|
pubmed:abstractText |
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 microM (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 microM), 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.
|
|
pubmed:affiliation |
Westfalische Wilhelms-Universität Münster, Medizinische Poliklinik, Experimentelle Nephrologie, Domagkstrasse 3a, D-48149 Münster, Germany. hirschi@uni-muenster.de
|
