

If the stretch sensitive BK channels act as a “biosensor” of cell membrane stretch and if swelling stretches the membrane then a BK response would be elicited. Whether cell volume changes is in fact a membrane phenomenon and whether membrane stretch really takes place during cell swelling have been of major debate. Groulx 2006 has shown that during moderate swelling (50% decrease in osmolarity) cells increase their surface area by 30% mainly by unfolding the surface membrane. In the other hand, under extreme hypotonic swelling (98% decrease in osmolarity) the cell prevents lysis by exocytotic insertion of membrane from intracellular pools or by stretching the membrane. However, in most physiological situations, Groulx indicates that membrane tension is unlikely to reach the level required to activate stretch regulated ion channels (Groulx et al., 2006).

Earlier studies have shown that stretch-induced activation of some K+ channels found at the single channel level is confirmed at the whole cell level through the increase of K+ currents in hypotonic solutions (Sackin, 1989;Filipovic & Sackin, 1992;Davidson, 1993;Allard et al., 2000;Christensen & Hoffmann, 1992). However, we have to remain critical to these results as in these studies, cells were exposed to more than 50% reduction in bath osmolarity (a hypotonic shock) resulting in an increase in cell volume to e.g. 66% (Sackin, 1989) whereas Grunnet et al. (2003) showed that oocytes exposed to osmotic challenges of -50 mOsm/l (27% reduction in bath osmolarity) increased about +8 % of the volume, which is much more closer to physiological ranges during “real life”. In fact, the use of unphysiologically large hypoosmotic shock may optimize the chance of seeing changes in mechanosensitive channel activity as a last line of defence against excessive cell swelling. Grunnet et al., 2003 considered KCNQ1 channels as precise sensors because they sense very small changes in volume. That means, theoretically speaking, that a hypoosmotic shock (>50% decrease in

PhD thesis Sofia Hammami osmolarity or more) that will provoke membrane tension due to unfolding and smoothing out of excess membrane area of the oocyte (in the form of microvilli and membrane folds) is not necessary to activate KCNQ1 channels, but very small volume changes are enough to activate them. This again points out that KCNQ1 sensitivity to these very low changes in cell volume is not membrane-mediated, but could be due to cell volume induced alteration of intracellular components, e.g. second messengers that subsequently affected the activity of the channels.

In another recent study, atomic force microscopy made it possible to “picture” the effect of cell volume expansion on the plasma membrane (Spagnoli et al., 2008). Unexpectedly, the study has shown that the cells get softer and not stiffer when they are swollen; hence no membrane stretch is taking place. This is due to the sponge like property of the cytoskeleton which bears most of the osmotic stress whereas little is attributed to the membrane.

From our study and the study by Spagnoli, we have proven that cell volume changes may not be confined to the cell membrane tension per se and hence the volume response of KCNQ1 is mediated through an alternative mechanism.

A recent study (Otway et al., 2007), reports a novel volume-responsive KCNQ1 mutation in a kindred with late-onset familial atrial fibrillation. The variant called R14C on the KCNQ1 gene having a cysteine instead of an Arginine at codon 14, is located within the short cytoplasmic N-terminus of the KCNQ1 protein. Functional studies have indicated a more marked response to cell swelling compared to wild type IKs channels: a higher increase in current, a more leftward shift in the voltage dependence of activation, faster acceleration of activation and slowing of deactivation were observed. This variant had a gain of function effect causing cardiac action potential shortening (Otway et al., 2007). However in this study they named this variant a stretch sensitive KCNQ1, though based on cell volume experiments and not on membrane stretch. Yet, it could be interesting to expose this R14C variant to real membrane stretch through pipette pressure in order to confirm or reject this supposed stretch sensitivity.

The key finding in manuscript I, is that membrane stretch and cell volume changes constitute independent ion channel regulators. This is true for KCNQ1 and BK channels used in the study. Whether the assumption is a general property relating to all mechanosensitive ion channels should be further examined. For this purpose e.g. the TRAAK and TREK channels belonging to the two-pore potassium channel family are particularly interesting, since they

PhD thesis Sofia Hammami are apparently sensitive to small changes in cell volume as well to membrane stretch (Patel et al., 2001). In addition, it seems well documented that the stretch sensitivity of the TREK channels is dependent on a single amino acid, namely a positively charged amino acid located in close proximity to the cell membrane at the inner part of transmembrane segment 2 (Honore et al., 2002). It would be of great interest to change the charged amino acid to a neutral amino acid by site directed mutagenesis, a procedure which has earlier been shown to eliminate the stretch sensitivity, and subsequently expose the mutated channels to cell volume changes and membrane stretch. If the channel is still volume sensitive then this will confirm our conclusion that cell volume and membrane stretch are two mechanisms regulating the same channel type by two different ways.

ATP release and cell volume changes

Many studies have shown that release of ATP induced by cell swelling can modulate chloride and potassium channels through G-protein coupled signalling pathways (Wang et al., 1996b;Roman et al., 1999;Perez-Samartin et al., 2000;Light et al., 2003;Darby et al., 2003;Hafting et al., 2006). In the case of the volume sensitive potassium channel KCNQ1, we demonstrated in manuscript II that released ATP upon changes in cell volume in KCNQ1±AQP1 injected oocytes does not contribute to the volume sensitivity of KCNQ1 channels indicating that ATP does not modulate KCNQ1 activity through a purinergic signalling pathway in our expression system.

Interestingly, previous studies have shown a coupling of the KCNQ1 and its auxiliary β- subunit KCNE1 to purinergic signalling in native tissues. The strial marginal cells and vestibular dark cell of the inner ear of rodents expresses both KCNQ1/KCNE1 and purinergic receptors (P2Y4) at the apical membrane having an important function in endolymph homeostasis and protection from overstimulation (Housley et al., 2009;Lee & Marcus, 2008). KCNQ1/KCNE1 channel activity and thus K+ secretion was shown to be modulated by 3 purinergic pathways in rodents when P2Y4 receptors are stimulated: 1) G protein-PLC activation leads to consumption of membrane PIP2 with the consequent reduction of K+ channel activity; 2) the DAG-PKC path decreases K+ channel activity directly via phosphorylation of the channel; and 3) the IP3/Ca2+ path decreases the channel activity directly via the effect of Ca2+ on channel activity (Lee & Marcus, 2008). Additionally, Honoré et al (1992) have reported that stimulation of purinergic receptors regulates the KCNQ1/KCNE1 channel activity in mouse heart. This study was concluded by injection of

PhD thesis Sofia Hammami cardiac polyA+ RNA from neonatal mouse heart into Xenopus oocytes. The RNA directed the expression of IKs channel as well the expression of purinergic P2 receptors. By stimulating these receptors it produced intracellular Ca2+ increase, DAG and thereby activating protein kinase C which alters IKs activity (Honore et al., 1992).

It seems obvious to consider a similar purinergic signaling mechanism taking place during changes in cell volume. Yet, extracellular added ATP that activate Gq receptors and stimulate

PKC activity did not have any effect on the stimulation of IKs current by hypoosmotic solution in guinea-pig ventricular myocytes (Missan et al., 2006).

The fact that we did not see any coupling of KCNQ1 channel during volume changes with purinergic signaling may be because there is indeed no link, or because of the expression system that we used. We cannot exclude that some components necessary to initiate or complete the purinergic signaling pathway are missing in the Xenopus oocyte expression system that are normally present in warm blooded animal cells. It may be therefore necessary to try in another expression system for example HEK cells or COS cells. Moreover, we are not sure that purinergic receptors are present in defolliculated oocytes (Discussed in manuscript II); however adenosine receptors are apparently present. Whether a signaling pathway via adenosine receptor can take place should be investigated. Moreover, according to the previous studies in native cells KCNE1 is implicated in the functional purinergic signaling pathway. In our study we did not include KCNE1. We may therefore need to repeat the same experimental protocol with KCNQ1/KCNE1 coexpression.

KCNQ1 association with KCNE1 and volume sensitivity

Besides being volume regulated, KCNQ1 is regulated by the β subunit KCNE1. Heteromeric association of KCNE1 with KCNQ1 induces a current with electrophysiological channel properties markedly different from that of the KCNQ1 channel itself. KCNE1 association give rise to a much larger current than the one seen by KCNQ1 alone. This increase in current is not mediated by an increase in the number of ion channels translocated to the membrane (N) as shown in Manuscript III however it may be due to an increase in the open state probability (Po) or the single channel conductance (γ). These findings are in agreement with previous studies which additionally show an increase in ion channel conductance upon coexpression (Yang & Sigworth, 1998;Pusch, 1998;Sesti & Goldstein, 1998).

PhD thesis Sofia Hammami

In relation to cell volume experiments, there is conflicting results as to whether KCNE1 is crucial for the volume sensitivity of KCNQ1. Studies on proximal convoluted tubule epithelial cells from KCNE1 knockout mice, show that KCNQ1 failed to respond to cell swelling, indicating that KCNE1 is essential for KCNQ1 volume sensitivity (Lock & Valverde, 2000) However, other studies indicate that the volume sensitivity is exclusively a KCNQ1 property, though slightly modulated by KCNE1 (Grunnet et al., 2003;Kubota et al., 2002) (Figure 4 B).

In principle, KCNQ1 channels could respond to cell volume changes by modulation of either (i) their open state probability, (ii) their single channel conductance, or (iii) by changes of the number of channels inserted in the plasma membrane. In Manuscript III, we used an enzyme-linked immunoassay and a HA-tagged version of the KCNQ1 channel in order to estimate the number of ion channels present in the plasma membrane in the presence and absence of KCNE1. This method is a plausible way to measure the number of ion channels at the cell membrane during resting and during swelling or shrinkage. For this purpose, oocytes need to be fixed at the different osmolarities, however preliminary trials with the formaldehyde as a fixative made the oocytes to shrink. Since the goal was to measure the number of ion channels during cell swelling and cell shrinkage, we could not proceed with this method.

Groulx postulated that exocytotic insertion only takes place under extreme hypoosomotic challenges; it seems that KCNQ1 current increase during modest cell swelling is then not due to alteration in the number of ion channels in the membrane surface. However, this can be further elucidated by the use of laser based total internal reflection fluorescence microscopy, a technique that visualizes events happening at the membrane surface such as vesicle trafficking and subsequent fusion and release of individual ion channels (which are fluorescently labelled) at the plasma membrane. This should allow us, in real time, to detect if channels are moved in and out of the plasma membrane of the Xenopus oocyte during cell volume changes.

