Ca2+ Dependent Inhibition of Cardiac Sodium Channel

Abstract

Cardiac sodium channels (Nav1.5) are principal molecular determinants responsible for myocardial conduction and maintenance of the cardiac rhythm. Intracellular calcium can trigger dual-mode regulation of the cardiac sodium channel but the mechanisms whereby intracellular Ca2+ may directly modulate Na+ channel function have yet to be identified.Whole-cell and perforated patch clamp experiments were performed to study the effects of NVP-1 on the hNav1.5. In the automated patch clamp Nav1.5 assay on the IonWorks Quattro system (perforated patch clamp mode), NVP-1 was inactive, producing only 13% inhibition of the Na+ current (INa) at 50 μM, while in conventional patch clamp experiments (in whole-cell patch clamp mode) it blocked the channel with an IC50 value of 5.45 μM. In conventional patch clamp experiments intracellular Ca2+ concentration ([Ca2+]i) was very low due to presence of 1 mM EGTA and no added Ca2+ in the intracellular solution. When [Ca2+]i was adjusted to 10 μM in whole-cell patch clamp mode and in perforated patch clamp experiments in which [Ca2+]i was near resting levels, NVP-1 had a small effect on the current (28% at 10uM). In perforated patch clamp experiments, when cells were incubated with BAPTA-AM (50 μM for 30 min) NVP-1 blocked the channel with an IC50 =7.2 μM. The inhibitory effect of amitriptyline was not dependent on [Ca2+]i and blocked the channel with an IC50 value of 1.2 μM in whole cell or perforated patch clamp experiments.Our data suggest that inhibition of Nav1.5 channels by NVP-1 is calcium dependent. It is proposed that interaction of NVP-1 with the channel involves the IQ-motif or CaMKII phosphorylation which is prevented in low [Ca2+]i. It is also possible that in low [Ca2+]i. NVP-1 has low affinity for binding or even can bind to EFL or IQ-motif instead of Ca2+/CaM/CaMKII.