Abstract 16845: Functional Regulation of Native Coronary Kv1 Channels by Pyridine Nucleotides

Abstract

Introduction: Recent studies indicate that vasodilation and enhanced blood flow to the heart following increases in myocardial O 2 demand are mediated by voltage-gated Kv1 channels in coronary smooth muscle cells (CVSM). Periods of high O 2 demand alter the intracellular pyridine nucleotide (PN) redox (e.g., elevated NADH:NAD + ), which, in heterologous systems, differentially modulates the gating properties of Kv1 channels by binding to intracellular Kvβ subunits. Hypothesis: Elevated levels of reduced PNs increase Kv1 channel activity in CVSM via the Kvβ complex. Methods: We performed patch clamp electrophysiology on CVSM freshly isolated from the left anterior descending coronary arteries (1 st and 2 nd order) of wild type and Kvβ2-null (Kvβ2 -/- ) mice. In the conventional whole cell configuration, we measured outward K + current density and the voltage-dependence of activation and inactivation (V 0.5, act, V 0.5,inact , respectively) in the absence and presence of extracellular lactate or pyruvate (10 mM), which alter intracellular PN levels. In inside-out membrane patches, we measured the open probability (nPo) of single K + -permeable channels that are sensitive to the Kv1 channel inhibitors 4-aminopyridine (4-AP; 1 mM) and psora-4 (500 nM), in the absence and presence of NADH (1 mM). Results: Treatment of wild type CVSM with lactate to elevate [NADH] I significantly increased whole cell Kv current density and shifted V 0.5, act to more negative membrane potentials (n=4, P<0.05). This response was effectively reversed upon increasing [NAD + ] i by treatment with pyruvate. Recordings performed in the inside-out configuration revealed K + currents that were sensitive to inhibition by psora-4 and 4-AP and had a unitary conductance of 70.2 ± 2.4 pS. Perfusion with a bath solution that contained 1 mM NADH lead to a significant increase (~2-fold) in the nPo of 4-AP-sensitive channels (n=25-30 cells, P<0.05). However, this effect was largely attenuated in membrane patches from Kvβ2 -/- CVSM. Conclusions: Native Kv1 channels in CVSM are functionally regulated by the intracellular NADH:NAD + ratio via their auxiliary Kvβ subunit complex.