Functional Coupling of Human β3-Adrenoreceptors to the KvLQT1/MinK Potassium Channel (2000)

The slow component of the delayed rectifier potassium current (IKs) plays an important role during repolarization in the human heart. Life-threatening arrhythmias can be triggered by sympathetic stimulation, presumably acting on IKs. The ion channel responsible for the IKs current is made of two proteins, the KvLQT1 protein and the MinK protein. In this study, we investigated the effects of adrenergic stimulation on the KvLQT1/MinK channel by coexpressing KvLQT1/MinK channels with the human β3-adrenoreceptor subunit heterologously inXenopus oocytes. Western blot experiments revealed that β3-adrenoreceptor proteins appear in the cell membrane ofXenopus oocytes, when the corresponding cRNA was injected. In electrophysiological measurements we found that stimulation with the β-adrenergic agonist isoproterenol increased the current amplitude of the β3/KvLQT1/MinK complex up to 237% with an ED50 of 8 nm, a value similar to that found on IKs in guinea pig cardiomyocytes. When oocytes with β3/KvLQT1/MinK were preincubated with cholera toxin (2 μg/ml), an activator of GS proteins, the basal current amplitude of the β3/KvLQT1/MinK complex was increased 3.1-fold, and the current amplitude increase by isoproterenol was drastically reduced, indicating that the signal transduction cascade was mediated via Gs proteins. The knowledge about functional coupling of the human β3-adrenoreceptor to KvLQT1/MinK channels reveals interesting aspects about the genesis and therapy of arrhythmias. The slow component of the delayed rectifier potassium current (IKs) plays an important role during repolarization in the human heart. Life-threatening arrhythmias can be triggered by sympathetic stimulation, presumably acting on IKs. The ion channel responsible for the IKs current is made of two proteins, the KvLQT1 protein and the MinK protein. In this study, we investigated the effects of adrenergic stimulation on the KvLQT1/MinK channel by coexpressing KvLQT1/MinK channels with the human β3-adrenoreceptor subunit heterologously inXenopus oocytes. Western blot experiments revealed that β3-adrenoreceptor proteins appear in the cell membrane ofXenopus oocytes, when the corresponding cRNA was injected. In electrophysiological measurements we found that stimulation with the β-adrenergic agonist isoproterenol increased the current amplitude of the β3/KvLQT1/MinK complex up to 237% with an ED50 of 8 nm, a value similar to that found on IKs in guinea pig cardiomyocytes. When oocytes with β3/KvLQT1/MinK were preincubated with cholera toxin (2 μg/ml), an activator of GS proteins, the basal current amplitude of the β3/KvLQT1/MinK complex was increased 3.1-fold, and the current amplitude increase by isoproterenol was drastically reduced, indicating that the signal transduction cascade was mediated via Gs proteins. The knowledge about functional coupling of the human β3-adrenoreceptor to KvLQT1/MinK channels reveals interesting aspects about the genesis and therapy of arrhythmias. In cardiac myocytes, repolarization of the action potential is produced by several potassium currents (1Barry D.M. Nerbonne J.M. Annu. Rev. Physiol. 1996; 58: 363-394Crossref PubMed Scopus (202) Google Scholar). One is the very slow activating and deactivating delayed rectifier potassium current IKs. This current represents the predominant repolarizing current during increased heart rate (2Jurkiewicz N.K. Sanguinetti M.C Circ. Res. 1993; 72: 75-83Crossref PubMed Google Scholar, 3Varnum M.D. Busch A.E. Bond C.T. Maylie H. Adelman J.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11528-11532Crossref PubMed Scopus (103) Google Scholar, 4Zeng J. Laurita K.R. Rosenbaum D.S. Rudy Y. Circ. Res. 1995; 77: 140-152Crossref PubMed Scopus (339) Google Scholar). Recent studies have revealed that the channel underlying IKs is formed by the assembly of two transmembrane proteins, the KvLQT1 and MinK protein (5Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romex G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1392) Google Scholar, 6Sanguinetti 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 (1515) Google Scholar). The KvLQT1 protein forms the pore of the channel, whereas the MinK protein seems to act as a regulator subunit (7Kaczmarek L.K. Blumenthal E.M. Physiol. Rev. 1997; 77: 627-641Crossref PubMed Scopus (50) Google Scholar, 8Romey G. Attali B. Chouabe C. Abitbol I. Guillemare E. Barhanin J. Lazdunski M. J. Biol. Chem. 1997; 272: 16713-16716Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 9Tristani-Firouzi M. Sanguinetti M.C. J. Physiol. (Lond.). 