Desensitization of Chemical Activation by Auxiliary Subunits
2008; Elsevier BV; Volume: 283; Issue: 33 Linguagem: Inglês
10.1074/jbc.m802426200
ISSN1083-351X
AutoresZhaobing Gao, Qiaojie Xiong, Haiyan Sun, Min Li,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoChemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the IKs current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native IKs current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation. Chemical openers for KCNQ potassium channels are useful probes both for understanding channel gating and for developing therapeutics. The five KCNQ isoforms (KCNQ1 to KCNQ5, or Kv7.1 to Kv7.5) are differentially localized. Therefore, the molecular specificity of chemical openers is an important subject of investigation. Native KCNQ1 normally exists in complex with auxiliary subunits known as KCNE. In cardiac myocytes, the KCNQ1-KCNE1 (IsK or minK) channel is thought to underlie the IKs current, a component critical for membrane repolarization during cardiac action potential. Hence, the molecular and pharmacological differences between KCNQ1 and KCNQ1-KCNE1 channels have been important topics. Zinc pyrithione (ZnPy) is a newly identified KCNQ channel opener, which potently activates KCNQ2, KCNQ4, and KCNQ5. However, the ZnPy effects on cardiac KCNQ1 potassium channels remain largely unknown. Here we show that ZnPy effectively augments the KCNQ1 current, exhibiting an increase in current amplitude, reduction of inactivation, and slowing of both activation and deactivation. Some of these are reminiscent of effects by KCNE1. In addition, neither the heteromultimeric KCNQ1-KCNE1 channels nor native IKs current displayed any sensitivity to ZnPy, indicating that the static occupancy by a KCNE subunit desensitizes the reversible effects by a chemical opener. Site-directed mutagenesis of KCNQ1 reveals that residues critical for the potentiation effects by either ZnPy or KCNE are clustered together in the S6 region overlapping with the critical gating determinants. Thus, the convergence of potentiation effects and molecular determinants critical for both an auxiliary subunit and a chemical opener argue for a mechanistic overlap in causing potentiation. Voltage-gated potassium channels are critical for membrane excitability. In cardiac tissue, potassium currents are important elements responsible for the repolarization of action potential. Among the different potassium current components, IKs and IKr are two key determinants for the duration of cardiac action potential (1Zeng J. Laurita K.R. Rosenbaum D.S. Rudy Y. Circ. Res. 1995; 77: 140-152Crossref PubMed Scopus (341) Google Scholar). Molecular and genetic studies have shown that the IKs component is likely formed by the heteromultimeric assembly of subunits encoded by KCNQ1 (Kv7.1) and KCNE1 (IsK or minK) (2Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1408) Google Scholar, 3Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1534) Google Scholar), whereas the current encoded by hERG (human ether-ago-go related gene) is responsible for IKr (4Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. 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Cell. 1999; 97: 175-187Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar, 14Piccini M. Vitelli F. Seri M. Galietta L.J. Moran O. Bulfone A. Banfi S. Pober B. Renieri A. Genomics. 1999; 60: 251-257Crossref PubMed Scopus (69) Google Scholar). Each KCNE subunit consists of a single transmembrane segment. All five KCNE members (KCNE1 to KCNE5) are capable of coassembly with KCNQ1 (15Abbott G.W. Goldstein S.A. Mol. Interv. 2001; 1: 95-107PubMed Google Scholar, 16Bendahhou S. Marionneau C. Haurogne K. Larroque M.M. Derand R. Szuts V. Escande D. Demolombe S. Barhanin J. Cardiovasc. Res. 2005; 67: 529-538Crossref PubMed Scopus (120) Google Scholar). In the case of KCNQ1 with KCNE1, the resultant heteromultimeric current is similar to IKs in cardiac tissue. Both KCNE1 and KCNE3 increase the maximum conductance of KCNQ1, whereas the association with KCNE2, KCNE4, and KCNE5 results in inhibition (2Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1408) Google Scholar, 3Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1534) Google Scholar, 16Bendahhou S. Marionneau C. Haurogne K. Larroque M.M. Derand R. Szuts V. Escande D. Demolombe S. Barhanin J. Cardiovasc. Res. 2005; 67: 529-538Crossref PubMed Scopus (120) Google Scholar, 17Schroeder B.C. Waldegger S. Fehr S. Bleich M. Warth R. Greger R. Jentsch T.J. Nature. 