Study Of EGF-induced Regulation Of Delayed Rectifier Potassium Currents

The delayed rectifier potassium currents played a key role in myocardial repolarization, which were composed of two different elements: slowly delayed rectifier potassium currents (IKs) and rapidly delayed rectifier potassium currents (IKr).α-subunit encoded by KCNQ1 heteromultimericly coassembled withβ-subunit encoded by KCNE1 to form IKs channels. hERG encodesα-subunit of IKr. Both KCNQ1 and hERG subunit consisted of six putative transmembrane domains (S1~S6) and an ion-selective P-loop. Fourα-subunits formed the outer pore and contain the selectivity filter. KCNE1 protein consisted of a single transmembrane domain, and altered the biophysical properties of the channels. Mutations in either IKs subunits or IKr subunits were associated with variants of the congenital long QT syndrome. Mutant subunits led to reduction of IKs or IKr by a loss-of-function mechanism, often with a dominant-negative effect. In the heart, reduced IKs or IKr led to prolongation of the cardiac action potential, lengthening of the QT interval, and increased risk of arrhythmia. Thus, the delayed rectifier potassium channels were involved in maintaining normal action potential as well as inducing arrhythmia. The delayed rectifier potassium channels were targets of many modulatory mechanisms.The epidermal growth factor receptor (EGFR) belonged to subclass I of the superfamily of the receptor tyrosine kinases. It was composed of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain possessing PTK activity. Binding of epidermal growth factor resulted in EGFR dimerization and subsequent activation of the intrinsic tyrosine kinase activity. These phosphorylated tyrosines functioned as docking sites for a variety of signaling molecules that regulated membrane-proximal steps of signal transduction cascades that ultimately brought about cellular responses to EGFR ligands. The EGFR controled a wide variety of biological processes such as cell proliferation, differentiation, and migration and modulation of apoptosis.Ion channels were targets of many intracellular signaling pathways, including protein phosphorylation and dephosphorylation. Constitutive IKs recorded from guinea-pig ventricular myocytes was suppressed by broad-spectrum tyrosine kinase (TK) inhibitors tyrphostin A23, tyrphostin A25 and genistein. The phosphotyrosyl phosphatase inhibitor orthovanadate almost completely reversed the suppression of IKs induced by broad-spectrum TK inhibitors. Basal IKs was strongly dependent on tyrosine phosphorylation of IKs channel (or channel-regulatory) protein.In the present study, we used Xenopus oocytes as expression system to observe effect of activation of EGFR on KCNQ1 alone, or KCNQ1/KCNE1 as well as hERG currents, and the mechanism of these effects. Besides this, we studied the effect of EGF on action potential of guinea pig papillary muscles to study the physiological significance of EGFR-mediated modulation of IKs and IKr.1. The characteristics of KCNQ1 and KCNQ1/KCNE1 currentsAim: To observe the characteristics of KCNQ1 current and KCNQ1/KCNE1 currents expressed in Xenopus oocyte.Methods: (1) Preparation of cRNA: KCNQ1 and KCNE1 cDNA were subcloned respectively into the pGEMHE plasmid vector. The sequences of all constructs were confirmed by sequencing. The plasmid DNA was amplified and extracted from E coli transformed with constructs. The plasmid was lineared by NheⅠrestriction endonuclease and purified with DNA fragment purification kit (TakaRa). All cRNAs were synthesized in vitro using RNA production systems-T7 kit (Promega), and were quantified after purification with RNA clean kit (Tiangen). (2) Preparation of Xenopus oocytes: Oocytes were surgically removed under iced anesthesia and placed into OR2 solution (mmol·L-1: NaCl 82.5,KCl 2,MgCl2 1,HEPES 5, pH7.4)containing 2g.L-1 collagenase