1998; 510: 37-45Crossref Scopus (136) Google Scholar). Only the coassembly of both proteins produces current kinetics resembling the native IK currents in cardiomyocytes (5Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romex G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1392) Google Scholar, 6Sanguinetti 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 (1515) Google Scholar). Mutations that map to the KvLQT1 and minKgenes account for more than 50% of an inherited cardiac disorder, the congenital long QT syndrome (10Splawski I. Firouzi-Tristani M. Lehmann M.H. Sanguinetti M.C. Keating M.T. Nat. Genet. 1997; 17: 338-340Crossref PubMed Scopus (675) Google Scholar). Most of these patients have a dominant negative effect on the KvLQT1 channel, resulting in a reduced current amplitude of IKs and prolongation of the QT interval in the surface electrocardiogram (11Wollnik B. Schroeder C. Kubisch C. Esperer H.K. Wieacker P. Jentsch T.J. Hum. Mol. Genet. 1997; 6: 1943-1949Crossref PubMed Scopus (165) Google Scholar). Through an unknown mechanism, this condition predisposes the myocardium to “torsades-de-pointes” polymorphic ventricular tachycardias that can degenerate into ventricular fibrillation and cause sudden cardiac death. The onset of “torsade-de pointes” tachycardias in patients with long QT syndrome is often triggered by adrenergic stress with sinus tachycardia, whereas therapeutic β-blockade prevents new syncopal episodes in ∼75% of patients (12Roden D.M. Lazzara R. Rosen M. Schwartz P.J. Towbin J. Vincent M. Circulation. 1996; 94: 1996-2012Crossref PubMed Scopus (566) Google Scholar). It has been shown that the IKs current amplitude in the heart is increased by catecholamines, presumably mediated by β-adrenoreceptors (13Duchatelle-Gourdon I. Hartzell H.C. Lagrutta A.A. J. Physiol. (Lond.). 1989; 415: 251-274Crossref Scopus (68) Google Scholar, 14Lo C.F. Numann R. Circ. Res. 1998; 83: 995-1002Crossref PubMed Scopus (56) Google Scholar, 15Sanguinetti M.C. Jurkiewicz N.K. Scott A. Siegl P.K.S. Circ. Res. 1991; 68: 77-84Crossref PubMed Scopus (283) Google Scholar). In the human heart, there is evidence for the presence of at least three β-adrenoreceptor populations, β1, β2, and β3, and a fourth as yet uncloned putative β4-adrenoreceptor subtype (16Gauthier C. Tavernier G. Charpentier F. Langin D. Le Marec H. J. Clin. Invest. 1996; 98: 556-562Crossref PubMed Scopus (402) Google Scholar, 17Kaumann A.J. Molenaar P. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 355: 667-681Crossref PubMed Scopus (166) Google Scholar, 18Lowe M. Grace A.A. Vandenberg J.L. Kaumann A.J. Nauyn-Schmiedeberg's Arch. Pharmacol. 1998; 124: 1341-1344Google Scholar, 19Strosberg A.D. Protein Sci. 1998; 2: 1198-1209Crossref Scopus (172) Google Scholar). Because the β1- and β2-adrenoreceptors in heart are linked to mechanical contractility with positive inotropy, the role of β3-adrenoreceptors is not well understood, especially since the naturally occurring catecholamines stimulate all three β-adrenoreceptors and surprisingly develop negative inotropic effects through β3-adrenoreceptors and shorten the human cardiac action potential through β3-adrenoreceptors (16Gauthier C. Tavernier G. Charpentier F. Langin D. Le Marec H. J. Clin. Invest. 1996; 98: 556-562Crossref PubMed Scopus (402) Google Scholar). So far, it is not known whether β3-receptors couple to the IK potassium current. By coexpressing the human clones for the components of IKs, KvLQT1 and MinK, together with human β3-adrenoreceptors in Xenopus oocytes, we demonstrate that human β3-adrenoreceptors can couple functionally to the KvLQT1/MinK potassium channel in theXenopus oocyte expression system. This might be an explanation for the shortening of the human cardiac action potential under β3-adrenergic stimulation (16Gauthier C. Tavernier G. Charpentier F. Langin D. Le Marec H. J. Clin. Invest. 1996; 98: 556-562Crossref PubMed Scopus (402) Google Scholar). Patch clamp measurements of guinea pig cardiomyocytes were performed in the whole-cell mode in a bath solution containing (in mm/liter) 3.5 KCl, 140 NaCl, 1.5 CaCl2, 1.4 MgSO4, and 10 HEPES (pH 7.4) plus additionally 1 μm Dofetilide, a blocker of the rapid component of the delayed rectifier potassium current IKr (20Kiehn J. Lacerda A.E. Wible B. Brown A.M. Circulation. 