Recently a potassium channel belonging to the BK family called Slick (Slo2.1) was shown to be volume sensitive (See table 1 and figure 2). This channel has a high conductance and therefore single channel events are easily detected with the patch clamp technique unlike the KCNQ1 channel. It would therefore be interesting to conduct single channel recordings on the slick channel in order to look after any alteration in Po or single channel conductance during changes in cell volume.

PhD thesis Sofia Hammami

The cytoskeleton

A rearrangement of the underlying cytoskeleton occurs during cell volume changes and in particularly actin filaments have been implicated in the modulation of ion channels during cell swelling and shrinkage (Pedersen et al., 2001). Treatment with cytochalasin D, which interferes with actin assembly has shown inhibition of KCNQ1 current in Xenopus oocytes (Grunnet et al., 2003) and in cardiomyocytes (Calloe et al., 2007) and subsequent abolishment of the regulatory volume decrease. This indicates that an intact actin cytoskeleton is important for activation of channels by cell swelling. For the KNCQ1 channels there is evidence that the interaction with the cytoskeleton takes place at the N-terminal of the channel protein.

In contradiction, similar studies where guinea-pig ventricular myocytes were treated with cytochalasin D had no effect on the response of IKs to hypoosomotic solution (Missan et al., 2006). Additionally, the volume sensitive Slick channel was not affected either by Cytochalasin treatment in Xenopus oocytes (Stolpe K. and Tejada M., personal communication). Therefore, the cytoskeleton may in some cases be involved in cell volume regulation of K+ channels, but it does on the other hand not seem to be a crucial player.

Intracellular calcium

It has been suggested that KCNQ1 can be inhibited (Shen & Marcus, 1998) or augmented (Kerst et al., 2001) by intracellular Ca2+, or indeed wholly Ca2+ insensitive unless coexpressed with KCNE1 (Boucherot et al., 2001). Apparently, a change in cytosolic calcium can not be detected in Xenopus oocytes during volume changes (Vandorpe et al., 1998;Grunnet et al.,

2002b). Moreover, in rat hepatocytes and colonic epithelial cells volume induced IKs current was shown to not be dependent on an increase in intracellular calcium (Lan et al., 2005) (Kunzelmann et al., 2001)

Cytosolic pH

There is evidence that upon cytosolic acidification, homomeric KCNQ1 channel current is inhibited whereas heteromeric KCNQ1/KCNE1 channel current is activated (Unsold et al., 2000;Heitzmann et al., 2007). In Xenopus oocytes a significant change in cytosolic pH

PhD thesis Sofia Hammami apparently does not occur upon the small changes in cell volume. Previous data have shown that the almost pH-insensitive Kir 4.1 and the strongly pH-sensitive Kir.4.1-Kir 5.1 responded almost identically to cell volume changes when expressed in Xenopus oocytes indicating no alteration in pH (Soe et al., 2009). It seems therefore unlikely that KCNQ1 volume response is due to changes in intracellular pH. However, this may be different in other expression systems or native cells. In fact, cardiac muscle tissue gets acidified down to a pH of 5.5-6.5 during heart ischemia and the fact that KCNQ1/KCNE1 current activates during acidification may have an important pathophysiological function (Heitzmann et al., 2007).

Membrane PIP2

A number of studies suggest a role of the phospholipid PIP2 as an important regulator of K+ channels (Hilgemann, 1997) among them are the Kir channels (Logothetis et al., 2007) and KCNQ channels (Zhang et al., 2003) (Lan et al., 2005;Loussouarn et al., 2003;Park et al., 2005). Hepatocellular volume current mediated by KCNQ1/KCNE1 has been shown to be dependent on PIP2 (Lan et al., 2005;Loussouarn et al., 2003;Park et al., 2005).

The requirement of PIP2 for KCNQ1/KCNE1 activity raises the possibility of physiological regulation of the channel complex by receptor coupled PLC. This evidence is provided for vestibular dark cell of the inner ear where purinergic receptor signalling has been coupled to the modulation of KCNQ1/KCNE1 channel through a G protein-PLC pathway (Lee & Marcus, 2008) earlier mentioned.

Upon volume increase and decrease in Ehrlich ascites cells, a respective decrease and increase in membrane PIP2 was observed (Nielsen et al., 2007). Given that KCNQ1/KCNE1 activity decreases upon consumption of PIP2 shown in earlier studies (Lee & Marcus, 2008;Matavel & Lopes, 2009), the increase in KCNQ1 activity that we see during cell swelling in Xenopus oocytes does not coincide with a PIP2 regulation.

Kinases

Protein kinase A and C are also known to be modulators of KCNQ1 channels (Boucherot et al., 2001;Grunnet et al., 2003;Schroeder et al., 2000;Kunzelmann et al., 2001). An earlier study, reinvestigated the possible involvement of PKA, PKC and Tyrosine kinase in the

PhD thesis Sofia Hammami

hypoosmotic stimulation of cardiac IKs in guinea-pig ventricular myocytes. They demonstrated that neither PKA, PKC (also in agreement with Grunnet 2003), PKG nor PI3- K is mediating KCNQ1/KCNE1 cell swelling response. However, Tyrosine kinase did have an effect (Zhou et al., 1997;Missan et al., 2006;Missan et al., 2008) coinciding with the presence of 7 tyrosine phosphorylation sites on KCNQ1 (Missan et al., 2006).

Specific residues for the volume sensitive potassium channels

At present, sensitivity to small changes in cell volume can be assigned to several potassium channels. This is a new regulatory mechanism along with voltage gated and ligand gated potassium channels. Whether this observable phenotype is related to a specific genotype and whether these potassium channel proteins have common conserved/specific residues for volume sensitivity is just beginning to be revealed. For the case of KCNQ1, it seems obvious that the specific residue responsible for the channel volume sensitivity lies within the N- terminal site. When the N-terminal end of 95 AA is deleted, the channel loses its ability to respond to changes in cell volume (Grunnet et al., 2003). Additionally, the mutated variant R14C on the KCNQ1 gene located within the cytoplasmic N-terminus was demonstrated to cause a marked increase in activation in response to cell swelling compared to wild type IKs channels (Otway et al., 2007). We have preliminary evidence that the 17 amino acids closest to the cell membrane are crucial (personal communication with Dan A. Klærke). By extensive mutagenesis in this region, it will be possible to determine exactly which amino acids are involved.

As mentioned in the introduction, we have recently found that two other, homologous channels, slo2.1 (Slick) and slo2.2 (Slack) show different responses to changes in cell volume, Slick is sensitive, whereas Slack is not (Figure 2). A closely related potassium channels to KCNQ1, the KCNQ2 does also exert non-volume sensitivity (Figure 2). This situation provides us the opportunity to construct chimeras between KCNQ1 and KNCQ2 channels or Slick and Slack, co-express the chimeras with AQP1 in Xenopus oocytes, and test their ability to be regulated by changes in cell volume in order to identify what specific region in the channel that is responsible for the volume sensitivity. These studies might help us to identify a sequence motif that might be common to all volume regulated potassium channels.

PhD thesis Sofia Hammami

Figure 7 Possible mechanisms for KCNQ1 cell volume sensitivity

PhD thesis Sofia Hammami

CONCLUSION

Figure 7 summarizes the above discussed possible mechanisms for volume sensitivity of KCNQ1 channel. These events may also be true for other potassium channels.

By looking at the figure, cell swelling seems to be an unspecific stimulus. Many processes can take place and affect the membrane embedded ion channels. Physical changes of the membrane such as membrane stretch, unfolding of membrane invaginations or insertion of extra membrane reserves may occur, as well as the rearrangement of the underlying cytoskeleton. Alteration in the intracellular calcium concentration and pH, ATP release, activation of purinergic signalling cascades and diverse kinases are some of the many processes that takes place modulating ion channel activity. These processes can vary from one cell type to another, having different effect on a specific type of channel. They can be interacting events occurring simultaneously, so interpreting cause and effect regarding the volume sensitivity of a particular ion channel can be difficult. However, we can only understand the mechanism by breaking it down to simpler components which are much manageable to study. For the case of KCNQ1 channel, we have come to the conclusion that membrane stretch and ATP release are not mediating cell volume sensitivity of the channel, at least in our expression system. However, it became clear that we need to look into the N- terminal end of the channel which is in contact with the actin cytoskeleton in order to identify the specific sequence motif that might be responsible for the regulation of the KCNQ1 channel by small changes in cell volume.

PhD thesis Sofia Hammami

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120. Sesti F & Goldstein SA (1998). Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome. J Gen Physiol 112, 651-663.

121. Shen Z & Marcus DC (1998). Divalent cations inhibit IsK/KvLQT1 channels in excised membrane patches of strial marginal cells. Hear Res 123, 157-167.

122. Shennan DB & Gow IF (2000). Volume-activated K(+)(Rb(+)) efflux in lactating rat mammary tissue. Biochim Biophys Acta 1509, 420-428.

123. Soe R, MacAulay N, & Klaerke DA (2009). Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by small changes in cell volume. Neurosci Lett 457, 80-84.

124. Spagnoli C, Beyder A, Besch S, & Sachs F (2008). Atomic force microscopy analysis of cell volume regulation. Phys Rev E Stat Nonlin Soft Matter Phys 78, 031916.

125. Suadicani SO, Brosnan CF, & Scemes E (2006). P2X7 receptors mediate ATP release and amplification of astrocytic intercellular Ca2+ signaling. J Neurosci 26, 1378-1385.

126. Sukharev SI, Blount P, Martinac B, Blattner FR, & Kung C (1994). A large- conductance mechanosensitive channel in E. coli encoded by mscL alone. Nature 368, 265-268.

127. Sukharev SI, Martinac B, Arshavsky VY, & Kung C (1993). Two types of mechanosensitive channels in the Escherichia coli cell envelope: solubilization and functional reconstitution. Biophys J 65, 177-183.

128. Tapper AR & George AL, Jr. (2000). MinK subdomains that mediate modulation of and association with KvLQT1. J Gen Physiol 116, 379-390.

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129. Tinel N, Diochot S, Borsotto M, Lazdunski M, & Barhanin J (2000). KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel. EMBO J 19, 6326-6330.

130. Unsold B, Kerst G, Brousos H, Hubner M, Schreiber R, Nitschke R, Greger R, & Bleich M (2000). KCNE1 reverses the response of the human K+ channel KCNQ1 to cytosolic pH changes and alters its pharmacology and sensitivity to temperature. Pflugers Arch 441, 368-378.

