Regulation of the Hyperpolarization-activated K + Channel in the Lateral Membrane of the Cortical Collecting Duct

An intermediate-conductance K + channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K + channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCI in the pipette and 5 mM KC1, 140 mM NaCI (NaCI Ringer's solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KC1 solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/ Na + was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann's equation: Po = 1/[1 + e x p ( V V1/i)zF/R~, with z = 1.2 and V1/2 = 4 0 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K + channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKA~; 20 U/ml) and 100 mM MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKA, increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K + channel in the basolateral membrane of CCD. I N T R O D U C T I O N The cortical collecting duct (CCD) is a neph ron segment impor tan t for K § secretion and hormoneregula ted Na + reabsorpt ion (Stanton and Giebisch, 1992; Breyer and Ando, 1994). Two types of cells, principal and intercalated cells, have been identified in the CCD (O'Neil and Hayhurst , 1985). It is well established that This work was performed with the technical support of Ming Lu. Address correspondence to Wen-Hui Wang, Department of Pharmacology, BSB Room 501, New York Medical College, Valhalla, NY 10595. J. GEN. PHYSIOL. @ The Rockefeller University Press 9 0022-1295/95/07/25/19 $2.00 Volume 106 July 1995 25--43 25 on A ril 5, 2016 jgp.rress.org D ow nladed fom Published July 1, 1995