2000; 403: 196-199Crossref PubMed Scopus (424) Google Scholar). The effects of KCNE1 on KCNQ1 include increasing overall current, slowing the activation and deactivation kinetics, and removal of inactivation (2Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1408) Google Scholar, 3Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1534) Google Scholar). There is also evidence suggesting an increase in the single channel conductance of KCNQ1 (18Pusch M. Pflugers Arch. 1998; 437: 172-174Crossref PubMed Scopus (63) Google Scholar, 19Sesti F. Goldstein S.A. J. Gen. Physiol. 1998; 112: 651-663Crossref PubMed Scopus (192) Google Scholar, 20Yang Y. Sigworth F.J. J. Gen. Physiol. 1998; 112: 665-678Crossref PubMed Scopus (115) Google Scholar). KCNE3 stabilizes KCNQ1 in the open state and augments current amplitude to a level comparable with that by KCNE1 (17Schroeder B.C. Waldegger S. Fehr S. Bleich M. Warth R. Greger R. Jentsch T.J. Nature. 2000; 403: 196-199Crossref PubMed Scopus (424) Google Scholar). Several residues in the KCNQ1 S6 domain (e.g. Ser338, Phe339, and Phe340) critical for the augmentation by KCNE1 and KCNE3 have been identified (21Panaghie G. Tai K.K. Abbott G.W. J. Physiol. 2006; 570: 455-467Crossref PubMed Scopus (86) Google Scholar, 22Melman Y.F. Um S.Y. Krumerman A. Kagan A. McDonald T.V. Neuron. 2004; 42: 927-937Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 23Tapper A.R. George Jr., A.L. J. Biol. Chem. 2001; 276: 38249-38254Abstract Full Text Full Text PDF PubMed Google Scholar), although the interaction mechanism still remains elusive. Recently, several KCNQ-activating compounds have been identified, some of which are in clinical trials for anti-convulsive applications (24Blackburn-Munro G. Dalby-Brown W. Mirza N.R. Mikkelsen J.D. Blackburn-Munro R.E. CNS Drug Rev. 2005; 11: 1-20Crossref PubMed Scopus (116) Google Scholar, 25Fatope M.O. Drugs. 2001; 4: 93-98PubMed Google Scholar, 26Miceli F. Soldovieri M.V. Martire M. Taglialatela M. Curr. Opin. Pharmacol. 2008; 8: 65-74Crossref PubMed Scopus (127) Google Scholar, 27Porter R.J. Nohria V. Rundfeldt C. Neurotherapeutics. 2007; 4: 149-154Crossref PubMed Scopus (72) Google Scholar, 28Porter R.J. Partiot A. Sachdeo R. Nohria V. Alves W.M. Neurology. 2007; 68: 1197-1204Crossref PubMed Scopus (194) Google Scholar). These compounds are interesting in several ways. First, their structures are sufficiently distinct and appear to affect different aspects of channel properties that lead to more active channels (29Peretz A. Degani N. Nachman R. Uziyel Y. Gibor G. Shabat D. Attali B. Mol. Pharmacol. 2005; 67: 1053-1066Crossref PubMed Scopus (180) Google Scholar, 30Xiong Q. Gao Z. Wang W. Li M. Trends Pharmacol. Sci. 2008; 29: 99-107Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Second, mutagenesis studies revealed that they indeed recognize different "agonistic" sites on KCNQ channels (31Schenzer A. Friedrich T. Pusch M. Saftig P. Jentsch T.J. Grotzinger J. Schwake M. J. Neurosci. 2005; 25: 5051-5060Crossref PubMed Scopus (226) Google Scholar, 32Wuttke T.V. Seebohm G. Bail S. Maljevic S. Lerche H. Mol. Pharmacol. 2005; 67: 1009-1017Crossref PubMed Scopus (234) Google Scholar). Furthermore, one KCNQ channel complex is capable of interacting with more than one class of chemical openers. As a result, the tripartite complex displays a hybrid response, tunable by different concentrations and/or ratios of the chemical openers (33Xiong Q. Sun H. Zhang Y. Nan F. Li M. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 3128-3133Crossref PubMed Scopus (40) Google Scholar). Specificity for either isoform or subunit composition is a topic critical for the molecular understanding of these cation channel openers. Because the KCNQ2–5 subunits are more commonly found in the nervous system, whereas KCNQ1 is predominantly localized in cardiac and other non-excitable tissues where they are in complex with KCNE subunits, it is particularly relevant to investigate subunit specificity among KCNQ1, KCNQ1-KCNE, and KCNQ2–5 channels. We have reported that bis(1-hydroxy-2(1H)-pyridineselonato-O,S) zinc, commonly known as zinc pyrithione (ZnPy), 2The abbreviations used are:ZnPyzinc pyrithioneCHOChinese hamster ovary. is a potent activator of KCNQ 1, 2, 4, and 5 channels but not KCNQ3 (34Xiong Q. Sun H. Li M. Nat. Chem. Biol. 2007; 3: 287-296Crossref PubMed Scopus (101) Google Scholar). The effects of ZnPy on neuronal KCNQ channels include both a hyperpolarizing shift in the voltage dependence of activation and an increase in current amplitude. Of particular interest, mutagenesis studies have revealed that KCNQ2(A306T) in the S6 segment significantly reduced current augmentation of ZnPy (34Xiong Q. Sun H. Li M. Nat. Chem. Biol. 2007; 3: 287-296Crossref PubMed Scopus (101) Google Scholar). The corresponding region of KCNQ2(A306T) in KCNQ1 was implicated for interactions with KCNE1 (21Panaghie G. Tai K.K. Abbott G.W. J. Physiol. 2006; 570: 455-467Crossref PubMed Scopus (86) Google Scholar, 22Melman Y.F. Um S.Y. Krumerman A. Kagan A. McDonald T.V. Neuron. 2004; 42: 927-937Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 23Tapper A.R. George Jr., A.L. J. Biol. Chem. 2001; 276: 38249-38254Abstract Full Text Full Text PDF PubMed Google Scholar). We thus hypothesized that the coassembly of KCNQ1 with KCNE1 subunits may lead to a distinct molecular specificity or pharmacological response, which would be relevant to finding compounds specific for either neuronal or cardiac KCNQ channels. Using a combination of electrophysiological analyses and site-directed mutagenesis, we have evaluated channel properties affected by ZnPy, tested whether KCNE auxiliary subunits affect ZnPy sensitivity, and investigated the molecular determinants critical for KCNE modulation and KCNQ1 pharmacology to chemical openers. zinc pyrithione Chinese hamster ovary. cDNAs and Mutagenesis—KCNQ1 and KCNE1 cDNA were gifts from Dr. M. C. Sanguinetti (University of Utah). KCNE3 cDNA was a gift from Dr. T. V. McDonald (Albert Einstein School of Medicine). Point mutations in the KCNQ1 channel were introduced by using the QuikChange II site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing. Cell Culture and Transfection—Chinese hamster ovary (CHO) cells were grown in 50/50 Dulbecco's modified Eagle's medium/F-12 (Cellgro, Manassas, VA) with 10% fetal bovine serum and 2 mm l-glutamine (Invitrogen). To express KCNQ1 and KCNQ1-KCNE1, cells were split at 24 h before transfection, plated in 60-mm dishes, and transfected with Lipofectamine2000™. After transfection, cells were split and replated onto coverslips coated with poly-l-lysine (Sigma-Aldrich). Plasmid expressing CD4 as a marker was cotransfected. Prior to recording, anti-CD4 Dynabeads (Invitrogen) were added to the medium to allow for identification of the transfected cells. To coexpress KCNQ1 and KCNE3, cells were electroporated with a Nucleofector™ kit for CHO-K1 cells (Amaxa, Gaithersburg, MD) according to the manufacturer's instruction. A green fluorescent protein cDNA (Amaxa) was cotransfected to allow identification of the transfected cells with the fluorescent microscope. The cDNA concentration of KCNQ1 or KCNE was 200 ng/μl, and the molar ratio of KCNE/KCNQ1 was 1:1. Electrophysiological Recording in CHO Cells—Whole-cell voltage clamp recording was carried out using cultured CHO cells at room temperature by an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA). The electrodes were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL). When filled with the intracellular solution, the electrodes have resistances of 3–5 MΩ. Pipette solution contained (in mm) 145 KCl, 1 MgCl2, 5 EGTA, 10 HEPES, and 5 MgATP (pH 7.3 with KOH). During the recording, constant perfusion of extracellular solution was maintained using a B-channel valve BPS-8 model perfusion system (ALA Scientific Instruments, Westburg, NY). Extracellular solution contained (in mm) 140 NaCl, 3 KCl, 2 CaCl2, 1.5 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). Signals