and were rocked for 1.5 to 2 hours. (3) StageⅤ-Ⅵoocytes were selected and injected with cRNA containing 5ng KCNQ1 with or without 1ng KCNE1 and were incubated at 18℃in ND96 solution (mmol·L-1:NaCl 96, KCl 1, CaCl2 1.8, MgCl2 1, HEPES 5, pH 7.4) plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique.Results: (1) The lineared plasmid DNA ran slower than the circular plasmid in agarose electrophoresis. Compared with DNA marker, KCNQ1 and KCNE1 plasmid DNA were 5Kb and 3.4Kb, respectively. KCNQ1 and KCNE1 cRNA were verified by agarose electrophoresis. (2) KCNE1 increased KCNQ1 current amplitude. The tail current of KCNQ1 at -50mV following by repolarization to 40mV was (0.09±0.01)μA, whereas KCNQ1/KCNE1 currents at same voltage was (0.39±0.03)μA (P<0.01, n=6). Thus, KCNE1 increased KCNQ1 current over fourfold. (3) KCNE1 altered kinetics characteristics of KCNQ1 current. From a holding potential of -80mV, oocytes were depolarized for 4 s to test potentials between -60mV to 50mV in 10mV steps followed by repolarization to -50mV. Normalized tail current-voltage curves were fitted with Boltzmann equation, and the half activation voltage (V1/2) of KCNQ1 and KCNQ1/KCNE1 currents were (-22.8±1.7) mV and (30.9±0.8) mV, respectively. Thus, KCNE1 shifted KCNQ1 activation voltage to more positive potentials. The activated current trace at +30mV and the tail current trace at -50mV following depolarization to +30mV were fitted with monoexponential function to acquire the time constants of activation and deactivation, respectively. The activation time constants of KCNQ1/KCNE1 was increased significantly to (3.84±0.52s), from (0.89±0.05s) of KCNQ1 alone (P<0.01), suggesting KCNE1 slowed KCNQ1 current activation. The deactivated time constants of KCNQ1/KCNE1 (1.01±0.02s) was lowered significantly, compared with that of (2.29±0.39s) of KCNQ1 alone, suggesting KCNE1 accelerated KCNQ1 current deactivation. KCNQ1 tail currents display a"hook", indicating that KCNQ1 inactivates to some extent. KCNE1 eliminated inactivation of KCNQ1 current. (4) Coexpression of KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B, a selective blocker of IKs. The KCNQ1 current inhibition rates with or without KCNE1 by Chromanol 293B were (79.9±0.6)% and (65.5±5.8)%.Conclusion: KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B. 2. Effect of EGF on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes.Aim: To investigate the effect of activation of EGFR on KCNQ1 and KCNQ1/KCNE1 currents expressed in Xenopus oocytes, and to explore the underlying mechanism. To observe the effect of EGF on action potentials of guinea pig papillary muscles.Methods: A full-length KCNQ1, KCNE1, EGFR cDNA were subcloned into the pGEMHE plasmid vector, respectively. Complementary RNAs (cRNAs) from all contructs were prepared in vitro from NheⅠ-lineared DNA templates using RibomaxTM large scale RNA production systems-T7 kit. cRNAs size and concentration were estimated by comparison with RNA ladder in agarose gel electrophoresis following purification of cRNAs. StageⅤ-Ⅵoocytes were injected with cRNA containing 5ng KCNQ1, 5ng EGFR with or without 1ng KCNE1 and incubated at 18℃in ND96 solution plus 2.5 mM pyruvic acid and 50mg.L-1 gentamicin. Currents were recorded at room temperature 2-6d after injection using two-microelectrode voltage-clamp technique. The effect of EGF on action potentials from guinea pig right ventricular papillary muscle was observed with a conventional intracellular recording technique. Site-directed mutagenesis and immunoprecipitation technique were used to explore mechanism underlying effect of activation of EGFR on KCNQ1/KCNE1 currents.Results: (1) EGF suppressed KCNQ1/KCNE1 currents amplitudes in a dose-dependent manner. EGF at 3,10,30,100,300ng/ml inhibited KCNQ1/KCNE1 currents by (6.0±2.1)%, (20.8±3.4)%, (38.9±2.7)%, (57.3±1.5)%, and (65.9±2.8)%, respectively; The