1996; 94: 2572-2579Crossref PubMed Scopus (187) Google Scholar) and 10 μm nisoldipine to block calcium currents. The holding potential was −40 mV to inactivate fast sodium currents. The pipette solution contained (in mm/liter) 140 KCl, 0.5 CaCl2, 1.5 MgCl2, 5 KATP, 1 EGTA, and 10 HEPES (pH 7.4). Two microelectrode voltage clamp measurements of Xenopusoocytes were performed in a low K+ solution containing (in mm) 5 KCl, 100 NaCl, 1.5 CaCl2, 2 MgCl2, and 10 HEPES (pH 7.3). All measurements were carried out at room temperature (20 °C). Data were low-pass filtered at 0.2 kHz (−3 db, 4-pole Bessel filter) before digitalization at 1 kHz. Activation curves were fitted with a Boltzmann distribution as follows: G (V) =G max/(1 −exp((V12 −V)/k)), where V is the test pulse potential; V12 is the half-maximal activation potential, and k is the slope of the activation curve. Statistical data are presented as mean ± S.E. Statistical significance was evaluated using the paired Student's ttest. Differences were considered significant when a p value <0.05 was reached. The human cDNA clone pBC12BI-β3-adrenoreceptor was a gift from R. J. Lefkowitz. The restriction fragment BamHI-EcoRI (∼1.5 kilobase pairs) containing the β3-adrenoreceptor coding region was subcloned into the pSP72 transcription vector (Promega). cDNA was verified by DNA sequencing (Sequence Laboratories, Göttingen, Germany). The cDNA clone encoding human KvLQT1 was a gift from M. T. Keating. The cDNA clone encoding human MinK was a gift from A. M. Brown. cRNAs were prepared and injected (46 nl in each oocyte) as described previously (21Kiehn J. Karle C. Thomas D. Yao X. Brachmann J. Kübler W. J. Biol. Chem. 1998; 273: 25285-25291Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The concentrations of the injected cRNAs were unless otherwise stated: KvLQT1 (500 ng/μl), MinK (200 ng/μl), and β3(200 ng/μl). Xenopus oocytes injected with cRNA of β3-adrenoreceptors (1000–2000 ng/μl) were used to isolate crude membrane fractions by centrifugation in two steps in a homogenization buffer containing 0.3 m sucrose, 10 mm sodium phosphate plus a protease inhibitor mixture tablet (Roche Molecular Biochemicals) (pH 7.4). All steps were done at 4 °C. The membrane proteins (∼30 μg/sample) were boiled in SDS-polyacrylamide gel electrophoresis sample loading buffer for 5 min and electrophoresed on a 7.5% polyacrylamide SDS gel, afterward electrophoretically transferred onto Hybond ECL nitrocellulose filters (Amersham Pharmacia Biotech). After transfer, the filters were blocked with 5% nonfat dry milk and 0.1% Tween 20 in phosphate-buffered saline (Western buffer) plus 0.025% sodium azide at 4 °C overnight. The filters were then incubated with goat anti-β3polyclonal antibodies (Santa Cruz Biotechnology) at 1:300 (β3) dilution for 1 h at room temperature. The filters were then washed with phosphate-buffered saline/Tween (PBST, 0.1% Tween 20). Afterward the filters were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) diluted 1:6000 in Western buffer for 1 h at room temperature. After washing with PBST, reactive bands were visualized with an ECL+ detection kit (Amersham Pharmacia Biotech). Slow activating delayed rectifier potassium currents (IKs currents) were measured in isolated guinea pig cardiomyocytes, and the effect of β-adrenergic stimulation with isoproterenol on IKs was investigated (Fig.1, A and B). Application of isoproterenol (10 μm) into the bath increased the IKs outward currents during depolarizing pulses and the IKs tail currents in the return pulses within 2 min. The increase in current amplitude was calculated on the IK tail current amplitude, as the most accurate way to determine IKs. The IKs tail current increase was between 53 and 401%, with a mean increase of 254 ± 53% (n = 6). The tail current amplitudes at −40 mV for control and after stimulation with isoproterenol are shown for a typical experiment in Fig. 1 C. When Xenopus oocytes were coinjected with cRNA for human MinK, KvLQT1, and cRNA for the human β3-adrenoreceptor, stimulation with isoproterenol (10 μm) increased β3/KvLQT1/MinK current amplitudes during depolarization and tail currents during repolarization (Fig. 1, D andF), similarly to IKs currents in the experiments with cardiomyocytes (Fig. 1, A and C). The increase in tail current amplitude of β3/KvLQT1/MinK currents was between 53 and 199% with a mean of 99 ± 12.7%, in four batches of oocytes (n = 3–5 oocytes each). The β3-adrenoreceptor-mediated increase of KvLQT1/MinK current was accompanied by a small shift of activation to more