Molecular and Functional Identification of Cyclic AMP-Sensitive BK Ca Potassium Channels (ZERO Variant) and L-Type Voltage-Dependent Calcium Channels in Single Rat Juxtaglomerular Cells

Abstract

This study aimed at identifying the type and functional significance of potassium channels and voltage-dependent calcium channels (Ca v ) in single rat JG cells using whole-cell patch clamp. Single JG cells displayed outward rectification at positive membrane potentials and limited net currents between −60 and −10 mV. Blockade of K + channels with TEA inhibited 83% of the current at +105 mV. Inhibition of K V channels with 4-AP inhibited 21% of the current. Blockade of calcium-sensitive voltage-gated K + channels (BK Ca ) with charybdotoxin or iberiotoxin inhibited 89% and 82% of the current, respectively. Double immunofluorescence confirmed the presence of BK Ca and renin in the same cell. cAMP increased the outward current by 1.6-fold, and this was inhibited by 74% with iberiotoxin. Expression of the cAMP-sensitive splice variant (ZERO) of BK Ca was confirmed in single-sampled JG cells by RT-PCR. The resting membrane potential of JG cells was −32 mV and activation of BK Ca with cAMP hyperpolarized cells on average 16 mV, and inhibition with TEA depolarized cells by 17 mV. The cells displayed typical high-voltage activated calcium currents sensitive to the L-type Ca v blocker calciseptine. RT-PCR analysis and double-immunofluorescence labeling showed coexpression of renin and L-type Ca v 1.2. The cAMP-mediated increase in exocytosis (measured as membrane capacitance) was inhibited by depolarization to +10 mV, and this inhibitory effect was blocked with calciseptine, whereas K + -blockers had no effect. We conclude that JG cells express functional cAMP-sensitive BK Ca channels (the ZERO splice variant) and voltage-dependent L-type Ca 2+ channels.