concentration for half maximal inhibition (IC50) was (24.1±1.8)ng/ml. (2) EGF at 100ng/ml increased KCNQ1 current. KCNQ1 current before and after EGF perfusion were (0.27±0.02)μA and (0.42±0.04)μA, respectively. (3) EGF voltage-dependently suppressed KCNQ1/KCNE1 currents amplitudes at membrane potentials between 0 and +90mV, with increased voltages, inhibition decreased. (4) EGF altered kinetics characteristic of KCNQ1/KCNE1 currents. EGF at 100ng/ml shifted V1/2 of KCNQ1/KCNE1 currents activation by 7mV toward more positive potentials, and significantly increased the activating time constant at 10mV (from 4.8±0.6s to 7.1±0.7s), increased slow deactivation time constant (from 1063±102ms to 1251±138ms), and fast deactivation time constant (from 339±34ms to 429±28ms), indicating EGF slowed activation and deactivation of KCNQ1/KCNE1 currents. (5) The signaling associated with effect of EGF on KCNQ1/KCNE1 currents was analyzed. PP2(200nM), a blocker of Src kinase, and Ca2+ chelator EGTA(5μM) did not affect EGF-induced inhibition of KCNQ1/KCNE1 currents. Genistein(200μM), an inhibitor of tyrosine kinase, eliminated the effect of EGF on KCNQ1/KCNE1 currents(from 43.8±4.2% to 4.8±2.9%), suggesting tyrosine phosphorylation of channel or channel modulator was involved. (6) The mechanism of EGF-induced activation of KCNQ1 current was analyzed. EGTA(5μM) or PLC inhibitor U73122(3μM) reduced EGF-induced activation of KCNQ1 current (from 66±2.0% to 25±1.8%, from 76±10% to 26±9%, respectively), indicating involvement of PLC-PIP2-IP3-Ca2+ signaling. (7) Mutation of KCNE1 (Y81A) or (S102A) did not affect EGF-induced modulation of KCNQ1/KCNE1 currents. However KCNQ1/KCNE1(Y81A) became insensitive to Chromanol 293B inhibition. KCNQ1/KCNE1(Y81A) currents were only inhibited by 38.3±8.7% by Chromanol 293B, as compared with a 69.9±2.6% inhibition for wild type KCNQ1/KCNE1. KCNE1(Y81A) also altered activation kinetics, shifting activation potentials to more positive direction. KCNQ1/KCNE1(Y81A) currents did not activate until a membrane depolarization to +40mV. These results suggested tyrosine in KCNE1 81 was important in modulating KCNQ1/KCNE1 currents amplitude and gating. (8) EGF lengthened the action potentials repolarization of guinea pig papillary. EGF at 100ng/ml increased APD50 (from 127±21ms to 148±24ms) and APD90 (from 189±16ms to 205±13ms), slowed the maximal rate of rise of AP (from 149±28V/s to 111±30V/s), suggesting EGF inhibited both delayed rectifier K+ channels and Na+ channel. (9) Results of immunoprecipitation showed EGF increased tyrosine phosphorylation of KCNE1, but did not affect tyrosine phosphorylation of KCNQ1, suggesting KCNQ1/KCNE1 currents amplitude and gating were modulated through tyrosine phosphorylation of KCNE1Conclusion: Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance. 3. Effect of EGF on hERG channel current expressed in Xenopus oocytesAim: To investigate the effect of activation of EGFR on hERG current expressed in Xenopus oocytes.Methods: EGFR cDNA was subcloned into the pGEMHE plasmid vector. EGFR cRNA was prepared in vitro from NheⅠ-lineared DNA template using RibomaxTM large scale RNA production systems-T7 kit. hERG cDNA was subcloned into pSP64 vector. hERG cRNA was synthesized with RibomaxTM large scale RNA production systems-SP6 kit from EcoRⅠ-lineared DNA template. Mixture of 5ng EGFR cRNA and 5ng hERG cRNA were injected into oocytes. Effect of activation of EGFR on hERG current was analyzed using two electrode voltage-clamp technique.Results: hERG current possessed characteristic of voltage-dependent fast activating and fast inactivating outward current sensitive to IKr blocker E-4031. (1) hERG current was elicited using a protocol in which the potential was first held at -80mV and then depolarized to 0mV followed by repolarization to -50mV. Tail current was measured upon repolarization to -50mV. EGF at 