negative potentials from V12 = 39 ± 1.5 toV12 = 33.3 ± 1.1 mV (n = 6) upon stimulation with isoproterenol (10 μm). The slope of the activation curve and the deactivation time constant remained unchanged (τcontrol = 874 ± 28 ms,k control = 20.9 ± 1.9 mV versusτIsoproterenol = 821 ± 23 ms,k Isoproterenol = 18.7 ± 1.0 mV). The time course of the isoproterenol effect on KvLQT1/MinK currents showed fast kinetics. When isoproterenol was washed into the bath, the stimulating effect developed within 3–8 min and could be washed out almost completely within 20 min (Fig.2 A). In guinea pig cardiomyocytes, the onset of effect and washout occurred within a similar time scale of 1.5–2 min (data not shown). The increase in tail current amplitude by isoproterenol of β3/KvLQT1/MinK channels was calculated with a dose-response curve to be ED50 = 8 nm (n = 3–5 at each concentration) (Fig. 2 B). The ED50 of isoproterenol on IK tail currents in isolated guinea pig cardiomyocytes was similar (ED50 = 0.4 nm (n = 3–5)). Furthermore, we investigated the effect of different β3-cRNA concentrations (1–200 ng/μl) on the tail current stimulation with isoproterenol (10 μm). Different cRNA concentrations for β3 (1–200 ng/μl) did not alter basal tail current amplitudes without isoproterenol significantly (data not shown). During stimulation with isoproterenol (10 μm), a concentration dependence of the maximal increase in KvLQT/MinK current amplitude on the amount of injected β3-cRNA could be demonstrated (Fig. 2 C). With 1 ng/μl, the current increase by isoproterenol (10 μm) was 11.6 ± 11.5% (n = 3); with 100 ng/μl cRNA the current was increased by 110 ± 20% (n = 11), respectively. Fig. 2 C shows the dose-response curve for various concentrations of β3-adrenoreceptor cRNA. When KvLQT1/MinK without β3 was injected intoXenopus oocytes, the current amplitude was not changed significantly by isoproterenol (10 μm) in three injection series (Fig. 2 D). Western blot analysis was performed on Xenopus oocytes expressing β3-adrenoreceptors (Fig.3). In this experiment, only one single band was detected in Western blot analysis, which corresponds to the predicted molecular mass of ∼42 kDa from the deduced amino acid sequence of the β3-adrenoreceptor protein. The control with uninjected oocytes showed no band (Fig. 3). Thus, Western blot analysis demonstrate that after injection of cRNA, Xenopusoocytes are able to express relevant amounts of human β3-adrenoreceptor proteins in their membrane. This result was found in three Western blot analyses. Xenopus laevis oocytes are capable of expressing small amounts of endogenous β-adrenoreceptors during certain phases of their maturation. To exclude potential effects on endogenous β-adrenoreceptors, we did measurements with uninjected oocytes. Only minimal unspecific tail currents could be elicited, which were in the magnitude of ∼0.1 μA. Stimulation with isoproterenol (10 μm) for up to 15 min did not alter these minimal unspecific currents significantly in three injection series (n = 3–5 oocytes). It is known that Xenopusoocytes can express small numbers of endogenous ionic channels, which could have been a target for the β3-adrenoreceptors. To exclude this possibility, we injected cRNA for β3-adrenoreceptors alone (200 ng/μl) into oocytes. Again we recorded minimal and unspecific tail currents, which were not changed by isoproterenol (10 μm) in three injection series (n = 3–5 oocytes). We investigated whether G-proteins were involved in the signal transduction pathway between the β3-adrenoreceptor and the KvLQT1/MinK potassium channel by preincubating oocytes injected with β3/KvLQT1/MinK in dishes for h with a solution containing cholera toxin (2 as an activator of Gs proteins Annu. Rev. PubMed Scopus Google Scholar, D. J. Biol. Chem. Full Text PDF PubMed Google Scholar) toxin E. R. J. Physiol. 