131. Vandorpe DH, Shmukler BE, Jiang L, Lim B, Maylie J, Adelman JP, de FL, Cappellini MD, Brugnara C, & Alper SL (1998). cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation. J Biol Chem 273, 21542-21553.

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139. Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T, & Logothetis DE (2003). PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor- mediated inhibition of M currents. Neuron 37, 963-975.

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APPENDIX

1 published article, a related press article and 2 manuscripts are included in the thesis:

I. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA (2009). Cell volume and membrane stretch independently control K+ channel activity. J Physiol 587, 2225-2231.

II. Hammami S., Willumsen NJ, Klaerke DA, & Novak I. (2010). KCNQ1 channel response to cell volume changes is not mediated by ATP release. (about to be submitted)

III. Hammami S, Klaerke DA & Willumsen NJ (2010). KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced plasma membrane expression (submitted)

Related paper: Cell swelling and membrane stretch – A common trigger of potassium channel activation?

PhD thesis Sofia Hammami

Manuscript I: Cell volume and membrane stretch independently control K + channel activity

PhD thesis Sofia Hammami

Manuscript II: KCNQ1 channel response to cell volume changes is not mediated by ATP release

Hammami S.1, Willumsen NJ1, Klaerke DA2, & Novak I1.

1Department of Biology, Faculty of Science, University of Copenhagen, Denmark;

2 Department of Physiology and Biochemistry, IBHV, Faculty of Life Sciences, University of

Copenhagen, Denmark

PhD thesis Sofia Hammami

Abstract

Objective: A number of K+ channels are regulated by small, fast changes in cell volume.

The mechanisms underlying cell volume sensitivity are not known. One frequently stated hypothesis is that cell volume sensitivity is mediated by membrane stretch. In a recent study we presented evidence against this assumption by showing that the highly volume sensitive KCNQ1 channel is not affected by membrane stretch. Another hypothesis is that cell volume could be mediated by an autocrine or paracrine mechanism in which ATP released from the cells in response to volume changes activates signaling pathways that subsequently lead to ion channel stimulation. Our aim was to investigate whether volume sensitivity of KCNQ1 is dependent on ATP release. Methods: KCNQ1 K+ channels were co-expressed with AQP1 in Xenopus laevis oocytes and currents were measured by

TEVC. Oocytes were subjected to volume changes by exposure to iso-, hypo- or hypertonic media with and without application of ATP. In other experiments the

ATP/ADP hydrolyzing enzyme apyrase was added. ATP release was also confirmed by a luciferin-luciferase assay in non-injected and KCNQ1±AQP1 injected oocytes before and after swelling and shrinkage. Results: In electrophysiological experiments it was shown that apyrase (7 U/ml) decreased all currents by about 50%. When oocytes were swelled or shrinked, the relative increase or decrease in current was not affected by apyrase nor application of extracellular ATP. Luminescence assay showed that there was an increase in ATP release in response to mechanical and hypotonic/hypertonic stimuli. Basal ATP release was also higher for the KCNQ1±AQP1 injected oocytes compared to the non- injected. Conclusion: Based on our data to date, we postulate that KCNQ1 does not seem to be responsive to ATP nor apyrase during cell volume changes in AQP expressing oocytes. This indicates that purinergic signaling is not involved in volume sensitivity/regulation of KCNQ1 channel.

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Introduction

Adenosine triphosphate (ATP) is a highly hydrophilic molecule responsible for energy storage inside the cells. In the last decades many studies have reported a continuous basal release of ATP into the extracellular medium where it can function as an autocrine/paracrine signal. Extracellular ATP interacts with purinergic receptors (ATP receptors) located at the cell membrane and modulates many cellular functions, such as regulation of tissue blood flow, growth, neuronal activity, epithelial transport and response to pathogens (Corriden & Insel, 2010). Various cell types, both secretory and non-secretory, have been shown to release ATP upon activation by neuronal and hormonal agonists (Abbracchio et al., 2009;Joseph et al., 2003;Novak, 2003), as well when exposed to mechanical stress and changes in cell volume. These studies also indicate an important role of ATP in cell volume regulation (see review (Franco et al.,

2008).

It is well known that a number of K+ channels are regulated by small, fast changes in cell volume and are involved in cell volume regulation (Jensen et al., 2005;Jorgensen et al.,

2003;Grunnet et al., 2003;Grunnet et al., 2002;Hougaard et al., 2004). Many theories have been proposed on how they are triggered during cell volume changes. A possible mechanism would be that volume-activation of ion channels could be mediated by an autocrine/paracrine mechanism in which the induced ATP release in response to cell volume changes activates signaling pathways that subsequently lead to ion channel stimulation as previously shown for different other ion channels (Hafting et al.,

2006;Franco et al., 2008;Corriden & Insel, 2010).

In our laboratory, ion channel activity during small and fast changes in cell volume is studied by coexpressing potassium channels with AQP1 in Xenopus oocytes, which are

PhD thesis Sofia Hammami natively devoid of water channels. Grunnet et al (2002) have shown that in non-AQP1 expressing oocytes, decreasing or increasing the osmolarity of the extracellular medium by 50 mOsm, changed cell volume by less than 0.2% after 50 s. In contrast oocytes expressing AQP1, they responded immediately with significant changes in volume; these oocytes swelled or shrank, respectively, by approximately 5% within 50 s and volume continued to change for more than 300 s (Grunnet et al., 2002). Particularly, the potassium channel KCNQ1 is very sensitive to changes in cell volume (Grunnet et al.,

2003) and is thought to have an important physiological role in cell volume regulation in cardiomyocytes (Calloe et al., 2007), mammary epithelial tissues (vanTol et al., 2007), liver cells (Lan et al., 2006)... Many theories have been proposed on how the channel activity is triggered during cell volume changes. One possibility was that cell volume sensitivity could be mediated by membrane stretch. We have recently disproved this hypothesis and shown that cell membrane stretch and cell volume change are two independent mechanisms that can regulate BK and KCNQ1 channels (Hammami et al.,

2009). In several studies it was shown that hypotonic cell swelling induces ATP release which can activate ion channels eg. Volume activated Cl- channel (Wang et al.,

1996;Roman et al., 1999;Perez-Samartin et al., 2000;Darby et al., 2003) and potassium channels (Hafting et al., 2006). Therefore, the goal of our study was to investigate whether ATP release is involved in volume mediated response of KCNQ1 channels. For this purpose we expressed KCNQ1 ± AQP1 in Xenopus oocytes.

Materials and methods

Expression in Xenopus laevis oocytes cDNAs coding for Aquaporin1 (AQP1) and KCNQ1 were subcloned into expression vectors and expressed in Xenopus laevis oocytes. Xenopus laevis oocytes were isolated

PhD thesis Sofia Hammami and defolliculated as previously described (Grunnet et al, 2002) or purchased from

Ecocyte Bioscience (Germany). Synthetic RNA was prepared by in vitro transcription (T3 and T7 mMessage machine kit from Ambion) from DNA templates (coding for AQP1 and KCNQ1) linearized with Pst1 and XbaI (New England Biolabs, Ipswich, MA, USA) for AQP1 and KCNQ1 respectively. RNA was extracted by MegaClear kit (Ambion). 50 nl of mRNA was injected in oocytes, which were then kept in Kulori medium (in mM:

90 NaCl, 1 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES-Tris, pH 7.4) at 19°C.

ATP release measurements

ATP released from individual defolliculated oocytes (non-injected, KCNQ1 injected and

KCNQ1+AQP1 injected) was monitored 3 days after RNA injection using luciferin- luciferase bioluminescence assay (FLAA, Sigma-Aldrich) similar to by Maroto and

Hamill 2001 with slight modifications. Individual oocytes were placed in a 96 well plate containing 45 μl Kulori and 5 μl of the Sigma ATP assay reagent in each well (1 mg/ml luciferin-luciferase mix). Oocyte handling and transfer to the well will elicit an increase in ATP release, therefore oocytes were left to rest in the well for 1 hour before ATP release was monitored using Victor luminometer (Perkin Elmer). After this first measurement, 22.5 μl of 50% Kulori + 2,5 μl luciferin-luciferase mix (LL-mix) or 22.5 μl

Kulori + 100 mM mannitol + 2.5 μl LL-mix was added carefully by pipetting in order to swell or shrink the oocyte respectively (∆50 mosmol). Immediately after, release of ATP was measured. Osmolarities of the solutions used here correspond to the same ones done with the two electrode voltage clamp measurements (see next section). For control, 22.5

μl Kulori +2.5 μl LL-mix was added in order to subtract any pipetting effect. For both standards and oocyte experiments the ATP induced light was measured over a 60 sec sampling period. The background signal (a blank) was measured and subtracted from samples. Standard curves were performed by plotting the log of luminescence intensity

PhD thesis Sofia Hammami

(relative luminescence units) against the log of ATP concentrations (moles/liters) with the different osmolarities using the Sigma ATP calibration standards. The ATP induced light was converted to moles/L of ATP concentration according to the standard curves prepared each day.

Electrophysiological measurements

All measurements were performed 3 days after RNA injection using a conventional two- electrode voltage-clamp set-up. The measurements were done in medium that was isotonic (65 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 50 mM mannitol, 5 mM

−1 Hepes, pH 7.4 (188 mosmol kg )), hypotonic (65 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1

−1 mM CaCl2, 5 mM Hepes, pH 7.4 (137 mosmol kg )) or hypertonic (65 mM NaCl, 1 mM

KCl, 1 mM MgCl2, 1 mM CaCl2, 100 mM mannitol, 5 mM Hepes, pH 7.4 (239 mosmol kg−1)). The chemicals Apyrase and ATP from Sigma Aldrich were added at the different conditions.

Data acquisition and analysis was performed with Clampex 10 and clampfit 10

(Molecular devices) software programs, respectively. GraphPad Prism 4 was used for preparing graphical displays.

Statistics

If nothing else mentioned, numerical data are presented as means ±SEM with n observations in different oocytes. Comparisons are made by using Student‟s two tailed, unpaired or paired t-test depending on the data.

Results

1) Basal ATP release at rest and during changes in cell volume

PhD thesis Sofia Hammami

In order to determine whether there is ATP release during cell volume changes in

Xenopus oocytes at the different osmotic conditions (50 mosmol difference), luciferin luciferase bioluminescence assay was applied. Figure 1A shows standard curves of bioluminescence assay in Kulori and after adding Kulori, hypotonic and hypertonic solution. The assay sensitivity was not reduced in the different solutions.

