Vasopressin stimulates action potential firing by protein kinase C-dependent inhibition of KCNQ5 in A7r5 rat aortic smooth muscle cells

Abstract

[Arg⁸]-vasopressin (AVP), at low concentrations (10–500 pM), stimulates oscillations in intracellular Ca²⁺concentration (Ca²⁺spikes) in A7r5 rat aortic smooth muscle cells. Our previous studies provided biochemical evidence that protein kinase C (PKC) activation and phosphorylation of voltage-sensitive K⁺(K_v) channels are crucial steps in this process. In the present study, K_vcurrents ( I_Kv) and membrane potential were measured using patch clamp techniques. Treatment of A7r5 cells with 100 pM AVP resulted in significant inhibition of I_Kv. This effect was associated with gradual membrane depolarization, increased membrane resistance, and action potential (AP) generation in the same cells. The AVP-sensitive I_Kvwas resistant to 4-aminopyridine, iberiotoxin, and glibenclamide but was fully inhibited by the selective KCNQ channel blockers linopirdine (10 μM) and XE-991 (10 μM) and enhanced by the KCNQ channel activator flupirtine (10 μM). BaCl₂(100 μM) or linopirdine (5 μM) mimicked the effects of AVP on K⁺currents, AP generation, and Ca²⁺spiking. Expression of KCNQ5 was detected by RT-PCR in A7r5 cells and freshly isolated rat aortic smooth muscle. RNA interference directed toward KCNQ5 reduced KCNQ5 protein expression and resulted in a significant decrease in I_Kvin A7r5 cells. I_Kvwas also inhibited in response to the PKC activator 4β-phorbol 12-myristate 13-acetate (10 nM), and the inhibition of I_Kvby AVP was prevented by the PKC inhibitor calphostin C (250 nM). These results suggest that the stimulation of Ca²⁺spiking by physiological concentrations of AVP involves PKC-dependent inhibition of KCNQ5 channels and increased AP firing in A7r5 cells.