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

1. K+ channels from the apical membrane of immortalized human kidney epithe lial (IHKE-1) cells were investigated in the cell-attached membrane configu ration as well as in excised membranes using the patch clamp technique. 2. In cell-attached membrane patches the open probability (P-o) of the K+ c hannel 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 pat ches the P-o 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 m M Ba2+ (cell-attached membrane: 78 +/- 8%, n = 9; excised: 80 +/- 4%, n = 2 6), 10 mM TEA(+) (excised inside-out: 48 +/- 5%, n = 34; excised outside-ou t: 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 n M. In the cell-attached membrane configuration the K+ channel was inhibited in a concentration-dependent manner by atrial natriuretic peptide (ANP). P -o was blocked equally well by 10 nM ANP (52 +/- 7%, n = 10), brain natriur etic 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 P-o of this K+ channel, by 70 +/- 10% (n = 5). 5. In excised membrane patches cGMP inhibited P-o of this K+ channel in a c oncentration-dependent manner. The first significant effects were measured at a concentration of 1 mu M (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 P-o of this K+ channel. ATP (0.1 mM) had a weak inhibitory effect (17 +/- 5%, n = 14). Addition of Mg-A TP to cGMP did not increase the inhibitory effect (30 +/- 4%, n = 14). KT58 23 (1 mu M), a specific inhibitor of cGMP-dependent protein kinases, did no t significantly alter the cGMP-induced reduction in P-o of the K+ channel i n three excised membrane patches. 6. The results present the first electrophysiological characterization of a mammalian K+ channel that is directly regulated by cGMP.