By monitoring ATP release just after handling and transfer of oocytes to the wells we noticed a very high ATP release due to the mechanical perturbations, which decreased with time (not shown). We therefore let the oocyte rest for one hour after they were transferred to the wells.

Basal ATP release was measured 1 hour after oocyte transfer to a well containing Kulori and luciferin luciferease mix. Figure 1B shows difference in basal ATP release between non-injected (CTRL) oocytes; AQP1, KCNQ1 and AQP1+KCNQ1 injected oocytes at rest. Surprisingly, AQP1 injected oocytes had nearly the same basal ATP release as the control oocytes, whereas basal ATP release for KCNQ1 injected oocytes was 3-4 fold higher.

We predicted that oocytes expressing AQP1, which are rapidly undergoing changes in cell volume, will release ATP to the surrounding medium at a higher rate within the first minutes compared to controls ones. In order to test for this hypothesis, ATP release was monitored before and after pipetting hypo - or hyperosmotic solution to each well. Since pipetting may induce mechanically ATP release, as control pipetting effect was also measured after adding Kulori. Pipetting had no significant effect on ATP release in non- injected control oocytes, whereas it had an effect on the injected KCNQ1 ± AQP1 as shown in Figure 2A.

PhD thesis Sofia Hammami

Figure 2B shows that in non-injected control oocytes swelling did not induce further release of ATP compared to oocytes subjected to Kulori in Figure 2A. In contrast, the injected oocytes KCNQ1+AQP1 had a 36 % further release of ATP to the extracellular medium when subjected to cell volume increase. Surprisingly, we did not expect ATP release for the oocytes expressing only KCNQ1 since these do not volume regulate. In another experiment, hyperosmotic exposure (Figure 3C.) and hence shrinkage also induced ATP release for KCNQ1+AQP1 and KCNQ1. A significant ATP release was also seen for the non-injected oocytes in response to shrinkage.

From these data we can conclude that during changes in cell volume there is indeed an

ATP release, but this is independent of AQP1. The question whether this increase in ATP release is coupled to the volume sensitivity of KCNQ1 channels is addressed in the following section.

2) Effect of apyrase on KCNQ1 currents

To investigate if the response of KCNQ1 to changes in cell volume involves the purinergic signaling pathway, we added the ATP/ADP hydrolyzing enzyme apyrase at the different osmotic conditions. Figure 3 shows that application of 7U/ml apyrase decreased the overall KCNQ1 current level. The current also decreased in increasing concentrations of Apyrase (not shown).We tested whether simply the presence of protein may have an effect on the current. Therefore we performed similar experiment with Bovine serum albumin at similar concentrations (not shown). However this did not have any effect. We propose that apyrase has an unspecific effect with the KCNQ1 channel. The decrease in current in all conditions with apyrase may also be due to the hydrolyzing effect on the basal ATP release close to the membrane of KCNQ1 injected oocytes.

PhD thesis Sofia Hammami

After normalization of the current to isotonic condition in figure 3 B. the relative percentage changes with hypo or hyper solution were the same with and without application of apyrase. This indicates that the volume sensitivity of KCNQ1 was relatively unchanged.

3) Effect of added extracellular ATP on KCNQ1 currents

In order to further test whether KCNQ1 current response to volume changes is mediated by extracellular release of ATP, 100 μM ATP was added to the extracellular solution (Iso, hypo and hyperosmotic solutions) and current was recorded at the end of + 40 mV depolarizing potential. Figure 4 shows that the KCNQ1 current did not decrease or increase in the presence of 100 μM extracellular ATP relative to control solutions. Higher concentrations of ATP were also tried with no effect (not shown). These data indicate that

ATP is not involved in the increase in current during hypoosmotic conditions or decrease during hyperoosmotic conditions.

Discussion

In the present study, we show that mechanical stimulation and changes in cell volume induces ATP release for oocytes expressing KCNQ1 with and without AQP1.

Extracellular given ATP and apyrase however, has no significant effect on KCNQ1 current response to changes in cell volume.

In this study, we measured ATP release at resting conditions and during changes in cell volume.

Basal ATP release: For non-injected control oocytes, basal release corresponded to an equivalent of 19 ± 2.65 femtomol of ATP in 50 μl Kulori. This value is in agreement with

PhD thesis Sofia Hammami previous results where basal ATP release for non-injected oocytes was around 20 femtomol (Maroto & Hamill, 2001). Note that this is very small concentrations as we are working in large volumes (50 μl); however this may be larger in close proximity to the oocyte surface. Another possible explanation for the low ATP concentrations, is the endogenous expression of the membrane protein CD39 which hydrolysis ATP and ADP

(Aleu et al., 2003).

The KCNQ1±AQP1 injected oocytes had a 4 fold higher basal ATP release than the

AQP1 injected oocytes. It seems like the type of membrane protein expressed may have different effects on the basal ATP release. Heterologous expression of KCNQ1 channel may have several consequences: 1) the oocyte is stressed due to a high synthetic rate of proteins and high rate of exocytosis, which may lead to an increase in the release of ATP present in the exocytotic vesicles; 2) KCNQ1 may be functionally interacting and upregulating the insertion of ATP transporters to the membrane; or 3) KCNQ1 may be permeable to ATP. In fact, several mechanisms have been suggested regarding how ATP is released. It can be released by a non-ionic process through mechanical induced constitutive release of vesicles (Maroto & Hamill, 2001) and through hormonal/neuronal regulated exocytosis (Lazarowski et al., 2003). Moreover release can take place by a conductive ionic process through membrane channels, such as mechanically gated ion channels, hemichannels such as connexins and pannexins, maxi anion channels, volume regulated anion channels, CFTR and P2X7 receptors (reviewed by (Corriden & Insel,

2010)). It is unlikely to consider the cation selective KCNQ1 channel as an ATP conductive channel, since ATP is negatively charged in physiological solutions (ATP4-).

ATP release during cell volume changes: In this study, ATP release was measured under iso-, hypo and hyperosmotic conditions for non-injected control oocytes and KCNQ1 ±

AQP1. Hypoosmotic induced ATP release is widely reported in many native and cultured

PhD thesis Sofia Hammami cells (Wang et al., 1996;Shinozuka et al., 2001;Boudreault & Grygorczyk, 2004;van der et al., 1999). However, previous studies where defolliculated, non-injected Xenopus oocytes were tested in hypotonic stress conditions (Δ140 mosmol/l) did not detect any release of ATP (Aleu et al., 2003;Maroto & Hamill, 2001). In contrast oocytes challenged with hypertonic solution (Δ300 mosmol/l) released ATP (Aleu et al., 2003). This behavior is also what we see for our control non-injected oocytes (cf. Figure 2 B and C).

In contrast to control oocytes we detect ATP release in the injected oocytes

(KCNQ1±AQP1) both during hypotonic and hypertonic conditions. We may assume that when expressing an exogenous channel, the exocytotic pathway is boosted and in parallel swelling the oocyte, may intensify the response thereby contributing to a higher release of vesicles containing both the channel proteins ready to be inserted to the membrane and the ATP molecules ready to be released to the outside. This explains the higher ATP and the increase in KCNQ1 current detected during volume increase.

During volume changes, we would expect that the AQP1+KCNQ1 expressing oocytes to be most volume sensitive and therefore we would expect to see a higher ATP release.

However we see a similar increase in ATP release in the KCNQ1 injected oocytes. This indicates that, larger volume changes in the presence of AQP1 do not induce a higher release of ATP and that the rate of release is independent on AQP1.

The released ATP in Xenopus oocytes during cell shrinkage was previously shown to be coupled to the activation of an inward current (Zhang & Hamill, 2000;Aleu et al., 2003).

There could be a possibility that different ATP release mechanisms operate under hypoosomotic and hyperosmotic in the injected oocytes through an exocytotic pathway during cell swelling and through a conductive channel during cell shrinkage.

Exogenous ATP and apyrase

PhD thesis Sofia Hammami

In another series of experiments ATP and Apyrase were added to the extracellular medium in order to investigate whether the response of KCNQ1 to changes in cell volume involves the purinergic signaling pathway. Neither ATP nor apyrase had any effect on the volume induced changes in KCNQ1 currents. A possible explanation for the absence of any effect could be that either ATP and apyrase does not reach the micro-environments of the channels (Joseph et al., 2003) or because of the extracellular ATP degradation by

CD39.

ATP exerts its function by activating purinergic receptors at the cell membrane that subsequently modulates ion channels through intracellular signaling pathways involving an increase in intracellular calcium concentration. However, the existence of endogenous purinergic receptors in Xenopus oocytes is puzzling. Many studies based on electrophysiological experiments have shown that follicular cell-enclosed oocytes are endowed with purinergic receptors that respond to ATP evoking a current response

(Lotan et al., 1986;Arellano et al., 1996;Arellano et al., 1998;Arellano et al., 2009;King et al., 1996a;King et al., 1996b) whereas defolliculated oocytes are devoid of purinergic receptors according to the fact that ATP failed to activate any current response. However these assumptions are based on studies done a few hours after defolliculation of oocytes and therefore the receptors could be desensitized or internalized. One single study however has shown the expression of endogenous Xenopus oocyte adenosine receptor at the membrane (Kobayashi et al., 2002). To our knowledge, there are no published data on purinergic receptor mRNA expression nor receptor protein expression on defolliculated oocytes. Moreover by using intracellular calcium changes as a functional indication of the presence of purinergic receptors seems to be problematic since Ca2+ signals are difficult to detect during volume changes in Xenopus oocytes (Vandorpe et al., 1998;Grunnet et al., 2002;Locovei et al., 2006)

PhD thesis Sofia Hammami

Whether purinergic receptors are present or not is unclear, but ATP seems not to induce

KCNQ1 current or volume sensitivity of the channel. It is unlikely that purinergic signaling is involved in KCNQ1 volume response in the Xenopus oocytes. A disadvantage in using Xenopus oocyte as an expression system is its amphibian origin and

“egg status” which may lack some components that normally present in warm blooded animal cells. Interestingly, previous studies have shown a coupling of the KCNQ1 to purinergic signaling in native tissues. The strial marginal cells and vestibular dark cell of the inner ear of rodents expresses KCNQ1, its auxiliary β-subunit KCNE1 and purinergic receptors (P2Y4) at the apical membrane which have been shown to have an important function in endolymph homeostasis and protection from overstimulation (Housley et al.,

2009;Lee & Marcus, 2008). KCNQ1/KCNE1 channel activity and thus K+ secretion was shown to be modulated by 3 purinergic pathways in rodents when P2Y4 receptors are stimulated: 1) G protein-PLC activation leads to consumption of membrane PIP2 with the consequent reduction of K+ channel activity 2) the DAG-PKC path decreases K+ channel activity directly via phosphorylation of the channel and 3) the IP3/Ca2+ path decreases the channel activity directly via the effect of Ca2+ on channel activity (Lee & Marcus,

2008).

