Biomedical subjects

Shimin Wang

Publications and source records attributed to Shimin Wang.

9 recordsLinked to original sources

PubMed2007

W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition.

Ca(+)-calmodulin (Ca(2+)-CaM)-dependent protein kinase II (Ca(2+)/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca(2+)-occupied CaM, and KN-93, an inhibitor of Ca(2+)/CaMKII, on the K(v)4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC(50) of 92.4 muM. The block was voltage dependent, with an effective electrical distance of 0.18 +/- 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of K(v)4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 muM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated K(v)4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 muM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca(2+)/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca(2+)-CaM, as well as a change in activity of Ca(2+)-CaM-dependent enzymes, including Ca(2+)/CaMKII.

Animals↗

PubMed2006

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4.

Voltage-gated K(+) channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K(+) currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At -60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K(+) channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K(+) channels.

Animals↗

PubMed2005

Time- and voltage-dependent components of Kv4.3 inactivation.

Kv4.3 inactivation is a complex multiexponential process, which can occur from both closed and open states. The fast component of inactivation is modulated by the N-terminus, but the mechanisms mediating the other components of inactivation are controversial. We studied inactivation of Kv4.3 expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Inactivation during 2000 ms pulses at potentials positive to the activation threshold was described by three exponents (46 +/- 3, 152 +/- 13, and 930 +/- 50 ms at +50 mV, n = 7) whereas closed-state inactivation (at potentials below threshold) was described by two exponents (1079 +/- 119 and 3719 +/- 307 ms at -40 mV, n = 9). The fast component of open-state inactivation was dominant at potentials positive to -20 mV. Negative to -30 mV, the intermediate and slow components dominated inactivation. Inactivation properties were dependent on pulse duration. Recovery from inactivation was strongly dependent on voltage and pulse duration. We developed an 11-state Markov model of Kv4.3 gating that incorporated a direct transition from the open-inactivated state to the closed-inactivated state. Simulations with this model reproduced open- and closed-state inactivation, isochronal inactivation relationships, and reopening currents. Our data suggest that inactivation can proceed primarily from the open state and that multiple inactivation components can be identified.

Animals↗

PubMed2005

Angiotensin II-induced increase of T-type Ca2+ current and decrease of L-type Ca2+ current in heart cells.

The effect of angiotensin II (Ang II) on the T- and L-type calcium currents (I(Ca)) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10(-7)M) increased the T-type calcium current and decreased the L-type I(Ca). The effect of Ang II on both types of currents was blocked by the AT1 peptidic antagonist, [Sar1, Ala8] Ang II (2 x 10(-7)M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca2+ currents differently, and this effect seems to be mediated by the PKC pathway.

Angiotensin II↗

PubMed2004

Activation properties of Kv4.3 channels: time, voltage and [K+]o dependence.

Rapidly inactivating, voltage-dependent K(+) currents play important roles in both neurones and cardiac myocytes. Kv4 channels form the basis of these currents in many neurones and cardiac myocytes and their mechanism of inactivation appears to differ significantly from that reported for Shaker and Kv1.4 channels. In most channel gating models, inactivation is coupled to the kinetics of activation. Hence, there is a need for a rigorous model based on comprehensive experimental data on Kv4.3 channel activation. To develop a gating model of Kv4.3 channel activation, we studied the properties of Kv4.3 channels in Xenopus oocytes, without endogenous KChIP2 ancillary subunits, using the perforated cut-open oocyte voltage clamp and two-electrode voltage clamp techniques. We obtained high-frequency resolution measurements of the activation and deactivation properties of Kv4.3 channels. Activation was sigmoid and well described by a fourth power exponential function. The voltage dependence of the activation time constants was best described by a biexponential function corresponding to at least two different equivalent charges for activation. Deactivation kinetics are voltage dependent and monoexponential. In contrast to other voltage-sensitive K(+) channels such as HERG and Shaker, we found that elevated extracellular [K(+)] modulated the activation process by slowing deactivation and stabilizing the open state. Using these data we developed a model with five closed states and voltage-dependent transitions between the first four closed states coupled to a voltage-insensitive transition between the final closed (partially activated) state and the open state. Our model closely simulates steady-state and kinetic activation and deactivation data.

Amino Acid Sequence↗

PubMed2003

Kv1.4 channel block by quinidine: evidence for a drug-induced allosteric effect.