were filtered at 1 KHz and digitized using a DigiData 1322A with pClamp 9.2 software (Molecular Devices). Series resistance was compensated by 60–80%. In the present study, three types of voltage protocols were used. The holding potential was −80 mV in all voltage protocols. To record the KCNQ1 current, the cells were stimulated by a series of 1,500-ms depolarizing steps from −70 mV to +50 mV in 10-mV increments. A testing step to −120 mV was applied to obtain tail currents. To record KCNQ1-KCNE1 currents, longer depolarizing steps (3,000 ms) were used to elicit the currents, and a −50-mV testing step was used to obtain tail currents. To record the KCNQ1-KCNE3 current, slightly different voltage protocols were used. The currents were elicited by a series of depolarizing steps from −100 to +80 mV in 20-mV increments. The tail current testing step was −50 mV. Isolation of Cardiac Myocytes and Native IKs Recording—Single myocytes were isolated from the left ventricle of adult guinea pig in a Langerdorff perfusion system as described previously (35Akao M. Ohler A. O'Rourke B. Marban E. Circ. Res. 2001; 88: 1267-1275Crossref PubMed Scopus (266) Google Scholar). Briefly, the hearts were removed quickly via midline thoracotomy and perfused with a Ca2+-free Tyrode's solution containing collagenase (6 mg/ml) and protease (0.1 mg/ml) for ∼5–6 min. Then the hearts were switched to Kraft-Bruhe solution perfusion for 5 min, and the ventricles were minced and gently triturated to single cells. The cells were stored at 4 °C in Kraft-Bruhe solution until use. Kraft-Bruhe solution contained the following (in mm): 50 l-glutamic acid, 80 KOH, 40 KCl, 3 MgSO4, 25 KH2PO4, 10 HEPES, 1 EGTA, 20 taurine, and 10 glucose (pH 7.4). Before recording, the myocytes were transferred to a recording chamber perfused with Tyrode's solution. Both Itotal and IKs were recorded with conventional configuration of patch-clamp technique using an Axopatch 200B amplifier (Molecular Devices). Pipettes were pulled from borosilicate glass capillaries (World Precision Instruments). When filled with the intracellular solution containing (in mm) 120 KCl, 10 KH2PO4, 1 MgSO4, 5 EGTA, and 5 HEPES (pH 7.2 with KOH), the pipettes have resistances of 3–5 MΩ. Itotal was elicited by 3-s depolarizing pulses from a holding potential of −80mV to various test potentials between −70 and +70 mV in 10-mV increments in Tyrode's solution perfusion. Tyrode's solution contained (in mm) 135 NaCl, 5.4 KCl, 1 MgCl2, 0.33 NaH2PO4, 5 HEPES, and 5 glucose (pH 7.4 with NaOH) and was oxygenated with 100% O2. To isolate for IKs, the external solution was switched to Na+-free solution containing (in mm) 132 N-methyl-d-glucamine (for sodium ion replacement), 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, 5 glucose, 0.05 lanthanum chloride to block IKr, and 0.005 nifedipine to block L-type calcium current (pH 7.4 with HCl). Modeling—Three-dimensional structural models for the KCNQ1 S5 and S6 domains were generated using the solved crystal structure of Kv1.2 (Protein Data Bank code 2A79) as a template. The corresponding domains between KCNQ1 and Kv1.2 were aligned with the DNASTAR MegAlign program using standard parameters. The KCNQ1 models were constructed using DeepView/SWISS-PDBViewer (36Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9690) Google Scholar). The structural representation was performed with the POV-Ray program. Data and Statistical Analysis—Patch-clamp data were processed using Clampfit 9.2 (Molecular Devices) and then analyzed in GraphPad Prism 4 (GraphPad, San Diego, CA). The activation curve was fitted by the Boltzmann Sigmoidal equation: G = Gmin+ (Gmax - Gmin)/(1 + exp(V - V½)/S)), where Gmax is the maximum conductance, Gmin is the minimum conductance, V½ is the voltage for reaching 50% of maximum conductance, and S is the slope factor. The dose-response curve was fitted by the Hill equation: E = Emax/(1 + (EC50/C)P), where EC50 is the drug concentration producing half of the maximum response, and P is the Hill coefficient. The activation and deactivation trace were fitted by the standard (or power) exponential equation using Clampfit 9.2. Data are