Additionally, Honoré et al (1992) have reported that stimulation of purinergic receptors regulates the KCNQ1/KCNE1 channel activity in mouse heart. This study was concluded by injection of cardiac polyA+ RNA from neonatal mouse heart into Xenopus oocytes.

The RNA directed the expression of IsK channel as well the expression of purinergic P2 receptors. By stimulating these receptors it produced intracellular Ca2+ increase, DAG and thereby activating protein kinase C which increased IsK activity (Honore et al.,

1992).

PhD thesis Sofia Hammami

These studies indicate that there is indeed a coupling of KCNQ1/KCNE1 channels with purinergic signaling. In our study where only KCNQ1 is expressed, KCNE1 is maybe necessary in order to elucidate whether purinergic signaling has an effect on KCNQ1 volume response.

In conclusion, we showed that oocytes release ATP in response to mechanical, hypo and hypertonic loads which may be due to different mechanisms. Also expression of KCNQ1 involves ATP release but AQP1 does not. Furthermore extracellular added ATP and apyrase had no effect on volume induced currents. These results indicated that purinergic signaling is not involved in volume mediated KCNQ1 activity in our expression system.

However we cannot exclude that other components e.g. KCNE1 could do the job.

Acknowledgements: The authors thank Ms. Z. Rasmussen for expert technical assistance. This work has been supported by FNU and Carlsberg Foundation (oocyte setup).

PhD thesis Sofia Hammami

Figure legends

Figure 1 A) Standard curves of the bioluminescence assay in Kulori, hypotonic and hypertonic solution. B) Basal ATP release difference from non RNA injected oocytes

(CTRL n= 116) and injected oocytes (AQP1 n=31, KCNQ1 n=97 and KCNQ1+AQP1 n=115) at resting condition. The data represent mean±SEM. Oocytes from 7 different frogs. *** P < 0.001; NS: non significant.

Figure 2 ATP release before and after exposing oocytes to (A) Kulori, (B) hyposmotic and (C) hyperosmotic solutions. In (A), pipetting had a slight increasing effect on ATP release in Q1 and Q1+AQP1 injected oocytes. In (B), hypososmolar solution induced a higher ATP release in the injected oocytes independent of whether AQP1 was present or not. In (C), hyperosmolar solutions had similar effect on ATP release in all cases. The data represent the mean ± S.E.M. (oocytes from 7 different frogs). (Kulori n= 35-40,

Hypo n= 36-44 and Hyper n= 26-34) NS: non significant, * P < 0.05, ** P < 0.01 and ***

P < 0.001.

Figure 3 Effect of application of extracellular apyrase on KCNQ1 current response to cell volume changes. A. Original KCNQ1+AQP1 current traces at 40 mV depolarizing potential during osmotic challenges with and without the presence of apyrase. B.

Columns show the changes of current at the end of a depolarization period (+40 mV) during osmotic challenges before (CTRL) and after addition of apyrase. The data represent the mean± S.E.M. n=5 oocytes.

Figure 4 Effect of extracellular ATP on KCNQ1 current response to cell volume changes

(coexpressed with AQP1). A. Original KCNQ1+AQP1 current traces at 40 mV depolarizing potential during osmotic challenges with and without the presence of 100

μM ATP. B. Columns show the changes of current at the end of a depolarization period

PhD thesis Sofia Hammami

(+40 mV) during osmotic challenges before (CTRL) and after addition of 100 μM ATP.

The data represent mean± S.E.M. n=6 oocytes.

PhD thesis Sofia Hammami

Figure 1

Figure 2

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Figure 3

Figure 4

PhD thesis Sofia Hammami

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Grunnet M, Jespersen T, MacAulay N, Jorgensen NK, Schmitt N, Pongs O, Olesen SP, & Klaerke DA (2003). KCNQ1 channels sense small changes in cell volume. J Physiol 549, 419-427.

Grunnet M, MacAulay N, Jorgensen NK, Jensen S, Olesen SP, & Klaerke DA (2002). Regulation of cloned, Ca2+-activated K+ channels by cell volume changes. Pflugers Arch 444, 167-177.

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Hougaard C, Klaerke DA, Hoffmann EK, Olesen SP, & Jorgensen NK (2004). Modulation of KCNQ4 channel activity by changes in cell volume. Biochim Biophys Acta 1660, 1-6.

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Jensen HS, Callo K, Jespersen T, Jensen BS, & Olesen SP (2005). The KCNQ5 potassium channel from mouse: a broadly expressed M-current like potassium channel modulated by zinc, pH, and volume changes. Brain Res Mol Brain Res 139, 52-62.

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Manuscript III: KCNE1-induced increase in KCNQ1 currents is not mediated through enhanced plasma membrane expression

1,2 2 1 Sofia Hammami , Dan A. Klaerke & Niels J. Willumsen

1Department of Biology, Faculty of Science, University of Copenhagen, Denmark;

2Department of Physiology and Biochemistry, IBHV, Faculty of Life Sciences, University of

Copenhagen, Denmark

Corresponding author:

Niels J. Willumsen [email protected]

Telephone: +45 35321635 and fax number: +45 3532 1567 University of Copenhagen Department of Biology The August Krogh Building

Universitetsparken 13

2100-Copenhagen Ø

Denmark

PhD thesis Sofia Hammami

Abstract

Association of the voltage activated potassium channel KCNQ1 with the accessory protein subunit KCNE1 gives rise to the cardiac IKs delayed rectifier potassium current.

Aside from altering the kinetic characteristics of the KCNQ1 channel current, KCNE1 also augments the KCNQ1 current. However it is debated whether this increase in macroscopic current is due to an increase in ion channel conductance (γ), the open state probability (Po) or an increase in the number of channels in the plasma membrane (N).

The latter can be quantified by measuring the level of KCNQ1 surface expression by using an enzyme-linked immunoassay. To do this, we employed a HA-tagged version of the KCNQ1 channel and expressed it with and without KCNE1 in Xenopus oocytes. The

HA-tag, which is located at the extracellular side of the protein, allowed us to “count” the number of KCNQ1 channels expressed in the cell membrane. In parallel, currents were measured with two electrode voltage clamp. The results show that the KCNQ1 surface expression is lower when KCNE1 is coexpressed compared to KCNQ1 alone despite the higher current for the heteromeric KCNQ1/KCNE1. This indicates that the overall increase of the KCNQ1 current when KCNE1 is coexpressed is not due to an increase in ion channel surface density but rather to an increase in single-channel conductance or in open state probability.

Keywords

Surface expression, KCNQ1, KCNE1, IKs current, Xenopus oocytes

PhD thesis Sofia Hammami

Introduction

KCNQ1 potassium channels are abundant in the cell membranes of cardiac muscle where they are involved in repolarisation of the cardiomyocyte membrane after an action potential.

In addition, KCNQ1 channels have been found in a number of epithelial tissues and have been demonstrated to be essential for transepithelial transport and for participating in potassium absorption and secretion in the inner ear, the kidney, lung, stomach, and intestine

[1]. KCNQ1 channel activity is regulated by many factors such as phosphorylation [2], cell volume [3] and regulation by β-subunits [1]. KCNQ1 associates with a number of accessory proteins such as KCNE1[4], KCNE2 [5], KCNE3 [6], KCNE4 [7], KCNE5[8] and corticosteroid hormone induced factor CHIF [9], which all have overlapping tissue distribution with KCNQ1. These auxiliary proteins which are small with only one- transmembrane-segment alter the function of the KCNQ1 by modifying its electrophysiological properties. Heteromeric association of KCNQ1 with KCNE1 (also denoted MinK or IsK) induces a current with electrophysiological channel properties markedly different from that of the KCNQ1 channel itself, but similar to the cardiac delayed

rectifier potassium current (IKs) which contribute to the repolarisation following a cardiac action potential.

When expressed alone, the KCNQ1 gives rise to a voltage dependent outward current upon depolarization which reaches a steady state within 1s. Subsequent repolarisation elicits a tail current with an initial increase in the amplitude (a „hook‟) before deactivation. This tail current hook is resulting from recovery of the channels from inactivation at a rate faster than deactivation [4]. However co-assembly of the regulatory β-subunit KCNE1 with KCNQ1 results in a significant change of the electrophysiological properties of the channel. The voltage activation threshold is shifted to a more positive potential, the deactivation is much slower and the inactivation is almost completely absent [4]. Moreover the current is much

PhD thesis Sofia Hammami larger than the one induced by KCNQ1 alone. This increase in macroscopic current can be due to an increase in single channel conductance, an increase in the number of functional

channel complexes in the membrane or increase in the channel open state probability Po.

Controversies have occurred when determining which of these parameters are altered.

Romey et al. [10] concluded that the effect of minK coexpression was twofold: a decreased single channel KCNQ1 conductance (from 7.6 to 0.6 pS) which was overruled by an increased channel density resulting in the overall enlargement of the macroscopic current.

However studies made by Yang and Sigworth (1998) [11] and Pusch (1998) [12] led to the opposite conclusion: the observed current increase upon coexpression with minK was contributed to an increase in single channel conductance. In noise analysis studies, these investigators demonstrated a larger single-channel conductance of heteromeric

(KCNQ1/KCNE1 complex; γ = 4.5 pS) compared to homomeric channels (KCNQ1 alone;

γ = 0.7 pS).

This present study focus on a comparison of the number of KCNQ1 channels in the plasma membrane with and without the presence of the auxiliary subunit KCNE1 through enzyme linked immunoassay on HA tagged KCNQ1 proteins. This allows us to determine if the number of ion channels (N) in the plasma membrane is altered when KCNQ1 is coexpressed with KCNE1.