100ng/ml decreased hERG tailed current from(0.44±0.07)μA to (0.28±0.04)μA. hERG tailed current recovered completely after washout of EGF. Thus, EGF inhibited reversibly hERG current. (2) Voltage-dependency of EGF action on hERG was tested in voltages from -70mV to 38mV. The results showed EGF suppressed hERG current in a voltage-dependent manner from -34mV to 14mV. (3) EGF shifted V1/2 of hERG current about 15 mV towards more positive potentials (from -8.0±0.6 mV to 6.7±0.8 mV). (4) EGF lengthened activation time constant of hERG current from (433±85)ms to (574±183)ms, suggesting EGF slowed activation of hERG current. EGF decreased deactivation time constant from (1500±224)ms to (810±138)ms, suggesting EGF promoted deactivation hERG current.Conclusion: Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation.4. Effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed in Xenopus oocytesAim: To investigate the effect of dipfluzine on KCNQ1/KCNE1 potassium currents expressed heterologously in Xenopus oocytes.Methods: Using Xenopus oocytes expression system, the current amplitude and kinetic characteristics of KCNQ1/KCNE1 were measured with the two electrode voltage-clamp technique before and after dipfluzine application.Results: Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. Dipfluzine at 0.3, 1, 3, 10, 30μmol·L-1 inhibited KCNQ1/KCNE1 currents by 6.0±0.9%, 11.6±0.8% , 25.7±2.9%, 45.6±2.5%, 63.5±1.6%, respectively. IC50 was 8.9±1.8μmol·L-1. The solvent (DMSO, 0.1% ) did not affect KCNQ1/KCNE1 currents. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant (from 3.7±0.6 s to 5.0±0.5 s), slowed KCNQ1/KCNE1 currents activation. Dipfluzine at 10μmol·L-1 significantly decreased the slow and fast deactivating time constants(from 1135±91 ms to 879±78 ms and 368±27ms to 313±45ms, respectively), enhanced KCNQ1/KCNE1 currents deactivation.Conclusion: Dipfluzine concentration-dependently and voltage- dependently inhibited KCNQ1/KCNE1 currents and modified kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.SUMMARY1. KCNQ1 current exhibited a rapidly activating, slow deactivating, partly inactivating characteristics. KCNE1 dramatically modulated KCNQ1 gating, slowing activation, removing inactivation, and shifting the voltage dependence of activation to more positive potentials. KCNE1 plus KCNQ1 had fourfold greater current amplitudes than KCNQ1 alone. KCNE1 increased sensitivity of KCNQ1 current to Chromanol 293B.2. Activation of EGFR had different effects on KCNQ1 current and KCNQ1/KCNE1 currents. EGF-induced activation of KCNQ1 current was possibly through PLC-PIP2-IP3-Ca2+ signaling pathway. EGF-induced inhibition of KCNQ1/KCNE1 currents was possibly mediated through tyrosine phosphorylation of KCNE1. EGF lengthened action potential repolarizion in guinea pig papillary. This would indicate modulation of delayed rectifier K+ channels by EGF may have physiological significance.3. Activation of EGFR inhibited hERG current amplitude and altered kinetics characteristics, shifting the curve of voltage-dependent activation to the right and slowing the activation and promoting the deactivation. Effect of EGF on action potential repolarization may involve modulation of hERG channel besides KCNQ1/KCNE1 channel.4. Dipfluzine concentration-dependently inhibited KCNQ1/KCNE1 currents. IC50 was (8.9±1.8)μmol·L-1. Dipfluzine-induced inhibition of KCNQ1/KCNE1 currents was voltage dependent at membrane potentials between -10 and 90 mV. Dipfluzine at 10μmol·L-1 shifted V1/2 of KCNQ1/KCNE1 currents activation by 3mV toward more positive potentials, and significantly increased the activating time constant, and decreased the slow and fast deactivating time constant. Dipfluzine inhibited KCNQ1/KCNE1 currents amplitudes and modifies kinetic characteristics of KCNQ1/KCNE1 activation and deactivation, which might be correlated with its antiarrhythmic effect.