presented as means ± S.E. Significance was estimated using a paired two-tailed Student's t test. Augmentation of KCNQ1 Channels by ZnPy—To examine ZnPy modulation on the KCNQ1 current, we first expressed the KCNQ1 cDNA in CHO cells and recorded the channel activity with a whole-cell voltage clamp. KCNQ1 displayed a characteristic outward current with visible inactivation (Fig. 1A). In the presence of 5 μm ZnPy, steady-state currents at different depolarizing voltages were greatly potentiated, and inactivation was no longer readily detectable during the depolarizing phase (Fig. 1A). The ZnPy-mediated potentiation was fully reversible upon removal of ZnPy (Fig. 1B), consistent with the idea of modulating channel activity instead of protein density on cell surface. Examination of current amplitude increase using steady-state currents at different concentrations of ZnPy revealed a half-maximal value (EC50) of 3.5 ± 1.1 μm (n > 3) (Fig. 1C). Previous work has shown that ZnPy-mediated potentiation of neuronal KCNQ2 channels involves both a hyperpolarizing shift of half-maximal activation voltage (V½) and an increase in overall conductance (Gmax) (34Xiong Q. Sun H. Li M. Nat. Chem. Biol. 2007; 3: 287-296Crossref PubMed Scopus (101) Google Scholar). To determine the ZnPy effects on the KCNQ1 homomultimer, we examined the G-V curve in the presence or absence of 5 μm ZnPy, which causes an ∼80% potentiation. In the absence of ZnPy, the V½ value was −23.2 ± 1.1 mV, similar to the value of the earlier reports (3Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1534) Google Scholar, 37Chouabe C. Neyroud N. Richard P. Denjoy I. Hainque B. Romey G. Drici M.D. Guicheney P. Barhanin J. Cardiovasc. Res. 2000; 45: 971-980Crossref PubMed Scopus (103) Google Scholar, 38Melman Y.F. Domenech A. de la Luna S. McDonald T.V. J. Biol. Chem. 2001; 276: 6439-6444Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) (Fig. 1D). Further experiments with a range of ZnPy concentrations revealed no significant change of V½ either (Fig. 1E). Hence, ZnPy increases overall conductance of KCNQ1 without affecting voltage dependence. Modulation of Kinetic Properties of KCNQ1 by ZnPy—To investigate ZnPy-mediated modulation on the close-open transition of the KCNQ1 channel, effects on both activation and deactivation were examined. The currents induced by depolarizing from −70 mV to +50 mV displayed a rapid activation followed by characteristic inactivation. In the presence of 5 μm ZnPy, the time constant of activation was slowed from 23.4 ± 2.4 ms to 74.0 ± 12.5 ms (n = 3; p < 0.01) (Fig. 2A). This effect was seen in a range of depolarizing voltages (Fig. 2B). In addition, ZnPy also induced slowing of deactivation in hyperpolarizing voltages (Fig. 2, C and D). The reduction of deactivation rate is consistent with the overall increase of current amplitude or Gmax. If ZnPy affects both activation and deactivation through the same interaction that causes the increase of current amplitude, one should see similar EC50 values. Indeed, when plotting the change of time constants for activation or deactivation against concentrations of ZnPy, we found EC50 values of 3.2 ± 0.2 μm (n > 3) for activation and 3.6 ± 0.1 μm (n > 3) for deactivation (Fig. 2, E and F). Thus, the EC50 values measured for current amplitude, activation, and deactivation time constants are essentially the same, providing evidence for the notion that one class of interaction (or binding site) is responsible for the activation and deactivation changes and for the current potentiation (Fig. 1C). Among different KCNQ channels, inactivation is a distinctive feature for KCNQ1. The overall increase in current amplitude could be contributed in part through inhibition of inactivation (Fig. 1A). Using the voltage pulse protocols outlined in Fig. 3A, the inactivation kinetics in the presence or absence of ZnPy was evaluated. In the absence of drug treatment, noticeable inactivation was observed (Fig. 3, A and B). However, in the presence of 5 μm of ZnPy, the inactivation was largely diminished. At a higher concentration of 10 μm, a similar effect was observed (data not shown). When examining the recovery from