A novel cGMP‐regulated K+ channel in immortalized human kidney epithelial cells (IHKE‐1)

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Abstract
1 K+ channels from the apical membrane of immortalized human kidney epithelial (IHKE-1) cells were investigated in the cell-attached membrane configuration as well as in excised membranes using the patch clamp technique. 2 In cell-attached membrane patches the open probability (Po) of the K+ channel was 0.42 ± 0.06 (mean ± s.e.m., n= 22) and its conductance was 94 ± 5 pS with 145 mM K+ in the pipette (n= 25). In excised membrane patches the Po of the channel was 0.55 ± 0.03 (n= 86) and its conductance was 65 ± 2 pS (n= 68) with 145 mM K+ on one side of the membrane and 3.6 mM K+ on the other. The I-V curve of the K+ channel was not rectifying. 3 The channel was inhibited by several blockers of K+ channels such as 1 mM Ba2+ (cell-attached membrane: 78 ± 8 %, n= 9; excised: 80 ± 4 %, n= 26), 10 mM TEA+ (excised inside-out: 48 ± 5 %, n= 34; excised outside-out: 100 ± 0 %, n= 26), 0.1 mM verapamil (excised: 73 ± 9 %, n= 12), and 10 nM charybdotoxin (excised outside-out: 67 ± 9 %, n= 9). 4 The K+ channel was activated by depolarization and rising cytosolic Ca2+. Half-maximal activity occurred at a cytosolic Ca2+ concentration of 200 nM. In the cell-attached membrane configuration the K+ channel was inhibited in a concentration-dependent manner by atrial natriuretic peptide (ANP). Powas blocked equally well by 10 nM ANP (52 ± 7 %, n= 10), brain natriuretic peptide (BNP; 37 ± 11 %, n= 6) and C-type natriuretic peptide (CNP; 44 ± 13 %, n= 8). 8-Bromoguanosine 3′,5′ cyclic monophosphate (8-Br-cGMP, 0.1 mM) also inhibited Poof this K+ channel, by 70 ± 10 % (n= 5). 5 In excised membrane patches cGMP inhibited Po of this K+ channel in a concentration-dependent manner. The first significant effects were measured at a concentration of 1 μm (22 ± 7 %, n= 6), and greatest effects were obtained at 0.1 mM (34 ± 5 %, n= 15). cAMP (0.1 mM, n= 5) as well as GTP (0.1 mM, n= 5) had no significant effects on Po of this K+ channel. ATP (0.1 mM) had a weak inhibitory effect (17 ± 5 %, n= 14). Addition of Mg-ATP to cGMP did not increase the inhibitory effect (30 ± 4 %, n= 14). KT5823 (1 μm), a specific inhibitor of cGMP-dependent protein kinases, did not significantly alter the cGMP-induced reduction in Po of the K+ channel in three excised membrane patches. 6 The results present the first electrophysiological characterization of a mammalian K+ channel that is directly regulated by cGMP.