PhD thesis Sofia Hammami

Materials and methods

Expression in Xenopus laevis oocytes

Xenopus laevis oocytes were isolated and prepared as previously described[13]. cDNAs coding for human KCNQ1, KCNQ1-HA [14] and KCNE1 were subcloned into pXOOM vectors and expressed in Xenopus laevis oocytes [15]. KCNQ1-HA has two hemagglutinin epitopes inserted in the extracellular site between the S3 and S4 segment of the KCNQ1 protein (insert, figure 1B). Synthetic RNA was prepared by in vitro transcription (T7 mMessage machine kit from Ambion) from DNA templates linearized with XbaI and NheI (New England Biolabs, Ipswich, MA, USA) for KCNQ1-HA and

KCNE1 respectively. RNA was extracted by MegaClear kit (Ambion). 50 nl of mRNA was injected in oocytes which were then kept in Kulori medium (in mM: 90 NaCl, 1 KCl,

1 MgCl2, 1 CaCl2, 5 HEPES-Tris, pH 7.4) at 19°C. For coexpression of KCNQ1(-HA) and KCNE1 subunit, the mRNAs were mixed in equal molar ratios. We also injected the same amount of water than KCNE1 to have the same diluted volume of KCNQ1.

Electrophysiological measurements

All measurements were performed 4 to 6 days after RNA injection using a conventional two electrode voltage clamp setup. The measurements were done in Kulori medium.

Surface expression

Measurements of surface expressed proteins through enzyme immunoassay were conducted right after current measurements on the same oocytes. The hemagglutinin epitopes which are inserted in the extracellular site between the S3 and S4 segment of the

KCNQ1 protein allows us to measure the amount of surface expressed protein through enzyme immunoassay. The procedure was conducted as previously described [16] with slight modifications: Oocytes were placed in Kulori with 1% bovine serum albumin

PhD thesis Sofia Hammami

(BSA) for 1 hour to block nonspecific binding. They were then incubated for 60 min in

0.5 µg/ml primary antibody (3F10 anti HA from Roche molecular Biochemicals) solution prepared in Kulori-BSA(1:200), then washed with Kulori-BSA six times by moving the oocytes from one petri dish to another changing to a new pasteur pipette at each time.

Oocytes were then incubated with the secondary antibody solution (goat anti-rat F(ab´)2 fragments (Jackson Immunosearch Laboratories) at a 1:400 dilution) also prepared in kulori-BSA for 1 hour, then washed 4 times with kulori-BSA and 4 times without BSA.

Five oocytes were then placed in 200 µl OPD luminescence solution (OPD tablets from sigma) in a well of a 96 well plate. They were incubated for one hour and subsequently

100 µl of the solution was moved to another plate where absorbance measurement was conducted on the absorbance reader Victor (Perkin Elmer) at 450 nm. All steps were done on ice and 4ºC solutions in order to minimize endocytosis. Background noise from injected oocytes expressing non-tagged protein has been subtracted. The light emitted by the OPD reaction was expressed as relative light units (RLU) reflecting the amount of surface expressed proteins.

PhD thesis Sofia Hammami

Results

Comparison between KCNQ1 and KCNQ1-HA

KCNQ1-HA has two HA epitopes located in the second extracellular loop, i.e. between the third and fourth transmembrane segment of the channel protein (see insert in figure

1B). In initial experiments we examined if the HA-tags had any influence on the current kinetics of the channel by comparing it with the original non-tagged channel KCNQ1.

The HA-tagged channel exhibited almost identical electrophysiological properties. Figure

1 shows that although overall current was lower for KCNQ1-HA than for KCNQ1, the current showed the same kinetic behavior: an outward rectifying current which reaches a plateau phase within ~1 sec and the characteristics tail current with the hook. For

KCNQ1 and KCNQ1-HA the half maximal V½ was -10.6 ± 0.9 mV and -8.1 ± 0.9 mV, respectively, and the slope factor was 17.8 ± 0.9 mV and 27.9 ± 1.0 mV, respectively, indicating similar activation behaviors (Figure 1C, upper panel). It has earlier been shown that the HA-tags do not interfere with channel trafficking [14].

When KCNQ1-HA was coexpressed with KCNE1 it induced a slowly activating current as seen for the non-tagged protein however with minor differences compared to KCNQ1

+ KCNE1 (Figure 1C, lower panel). Recent studies which determined the KCNE1 structure by solution NMR and subsequently experimentally restrained docking of the

KCNE1 transmembrane domain (TMD) into the KCNQ1 channel, have revealed that the curved nature of KCNE1 enables the TMD to form extensive contacts with a cleft formed between the upper part of S3 on the voltage sensor of one KCNQ1 subunit and the S5 and

S6 segments of another KCNQ1 subunit [17]. As the HA tags in our construct are placed between the S3 and S4 domain (where KCNE1 actually make contact), it is likely that they affect the interaction between KCNE1 and KCNQ1. This can possibly explain the difference in the I/V relationship, we see when KCNQ1-HA is coexpressed with KCNE1

PhD thesis Sofia Hammami as we see no differences between KCNQ1-HA and wild type KCNQ1 when expressed alone (figure 1C).

The steady state current was also lower for KCNQ1-HA + KCNE1 than for KCNQ1 +

KCNE1, but most importantly, we observed an almost identical effect of KCNE1 on the fractional increase in current when coexpressed with either KCNQ1-HA or KCNQ1 (Cf.

Fig 1A + B). Thus, it seems reasonable to measure the level of KCNQ1-HA surface expression by enzyme-linked immunoassay in order to determine whether the number of surface expressed KCNQ1 channels is increased or decreased when coexpressed with

KCNE1 β-subunit.

Surface expression over time, a test for the sensitivity of the HA-tag system

In another series of experiments the sensitivity of the enzyme-linked immunoassay was examined. For oocytes injected with mRNA coding for the HA-tagged KCNQ1 channels, steady state current as well as the HA-signal were measured on the 4th and 6th day after injection. As expected, current increased from day 4 to day 6 after RNA injection indicating that an increased number of ion channels were expressed in the plasma membrane (Fig. 2, black columns). In consistence with this, parallel experiments showed an increased signal from exposed HA-tags (Fig. 2, scattered columns). From day 4 to day

6, the steady state current increased 44 % (from 0.93 ± 0.15 to 1.34 ± 0.16 µA) and the

HA-tag signal simultaneously increased 54% (from 0.0076 ± 0.0008 to 0.0118 ± 0.0024

RLU ). These results indicate that for the HA-tagged KCNQ1 channels, the number of channels expressed in the plasma membrane is reflected with reasonable precision in the signal from the the enzyme-linked immunoassay. Therefore, this assay seems to be a relieable and sensitive method to “count” the number of channels in the plasma membrane.

PhD thesis Sofia Hammami

Surface expression versus current. KCNE1coexpression does not increase the number of KCNQ1 ion channels translocated to the membrane surface

In order to investigate whether the current induced by KCNE1 is affecting the number of ion channels that are translocated to the membrane, oocytes were injected with mRNA coding for KCNQ1-HA + water or a mixture of mRNA coding for KCNQ1-HA and

KCNE1. Four days after the injection, the current and surface expression for KCNQ1-

HA+ KCNE1 and KCNQ1-HA + H2O was measured. The measurements show that, the current amplitudes are close to equal for KCNQ1-HA + H2O (0.54 ± 0.07 µA) and

KCNQ1-HA+ KCNE1 (0.59 ± 0.11 µA), whereas the surface expression for the homomeric channel is significantly higher (0.0048 RLU) than for the heteromeric channel

(0.0014 RLU). The experiments shown in Figure 2 indicate that the surface expression measurements to some extent can be considered quantitative. Thus, the data in Figure 3 suggests that the membrane expression for KCNQ1-HA + KCNE1 in the present studies is approximately 3.5-fold lower than for and KCNQ1-HA although the currents conducted by the channels are equal. This indicates that KCNE1 is not inducing an increased number of KCNQ1 channels in the membrane but is rather acting on the ion channel conductance or open probability.

PhD thesis Sofia Hammami

Discussion

The mechanism for the increased current seen in electrophysiological measurements when the KCNQ1 channel is coexpressed with KCNE1 has previously been investigated by fluctuation analysis in a number of studies [10-12]. However, the results have been conflicting, and it is at present not clear whether the presence of KCNE1 affects the membrane expression, the open probability or the single channel conductance.

In the present study, we address this unresolved question by employing an alternative approach, which relies on the detection of surface-bound antibodies using enzyme-linked immunoassay to elucidate whether the number of functional ion channels is altered after coexpression of KCNQ1 with KCNE1. By using an enzyme-linked immunoassay and

HA-tagged version of the KCNQ1 channel we were able to estimate the number of ion channels present in the plasma membrane in the presence and absence of KCNE1.

Our results show that co-expression of the KCNE1 subunit in fact decreases the membrane expression of the KCNQ1 alpha-subunit, but increases the current through each expressed channel by a factor of approximately 3.5 (Cf. results section and Fig. 3).

Our results do not allow us to determine whether this increase in current in mediated through increased open probability, increased single channel conductance or both.

However, currents for KCNQ1 as well as KCNQ1/KCNE1 are close to saturation after depolarisation to 60 mV for 4 s (Cf. Fig. 1B), and it could be assumed that the open state probabilities for homomeric and heteromeric ion channels are equal and close to unity at

60 mV. If that is the case, then our results indicate that the single channel conductance for the heteromeric KCNQ1/KCNE1 channels is approximately 3.5-fold higher than for the homomeric KCNQ1 channels. This estimate would agree with noise analysis studies made by Yang and Sigworth [11], Pusch [12] and Sesti and Goldstein [18] who all estimated a 3-4 fold increase in single channel conductance upon co-assembly of KCNE1

PhD thesis Sofia Hammami and KCNQ1. In contrast, our results are not consistent with Romey et al. (1997); these authors concluded that the interaction reduces KCNQ1 conductance from 7.6 to 0.6 pS and at the same time dramatically increases the KCNQ1 channel density in the plasma membrane.