inactivation, the effect was rather minimal (n > 4; p > 0.1) (Fig. 3, C and D). Thus, the inhibition of inactivation by ZnPy is a contributing factor for an overall increase of current amplitude. Coassembly with KCNE1 Desensitizes ZnPy-mediated Augmentation—Coassembly of KCNQ1 with KCNE1 is thought to form the native IKs (2Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1408) Google Scholar, 3Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1534) Google Scholar). The modulation of KCNQ1 by KCNE1 displays considerable similarity to ZnPy-induced effects. Both KCNE1 and ZnPy increase current amplitude, decrease inactivation, and slow activation and deactivation (Table 1), although their effects on the G-V curve are different. To test any potential alteration of sensitivity to ZnPy, we coexpressed KCNQ1 and KCNE1 (Fig. 4C). In the presence of 10 μm ZnPy, there were no obvious effects of drug-induced further potentiation (Fig. 4, C and E). It is known that the heteromultimeric KCNQ1-KCNE1 channels have a depolarizing V½ shift of 38.5 ± 1.5 mV compared with homomultimeric KCNQ1 channels (Fig. 4D). In the presence of different concentrations of ZnPy, the V½ values remain unchanged (Fig. 4F). In addition, no changes were detected for deactivation in the presence of ZnPy (Fig. 4G).TABLE 1Comparision of ZnPy, KCNE1, and KCNE3 effects on KCNQ1 G is the channel conductance in the presence of either ZnPy or auxiliary subunits, whereas G0 is the conductance of KCNQ1 without ZnPy or any auxiliary subunits (KCNE1 or KCNE3). Both ZnPy and KCNE increase the conductance ∼4-fold; ZnPy and KCNE1 slow the activation and deactivation, whereas KCNE3 accelerates the deactivation. In addition, ZnPy inhibits KCNQ1 inactivation, whereas KCNE completely abolishes the inactivation. Significance was estimated by using a paired t-test; n > 4. NA, not applicable.KCNQ1aThe KCNQ1 and KCNQ1-KCNE1 currents were elicited by depolarization to +50 mVZnPy (5 μm)aThe KCNQ1 and KCNQ1-KCNE1 currents were elicited by depolarization to +50 mVKCNQ1-KCNE1aThe KCNQ1 and KCNQ1-KCNE1 currents were elicited by depolarization to +50 mVKCNQ1-KCNE3bThe KCNQ1-KCNE3 currents were elicited by depolarization to +40 mVG/G01.04.2 ± 0.8c*, p < 0.05 compared with KCNQ14.3 ± 0.9c*, p < 0.05 compared with KCNQ14.4 ± 1.5c*, p < 0.05 compared with KCNQ1τ activation (ms)23.4 ± 4.973.7 ± 12.5c*, p < 0.05 compared with KCNQ1τ1, 1297.6 ± 432.5c*, p < 0.05 compared with KCNQ1; τ2, 218.0 ± 46.825.9 ± 2.8τ deactivation (ms)60.6 ± 6.2113.20 ± 15.41c*, p < 0.05 compared with KCNQ1243.8 ± 47.9c*, p < 0.05 compared with KCNQ121.6 ± 3.4c*, p < 0.05 compared with KCNQ1Inactivation (%)67.2 ± 3.714.2 ± 12.5c*, p < 0.05 compared with KCNQ1NANAa The KCNQ1 and KCNQ1-KCNE1 currents were elicited by depolarization to +50 mVb The KCNQ1-KCNE3 currents were elicited by depolarization to +40 mVc *, p < 0.05 compared with KCNQ1 Open table in a new tab It is believed that coassembly of KCNE1 and KCNQ1 forms the native IKs channels. Therefore, if KCNE1 indeed abolishes the KCNQ1 sensitivity to ZnPy, native IKs should display no sensitivity. To examine this possibility, ventricular cardiac myocytes from guinea pig were acutely isolated. Using the previously reported conditions (see "Experimental Procedures"), the IKs component was isolated and showed a characteristic inhibition by 100 μm chromanol 293B (Fig. 5, A–C) (39Busch A.E. Suessbrich H. Waldegger S. Sailer E. Greger R. Lang H. Lang F. Gibson K.J. Maylie J.G. Pflugers Arch. 1996; 432: 1094-1096Crossref PubMed Scopus (144) Google Scholar). In the presence of ZnPy, the IKs displayed no detectable change in macroscopic currents, voltage sensitivity, or deactivation kinetics (Fig. 5, D–G). Hence, neither recombinant KCNQ1-KCNE1 nor native IKs displays the sensitivity to ZnPy. This is in further agreement with KCNE1-mediated desensitization. The five known KCNE subunits may be divided according to their effects. Both KCNE1 and KCNE3 display potentiation when coassembled with pore-forming α subunits. In contrast, the other KCNE subunits display inhibitory effects. KCNE3 has a 35% identity with KCNE1. Coas
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