Effect of Cd2+ on Kv4.2 and Kv1.4 expressed in Xenopus oocytes and on the transient outward currents in rat and rabbit ventricular myocytes.

The aim of the present study was to compare the biophysical properties and Cd2+ sensitivity of Kv4.2 and Kv1.4 in Xenopus oocytes with those of native transient outward potassium currents in rat and rabbit ventricular myocytes. In Xenopus oocytes, Kv4.2 inactivated at hyperpolarized voltages (V(1/2)inact = -58.4 +/- 0.96 mV, n = 12) and recovered from inactivation rapidly (time constant = 224 +/- 23 ms, n = 3). Cd2+ induced large (approx. 30 mV with 500 microM Cd2+), concentration-dependent rightward shifts in Kv4.2 steady-state activation and inactivation. Kv1.4 inactivated over more depolarized voltages than Kv4.2 (V(1/2)inact = -49.3 +/- 1.4 mV, n = 12). Recovery from inactivation of Kv1.4 was dominated by a large slow component (time constant = 9,038 +/- 1,178 ms, n = 4). Cd2+ exerted only modest effects on Kv1.4 gating, with 500 microM Cd2+ shifting the voltage dependence of steady-state activation and inactivation by approximately 12 mV. We show that the biophysical properties and Cd2+ sensitivity of rat ventricular Ito resemble those of heterologously expressed Kv4.2. These findings support previous suggestions that Kv4.2 is an important molecular component of Ito in adult rat heart. In addition, our findings show that Ito in rabbit ventricular myocytes and Kv1.4-based currents in Xenopus oocytes share similar biophysical properties and sensitivity to Cd2+, suggesting that Kv1.4 may underlie Ito in rabbit ventricle. However, a number of discrepancies exist between the properties of native currents and their putative molecular counterparts, suggesting that additional proteins and/or modulatory factors may also play a role in determining the biophysical and pharmacological properties of these native currents.

Chloride channel inhibition blocks the protection of ischemic preconditioning and hypo-osmotic stress in rabbit ventricular myocardium.

The objective of this study was to examine the role of chloride (Cl-) channels in the myocardial protection of ischemic preconditioning (IP). Isolated rabbit ventricular myocytes were preconditioned with 10-minute simulated ischemia (SI) and 20-minute simulated reperfusion (SR) or not preconditioned (control). The myocytes then received 180-minute SI or 45-minute SI/120-minute SR. Indanyloxyacetic acid 94 (IAA-94, 10 micromol/L) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 1 micromol/L) was administered before IP or before SI or SI/SR to inhibit Cl- channels. Electrophysiological studies indicate that these drugs, at the concentrations used, selectively abolished Cl- currents activated under hypo-osmotic conditions (215 versus 290 mOsm). IP significantly (P<0.001) reduced the percentage of dead myocytes after 60-minute (30.8+/-1.3%, mean+/-SEM), 90-minute (35.3+/-1.3%), and 120-minute (39.2+/-1.7%) SI compared with controls (44.7+/-1.6%, 54.5+/-1.3%, and 58.9+/-1.8%, respectively) and after 45-minute SI/120-minute SR (36.3+/-0.6%) compared with control (56.6+/-2.2%). Hypo-osmotic stress also produced protection similar to IP. IAA-94 or NPPB abolished the protection of both IP and hypo-osmotic stress. In buffer-perfused rabbit hearts preconditioned with three 5-minute ischemia/10-minute reperfusion cycles given before the 40-minute long ischemia and 60-minute reperfusion, IP significantly (P<0.0001) reduced infarct size (IP+vehicle, 4.7+/-0.9%, versus control+vehicle, 26.6+/-3.3%; mean+/-SEM). Again, IAA-94 or NPPB abolished the protection of IP. Our results implicate Cl- channels in the IP protection of the myocardium against ischemic/reperfusion injury and demonstrate that hypo-osmotic stress is capable of preconditioning cardiomyocytes.

Preferential regulation of rabbit cardiac L-type Ca2+ current by glycolytic derived ATP via a direct allosteric pathway.

1. The activity of Ca2+ channels is regulated by a number of mechanisms including direct allosteric modulation by intracellular ATP. Since ATP derived from glycolysis is preferentially used for membrane function, we hypothesized that glycolytic ATP also preferentially regulates cardiac L-type Ca2+ channels. 2. To test this hypothesis, peak L-type Ca2+ currents (ICa) were measured in voltage-clamped rabbit cardiomyocytes during glycolytic inhibition (2-deoxyglucose + pyruvate), oxidative inhibition (cyanide + glucose) or both (full metabolic inhibition; FMI). 3. A 10 min period of FMI resulted in a 40.0 % decrease in peak ICa at +10 mV (-5.1 +/- 0.6 versus -3.1 +/- 0.4 pA pF-1; n = 5, P < 0.01). Similar decreases in peak ICa were observed during glycolytic inhibition using 2-deoxyglucose (-6.2 +/- 0.2 versus -3.7 +/- 0.2 pA pF-1; n = 5, P < 0.01) or iodoacetamide (-6.7 +/- 0.3 versus -3.7 +/- 0.2 pA pF-1; n = 7, P < 0.01), but not following oxidative inhibition (-6.2 +/- 0.4 versus -6.4 +/- 0.3 pA pF-1; n = 5, n.s.). The reduction in ICa following glycolytic inhibition was not mediated by phosphate sequestration by 2-deoxyglucose or changes in intracellular pH. 4. Reductions in ICa were still observed when inorganic phosphate and creatine were included in the pipette, confirming a critical role for glycolysis in ICa regulation. 5. With 5 mM MgATP in the pipette during FMI, peak ICa decreased by only 18.4 % (-6.8 +/- 0.6 versus -5.5 +/- 0.3 pA pF-1; n = 4, P < 0.05), while inclusion of 5 mM MgAMP-PCP (beta,gamma-methyleneadenosine 5'-triphosphate, Mg2+ salt) completely prevented the decrease in peak ICa (-6.9 +/- 0.3 versus -6.5 +/- 0.3 pA pF-1; n = 5, n.s.). 6. Together, these results suggest that ICa is regulated by intracellular ATP derived from glycolysis and does not require hydrolysis of ATP. This regulation is expected to be energy conserving during periods of metabolic stress and myocardial ischaemia.