Since it is well described that the presence of KCNE1 increases the whole cell KCNQ1- current, it may seem surprising that co-expression of KCNE1 in fact decreases the number of ion channels in the plasma membrane. However, as argued above, the explanation may be a large increase in single channel conductance induced by the presence of KCNE1. In the present study, the decrease in the number of channels in the plasma membrane is quite significant, and it cannot be excluded that this is in part due to

“saturation” of the expression system. However, since our results are based on the ratio between measured current and the presence of HA-tagged KCNQ1 in the plasma membrane, such a phenomenon would not affect our conclusions. In addition, the effect of co-expression of KCNE1 or KCNE2 on KCNQ1 channels was recently examined in another expression system (COS-7 cells). Also this study showed that cell surface expression of KCNQ1 was significantly lower as compared to expression of KCNQ1 alone [19]. It cannot be excluded that the presence of KCNE1 also in intact tissue may decrease the membrane expression of KCNQ1 although the opposite has also been shown to be the case [20]. This assumption is supported by the finding that, although the interaction between KCNQ1 and KCNE1 is dynamic, the assembly of KCNQ1 and

KCNE1 takes place in the secretion pathway before the channel complex reaches the plasma membrane [21;22]

The KCNE1 beta-subunit (also formerly called minK) may coassemble with endogenous

KCNQ1 channels from Xenopus oocytes creating a current similar to the slow delayed rectifier IKs identified in human heart. Thus the currents that are measured in the present

PhD thesis Sofia Hammami study (see. e.g. Fig 1B) could in principle be a combination of heterologeously expressed

KCNQ1-HA/KCNE1 and endogenous KCNQ1/KCNE1. Since the membrane expression is measured by means of the HA-tagged KCNQ1, this would give an apparent decrease in membrane expression in our experiments. However, data from our laboratories have shown that in such experiments the current arising from endogenous KCNQ1 channels in the Xenopus oocytes is negligible. Also in this context, as mentioned above, it should be kept in mind that when a different expression system (COS-7 cells, which do not express endogenous KCNQ1 channels) was used [19], a reduction in the number of ion channels upon co-expression with KCNE1 was measured.

Several studies have focused on the interaction of K+ channel α-subunits and accessory proteins, how they assemble and how they affect trafficking or gating. The influence of the different β-subunits on the α-subunits surface expression does not follow a uniform behavior. For example the Kvβ4 subunit increases the surface expression of Kv2.2 channels without affecting the kinetic properties of the channel [23]. Other reports have shown that KCNQ1 channel currents are inhibited by KCNE4 in mammalian cells and

Xenopus oocytes without modulating the membrane expression of KCNQ1 channels.

KChIPs 1-3 were shown to increase Kv4 current by promoting channel trafficking to the membrane. These findings indicate that β-subunits are capable of regulating ion channel

α-subunits by either affecting the number of ion channels, the ion channel conductance or the open channel probability. The dramatic effects we see of the association of KCNE1 with KCNQ1 on gating and permeation of the potassium current has led several investigators to conclude that KCNE1 directly modifies the conductive properties of

KCNQ1. Indeed, there is striking evidence that KCNE1 lies in close proximity to the

KCNQ1 pore and thereby influencing KCNQ1 conducting properties and pharmacology.

Studies have shown that KCNE1 directly interacts with the S5-P-S6 pore domain and sits

PhD thesis Sofia Hammami in a cleft between this pore domain and adjacent voltage sensor [17;24-26]. Moreover, some residues from the KCNE1 transmembrane domain modulates channel activation

[26;27] whereas the juxtamembrane C-terminal domain of KCNE1 is important in preventing channel inactivation [28;29]. Truncation of the KCNE1 C-terminus as well a point mutation (D76N) reduced IKs current but did not affect the number of surface expressed KCNQ1 channel proteins compared to wild type KCNE1 indicating either a change in open probability, Po, or unitary conductance [28]. These findings further support that an interaction of KCNE1 with KCNQ1 affects the gating and conductance rather than the number of ion channels translocated to the membrane.

The increasing understanding of the role of the auxiliary subunit KCNE1 and its impact on the pore forming subunit KCNQ1 has gathered substantial knowledge of the malfunctioning of IKs channel under pathological conditions such as Long QT syndrome.

This will hopefully lead to the development of safer and more specific therapeutic drugs.

Acknowledgement: The authors thank Ms. Z. Rasmussen for expert technical assistance.

This work has been supported by The Danish Council for Independent Research -

Medical Sciences (FSS), The Danish Council for Independent Research – Natural

Sciences (FNU), The Novo Nordic Foundation, The Carlsberg Foundation, Fonden til

Laegevidenskabens Fremme and The Fouger-Hartmann Foundation.

PhD thesis Sofia Hammami

Figure legends

Fig. 8. Expression of KCNQ1 or KCNQ1-HA with and without KCNE1 in Xenopus oocytes.

Current/time (I/t) traces showing KCNQ1 (A) and the HA-tagged version of the KCNQ1 (B) with

(lower panels) and without (upper panels) KCNE1 recorded with TEVC in Xenopus oocytes.

Currents were activated by a step protocol from a holding potential of -80 mV. Steps of 20 mV (4 sec duration) were applied from -80 mV to 60 mV. Insert shows the location of the double HA- epitope at the extracellular segment between S3 and S4 of KCNQ1. (C) Upper panel: Normalized isochronal activation curves fitted with Boltzmann function for oocytes injected with KCNQ1 (V½

= -10.6 ± 0.9 mV; slope = 17.8 ± 0.9; n= 18) and KCNQ1-HA (V½ = -8.1 ± 0.9 mV; slope = 27.9

± 1.0; n= 16). Lower panel: KCNQ1+ KCNE1 (V½ = 8.9 ± 2.0 mV; slope = 28.1 ± 2.3; n= 17) and

KCNQ1-HA+KCNE1 (V½ = 124.9 ± 34.7 mV; slope = 55.0 ± 7.0; n= 15).

Fig. 9. Current and surface expression of HA-tagged KCNQ1.

Xenopus oocytes were injected with RNA coding for the HA-tagged KCNQ1 channel and the resulting whole cell currents (black boxes) were measured with TEVC technique on day 4 and day 6 after injection. Current were measured at Vm=60 mV at the end of 4 s depolarization step.

Subsequently surface expression measurements were made on the same oocytes by the enzyme- linked immunoassay (scattered boxes). Data are mean±SEM. (N=5-10)

Fig. 10. Current and cell surface expression of HA-tagged KCNQ1 in presence and absence of KCNE1.

Xenopus oocytes were injected with RNA coding for HA-tagged KCNQ1 channels and water (left bars) or HA-tagged KCNQ1 channels and KCNE1 (right bars). Four days after injection whole cell currents were measured (black bars) and subsequently surface expression measurements

(scattered bars) were made on the same oocytes as described above. Data are from 5 independent experiments with 4-13 oocytes for each measurement. Data are mean±SEM. * P < 0.05, NS: Non significant.

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Figure 1

Figure 2

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Figure 3

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[8] K. Angelo, T. Jespersen, M. Grunnet, M.S. Nielsen, D.A. Klaerke, S.P. Olesen, KCNE5 induces time- and voltage-dependent modulation of the KCNQ1 current. Biophys.J. 83 (2002) 1997-2006.

[9] T. Jespersen, M. Grunnet, H.B. Rasmussen, N.B. Jorgensen, H.S. Jensen, K. Angelo, S.P. Olesen, D.A. Klaerke, The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels? Biochem.Biophys.Res.Commun. 341 (2006) 979-988.

[10] G. Romey, B. Attali, C. Chouabe, I. Abitbol, E. Guillemare, J. Barhanin, M. Lazdunski, Molecular mechanism and functional significance of the MinK control of the KvLQT1 channel activity. J.Biol.Chem. 272 (1997) 16713-16716.

[11] Y. Yang, F.J. Sigworth, Single-channel properties of IKs potassium channels. J.Gen.Physiol 112 (1998) 665-678.

[12] M. Pusch, Increase of the single-channel conductance of KvLQT1 potassium channels induced by the association with minK. Pflugers Arch. 437 (1998) 172-174.

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[13] M. Grunnet, N. MacAulay, N.K. Jorgensen, S. Jensen, S.P. Olesen, D.A. Klaerke, Regulation of cloned, Ca2+-activated K+ channels by cell volume changes. Pflugers Arch. 444 (2002) 167-177.

[14] S. Dahimene, S. Alcolea, P. Naud, P. Jourdon, D. Escande, R. Brasseur, A. Thomas, I. Baro, J. Merot, The N-terminal juxtamembranous domain of KCNQ1 is critical for channel surface expression: implications in the Romano-Ward LQT1 syndrome. Circ.Res. 99 (2006) 1076-1083.

[15] T. Jespersen, M. Grunnet, K. Angelo, D.A. Klaerke, S.P. Olesen, Dual-function vector for protein expression in both mammalian cells and Xenopus laevis oocytes. Biotechniques 32 (2002) 536-8, 540.

[16] M. Margeta-Mitrovic, Assembly-dependent trafficking assays in the detection of receptor-receptor interactions. Methods 27 (2002) 311-317.

[17] C. Kang, C. Tian, F.D. Sonnichsen, J.A. Smith, J. Meiler, A.L. George, Jr., C.G. Vanoye, H.J. Kim, C.R. Sanders, Structure of KCNE1 and implications for how it modulates the KCNQ1 potassium channel. Biochemistry 47 (2008) 7999-8006.

[18] F. Sesti, S.A. Goldstein, Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome. J.Gen.Physiol 112 (1998) 651-663.

[19] M. Jiang, X. Xu, Y. Wang, F. Toyoda, X.S. Liu, M. Zhang, R.B. Robinson, G.N. Tseng, Dynamic partnership between KCNQ1 and KCNE1 and influence on cardiac IKs current amplitude by KCNE2. J.Biol.Chem. 284 (2009) 16452-16462.

[20] S.M. Clancy, B. Chen, F. Bertaso, J. Mamet, T. Jegla, KCNE1 and KCNE3 beta- subunits regulate membrane surface expression of Kv12.2 K(+) channels in vitro and form a tripartite complex in vivo. PLoS.One. 4 (2009) e6330.

[21] K.D. Chandrasekhar, T. Bas, W.R. Kobertz, KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression. J.Biol.Chem. 281 (2006) 40015-40023.

[22] C.G. Vanoye, R.C. Welch, C. Tian, C.R. Sanders, A.L. George, Jr., KCNQ1/KCNE1 assembly, co-translation not required. Channels (Austin.) 4 (2010).

[23] M. Fink, F. Duprat, F. Lesage, C. Heurteaux, G. Romey, J. Barhanin, M. Lazdunski, A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression. J.Biol.Chem. 271 (1996) 26341-26348.

[24] G. Panaghie, K.K. Tai, G.W. Abbott, Interaction of KCNE subunits with the KCNQ1 K+ channel pore. J.Physiol 570 (2006) 455-467.

[25] Y.F. Melman, S.Y. Um, A. Krumerman, A. Kagan, T.V. McDonald, KCNE1 binds to the KCNQ1 pore to regulate potassium channel activity. Neuron 42 (2004) 927- 937.