We studied quinidine block of Kv1.4DeltaN, a K(+) channel lacking N-type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non-additively) by interventions that slow C-type inactivation: [K(+)](o) elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ~10-fold, but left the effective electrical distance unchanged at ~0.15. Following rapid open channel block, there were time-dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time-dependent aspect of quinidine block was similar to recovery from normal C-type inactivation. Manipulations that prevented the channel from entering the C-type inactivated state (i.e. high [K(+)](o) or the K532Y mutation) prevented the development of the time-dependent quinidine-induced inactivation. The concentration dependence of the rapid block and the time-dependent quinidine-induced inactivation were similar, but the time-dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage-sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C-type inactivated state in a time- and voltage-dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C-type inactivated state through a voltage-dependent conformational change.

Allosteric Regulation↗

PubMed2003

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells.

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.

Aniline Compounds↗

PubMed2003

Effects of BmkTXK beta on electrophysiological properties of rabbit atrial myocytes.

OBJECTIVE: To investigate the effects of BmkTXK(beta), a newly purified 'long chain' peptide inhibitor of K(+) channels from the Chinese scorpion Buthus martensi Karsch (BmK), on the electrophysiological properties of isolated rabbit atrial myocytes. METHODS: The standard whole-cell patch-clamp technique was used to study the effects of multiple concentrations of BmkTXK(beta) on potassium currents and action potentials. RESULTS: BmkTXK(beta) produced concentration-dependent prolongation of action potential duration at 20%, 50%, and 90% repolarization (APD(20,50,90)) without any use-dependence. Meanwhile, it had no significant effect on RMP, APA, or V(max) (n = 9). At a dose of 1 micro mol/L, BmkTXK(beta) decreased I(to) by 41.4% (n = 10, P < 0.01) at a membrane potential of +50 mV [from (13.63 +/- 0.87) pA/pF to (7.98 +/- 0.78) pA/pF]. I(to) was reduced significantly with an IC(50) value of 1.82 micromol/L (95% confidence interval: 1.47 - 2.17 micro mol/L), in a clear concentration-dependent manner. BmkTXK(beta) blocked I(Ks) and I(Ks),tail with an IC(50) of 20.15 micromol/L and a 95% confidence interval of 16.93 - 23.37 micromol/L. At a concentration of 10 micromol/L, BmkTXK(beta) blocked both I(Ks) (mean reduction 37.3% +/- 4.2%, P < 0.01, n = 7) and I(Ks), tail (mean reduction 35.8% +/- 4.1%, P < 0.01, n = 7). At 0 mV, 10 micromol/L BmkTXK(beta) inhibited both I(Kr) (mean reduction 40.5% +/- 2.6%, P < 0.01, n = 6) and I(Kr), tail (mean reduction 42.3% +/- 2.9%, P < 0.01, n = 6). Blocking of I(Kr) by BmkTXK(beta) occurred in a concentration-dependent manner, with an IC(50) of 17.21 micromol/L (95% confidence interval: 14.76-19.66 micromol/L). An absence of effects on I(K1) was observed for BmkTXK(beta), with no change in reversal-potential (n = 6, P > 0.05). CONCLUSIONS: BmkTXK(beta) exerts direct blocking effects on several potassium channels involved in cardiac repolarization, and has a strong effect on prolonging the repolarization of rabbit cardiomyocytes without reverse frequency dependence. This finding suggests that BmkTXK(beta) could be a promising class III drug for anti-arrhythmic therapy without the risk of proarrhythmia.

Action Potentials↗

PubMed2002

Kinetic properties of Kv4.3 and their modulation by KChIP2b.

KChIPs are a family of Kv4 K(+) channel ancillary subunits whose effects usually include slowing of inactivation, speeding of recovery from inactivation, and increasing channel surface expression. We compared the effects of the 270 amino acid KChIP2b on Kv4.3 and a Kv4.3 inner pore mutant [V(399, 401)I]. Kv4.3 showed fast inactivation with a bi-exponential time course in which the fast time constant predominated. KChIP2b expressed with wild-type Kv4.3 slowed the fast time constant of inactivation; however, the overall rate of inactivation was faster due to reduction of the contribution of the slow inactivation phase. Introduction of [V(399, 401)I] slowed both time constants of inactivation less than 2-fold. Inactivation was incomplete after 20s pulse durations. Co-expression of KChIP2b with Kv4.3 [V(399, 401)I] slowed inactivation dramatically. KChIP2b increased the rate of recovery from inactivation 7.6-fold in the wild-type channel and 5.7-fold in Kv4.3 [V(399,401)I]. These data suggest that inner pore structure is an important factor in the modulatory effects of KChIP2b on Kv4.3 K(+) channels.

Animals↗