1998; PubMed Google Scholar) as an inhibitor of After the basal current amplitude and effect of current stimulation with isoproterenol were We found that the basal current amplitude of the β3/KvLQT1/MinK complex was increased significantly from ± (n = in control to ± (n = with cholera toxin and to 1.9 ± with toxin (n = (Fig. 4 A). The were A similar result was found in three injection We investigated the stimulation of current amplitude by isoproterenol (10 μm) in the control the cholera and the β3/KvLQT1/MinK oocytes. After of isoproterenol for 20 the current amplitude increase in the tail current was ± (n = in the control ± (n = in the cholera and ± (n = in the (Fig. 4 B). The of the increase in current amplitude by isoproterenol was The was not significantly different from the control This result that Gs proteins are involved in the coupling of the β3-adrenoreceptor to the KvLQT1/MinK potassium channel inXenopus oocytes. This the functional coupling of the human β3-adrenoreceptor to the human KvLQT1/MinK channel in theXenopus oocyte expression system. The current amplitude of KvLQT1/MinK currents was increased by stimulation with the This increase in current amplitude was similar to the increase in native IKs current in isolated guinea pig cardiomyocytes. The concentration dependence upon stimulation with isoproterenol was similar as channels an ED50 of 8 In guinea pig cardiomyocytes the ED50 was 0.4 The small in and onset of effect in Xenopus oocytes be to the membrane of the oocytes, which is known to be a for concentrations of isoproterenol for the effects demonstrate coupling of the β3-adrenoreceptor to the KvLQT1/MinK channel, indicating that the signal transduction cascade was The coupling of the β3-adrenoreceptor to the KvLQT1/MinK potassium channel This was demonstrated in experiments where we used cholera which basal current amplitudes of the β3/KvLQT1/MinK complex in to the control (Fig. 4 A). The increase in current amplitude by isoproterenol was significantly reduced by with cholera toxin (Fig. 4 B). The most explanation of the is that cholera toxin Gs proteins and the current amplitude of the β3/KvLQT1/MinK Application of isoproterenol to the cholera oocytes could not increase the current amplitude, Gs proteins have been by cholera proteins to a the can be in the The basal current amplitude was increased by toxin proteins were (Fig. 4 A). The current in the could be by isoproterenol Gs proteins are through the Gs protein toxin is known to the of the of the and activating the protein Annu. Rev. PubMed Scopus Google Scholar, T. Biol. 1993; 17: PubMed Scopus Google Scholar). evidence that the Gs are involved in the signal transduction cascade from β3-adrenoreceptor to the KvLQT1/MinK potassium To determine which of and of G-proteins are involved in the signal transduction cascade is very complex and In human cardiac myocytes, it has been demonstrated that β3-adrenoreceptors are and functional and that stimulation of these is responsible for a negative inotropic effect and a shortening of the human cardiac action potential (16Gauthier C. Tavernier G. Charpentier F. Langin D. Le Marec H. J. Clin. Invest. 1996; 98: 556-562Crossref PubMed Scopus (402) Google Scholar). It has been that this shortening is to an increase of a unknown potassium current (16Gauthier C. Tavernier G. Charpentier F. Langin D. Le Marec H. J. Clin. Invest. 1996; 98: 556-562Crossref PubMed Scopus (402) Google Scholar, 17Kaumann A.J. Molenaar P. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 355: 667-681Crossref PubMed Scopus (166) Google Scholar). demonstrate the functional coupling of the human KvLQT1/MinK potassium channel to the human β3-adrenoreceptor, a molecular explanation for the shortening of human cardiac action potential under adrenergic The coupling of the KvLQT1/MinK channel to the β3-adrenoreceptor have important for in the heart. Mutations in the and have been to be responsible for certain forms of the inherited long QT which can to triggered by sympathetic stress with a of catecholamines (12Roden D.M. Lazzara R. Rosen M. Schwartz P.J. Towbin J. Vincent M. Circulation. 1996; 94: 1996-2012Crossref PubMed Scopus (566) Google G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. J. M. C. 1993; Google Scholar, M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, J.P. H. J. PubMed Scopus Google Scholar). The role of β-adrenoreceptors in the genesis of long QT arrhythmias is by the that therapeutic β-blockade is in the of sudden cardiac death. stimulation of adrenergic can arrhythmias in cardiac The of cardiac is by two of accompanied by arrhythmias. is after stimulation of and a that is by E. Physiol. Rev. PubMed Scopus Google Scholar, A.J. Naunyn-Schmiedeberg's Arch. Pharmacol. 1993; PubMed Scopus Google Scholar). the effects of catecholamines to the KvLQT1/MinK as one repolarizing potassium current in the heart. The resulting and shortening of cardiac action potentials is to the repolarization arrhythmias. into the coupling of β3-adrenoreceptors to KvLQT1/MinK channels new for the and of cardiac arrhythmias. We A. M. Brown for the MinK R. J. for the β3-adrenoreceptor and M. T. Keating for the KvLQT1 We the of and S.