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[26] Y.F. Melman, A. Krumerman, T.V. McDonald, A single transmembrane site in the KCNE-encoded proteins controls the specificity of KvLQT1 channel gating. J.Biol.Chem. 277 (2002) 25187-25194.

[27] H. Chen, S.A. Goldstein, Serial perturbation of MinK in IKs implies an alpha- helical transmembrane span traversing the channel corpus. Biophys.J. 93 (2007) 2332-2340.

[28] J. Chen, R. Zheng, Y.F. Melman, T.V. McDonald, Functional interactions between KCNE1 C-terminus and the KCNQ1 channel. PLoS.One. 4 (2009) e5143.

[29] A.R. Tapper, A.L. George, Jr., MinK subdomains that mediate modulation of and association with KvLQT1. J.Gen.Physiol 116 (2000) 379-390.

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Related paper: Cell swelling and membrane stretch – A common trigger of potassium channel activation?

Ion channels are sensitive to mechanical stimuli such as cell volume changes and membrane stretch but whether ion channel activation upon cell swelling is a result from membrane stretch is controversial.

Mechanosensitive ion channels play important roles in many basic cellular functions. For example, changes in volume and shape during growth division and migration subject cells to compression, shearing and stretch. In addition, cells are constantly exposed to mechanical stimuli from their external environment including osmotic stress, touch, sound or gravity.

Mechanical stimuli are quantified as force per area (i.e. N/m2 or pascal, Pa), and the applied force is detected either by membrane-bound or –associated sensor molecules that initiate intracellular signal transduction pathways. Sensor molecules may be located either in the general plasma membrane or in membranes of specialized sensory structures (or „antennae‟), e.g. kinocilia and primary cilia. An important heterogenous group of membrane proteins that include a number of mechanosensitive members is ion channel proteins, and most, if not all, major ion channel families include mechanosensitive members. A substantial amount of knowledge has been gathered through the later years about the nature and function of mechanosensitive ion channels, and about their roles in a number of human diseases. The increasing focus on mechanosensitive ion channels is reflected in the fact that the number of scientific publications on the subject has increased more than 10-fold over the last three decades.

Mechanosensitive ion channels act as mechanosensors by altering their ion transport properties in response to the magnitude of the applied force. These properties include the number of ion channels present in the membrane (N), open-state probability (Po), and single-channel conductance (γ). Ion channel activation constitutes either a direct link that transduces mechanical force into electrical or chemical intracellular signals, or they are activated secondary to mechanical stimulation of other membrane-associated proteins.

Animal cells do not possess a rigid/stiff wall which can resist turgor pressure as in the case of plant cells. Nevertheless, animal cells are able to withstand exposure to highly hypoosmotic conditions that would result in a high hydrostatic pressure in a noncompliant

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PhD thesis Sofia Hammami cell. Generally cells have excess membrane in the form of membrane invaginations and microvilli or they may reduce their surface-to-volume ratio by attaining a more spherical shape. During volume expansion cells increase their surface area either 1) by recruiting excess membrane from membrane folds and smoothing out surface membrane projections or 2) by exocytotic insertion of membrane from intracellular pools or 3) by stretching of the membrane.

The controversy: related or independent mechanisms?

Implicitly it has been assumed that cell swelling is associated with parallel changes in membrane stretch or tension, and that membrane stretch is the factor that elicits activation of volume sensitive ion channels. According to this view, volume increase and membrane stretch constitute a common mechanism in the regulation of ion channels, and, consequently, cell swelling experiments have frequently been used as surrogate model for membrane stretch. Experimentally, cells may be swollen osmotically in which case the entire membrane is affected or a membrane patch may be exposed to stretch by application of suction to a patch pipette in which case the effect is highly local. Characteristics of membrane stretch and cell volume changes are presented in figure 1 and 2, respectively, as well as examples of their effects on ion channels.

We have aimed to clarify whether regulation of K+ channels by small changes in cell volume and by membrane stretch indeed represents a common mechanism or whether they can be considered independent regulatory mechanisms. This question was addressed by taking a simple approach (Hammami et al., 2009): we studied the mechanosensitive properties of two types of potassium channels with patch clamp technique, one which is known to be regulated by cell volume (the KCNQ1 channel) and another, which is known to be regulated by membrane stretch (the BK channel). The strategy was simply to test the volume-sensitive KCNQ1 channel for stretch sensitivity, and the stretch-activated BK channel for volume sensitivity. It was demonstrated that KCNQ1 activation in response to cell swelling is not caused by membrane stretch, and that BK stretch-activation is not mimicked by cell swelling (figure 3). These findings demonstrate that cell swelling is not necessarily associated with membrane stretch, and, consequently, the two mechanisms represent independent ways of activating mechanosensitive ion channels.

In our experiments, Xenopus oocyte volumes were varied within a physiological range, ± approximately 5 % (27% decrease in osmolarity). Groulx (Groulx et al., 2006) has shown

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PhD thesis Sofia Hammami that during moderate swelling (50% decrease in osmolarity) cells (human bronchial epithelial cells, CHO cells and human lung carcinoma cells) increase their surface area by 30% mainly by unfolding the surface membrane. Only under extreme hypotonic swelling (98% decrease in osmolarity) exocytotic insertion of membrane from the intracellular pool and membrane stretch occurs. However, whether non stretch-activated ion channels react upon this extreme membrane stretching is a question. In fact, the use of unphysiologically large hypoosmotic shock may optimize the chance of seeing changes in ion channel activity as a last line of defense against excessive cell swelling. Recently Spagnoli et al. (Spagnoli et al., 2008) have shown that osmotic stress is not confined to the cell surface /cell membrane but is distributed throughout the cell. By using atomic force microscopy it was shown that the cell membrane did not stiffen upon cell swelling but it became softer which was explained by the role of the cytoskeleton as a dynamic lattice with gel-like properties.

The controversy of whether stretch- and volume-activation represent a common mechanism is relevant to all mechanosensitive ion channels. Our data indicated that the two mechanisms are indeed independent regulatory mechanisms. However, some ion channels e.g. the two pore family of K+ channels TRAAK and TREK are intriguing, since they are apparently sensitive to small changes in cell volume as well as membrane stretch (Patel et al., 2001).

In some ion channels, stretch- and volume-sensitivity has been identified to rely on certain amino acids in the channel-forming protein. For example, it seems well documented that stretch sensitivity of the TREK channels is dependent on a single positively charged amino acid located close to the cell membrane at the inner part of transmembrane segment 2 (Honore et al., 2002). Likewise deletion of the cytoplasmic c- terminus, the so called Stress-axis Regulated Exon (STREX), of BK channels cloned from chick hearts, abolishes stretch-sensitivity of the channel (Qi et al., 2005). This finding suggests that STREX constitutes a part of the mechano-sensing apparatus of the channel.

Consequently, future studies based on site-directed mutagenesis and construction of chimeras can likely provide insight in the roles of specific amino acids and constitute a key to the understanding of regulatory properties of mechanosensitive ion channels. Also

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PhD thesis Sofia Hammami these studies may finally establish whether the two mechanisms can be considered independent or interrelated.

Figures

Figure 1. Membrane stretch. A. Mechanical impact such as stretch or physical bending of the membrane leads to changes in membrane tension, thickness and curvature. These processes are characteristic of compliant epithelia like those of the lining of the gastrointestinal tract, airways, bladder, the endothelia, and of non-epithelial cell membranes like muscle cells. Also, cell types experiencing wounding such as epidermal cells, fibroblasts and migrating cells are subject to membrane stretch. B. Single channel traces of a stretch activated channel from frog skin gland at increasing negative pressure in the patch pipette. Three channels are present in the patch. Numbers indicate number of open channels. C denotes the closed state. The open state probability increases with increasing pressure (unpublished data). C. The sensitivity of ion channels to membrane stretch can be investigated in patch clamp experiments by application of negative pressure (suction) to a membrane patch.

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Figure 2. Cell volume change A. Changes in cell volume result from an imbalance between the intracellular and extracellular concentration of osmolytes that causes gain (swelling) or loss (shrinkage) of cellular water. Subsequently the cell regulates its volume back to the initial value by regulatory volume increase (RVI) or decrease (RVD). Changes in cell volume occur at different physiological processes such as secretion, cell migration, cell growth, hormone and transmitter release, synthesis or breakdown of macromolecules. B. Time course of potassium current in Xenopus oocytes expressing KCNQ1 and AQP1 following changes in extracellular osmolarity. The current increases in parallel with cell swelling and decreases with cell shrinkage (Grunnet et al., 2003). C. The volume sensitivity of ion channels can be studied in patch clamp experiments by exposing the cell to different extracellular osmolarities thus inducing cell swelling or shrinkage.

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Figure 3. Membrane stretch versus cell swelling. When exposing KCNQ1 expressing oocytes to extracellular hypoosmotic solution (cell swelling), current increases. Stretching the membrane by applying negative pressure in the patch pipette, does not affect the current. In contrast, cell volume changes have no effect on BK currents which are highly sensitive to membrane stretch. The results imply that cell swelling and membrane stretch constitute two independent ion channel regulatory mechanisms.

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Reference List

1. Groulx N, Boudreault F, Orlov SN, & Grygorczyk R (2006). Membrane reserves and hypotonic cell swelling. J Membr Biol 214, 43-56.

2. Grunnet M, Jespersen T, MacAulay N, Jorgensen NK, Schmitt N, Pongs O, Olesen SP, & Klaerke DA (2003). KCNQ1 channels sense small changes in cell volume. J Physiol 549, 419-427.

3. Hammami S, Willumsen NJ, Olsen HL, Morera FJ, Latorre R, & Klaerke DA (2009). Cell volume and membrane stretch independently control K+ channel activity. J Physiol 587, 2225-2231.

4. Honore E, Maingret F, Lazdunski M, & Patel AJ (2002). An intracellular proton sensor commands lipid- and mechano-gating of the K(+) channel TREK-1. EMBO J 21, 2968-2976.

5. Patel AJ, Lazdunski M, & Honore E (2001). Lipid and mechano-gated 2P domain K(+) channels. Curr Opin Cell Biol 13, 422-428.

6. Qi Z, Chi S, Su X, Naruse K, & Sokabe M (2005). Activation of a mechanosensitive BK channel by membrane stress created with amphipaths. Mol Membr Biol 22, 519- 527.

7. Spagnoli C, Beyder A, Besch S, & Sachs F (2008). Atomic force microscopy analysis of cell volume regulation. Phys Rev E Stat Nonlin Soft Matter Phys 78, 031916.

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