BASOLATERAL K-CHANNEL ACTIVATED BY CARBACHOL IN THE EPITHELIAL-CELL LINE T-84

Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the e lectrical,oradient ent favoring apical Cl efflux and allows K to recyc le at the basolateral membrane. We have used transepithelial short-cir cuit current (I-sc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T-84 cells. Carbachol had little effect on I-sc when added alone but produced large, transient currents if added to monolayers prestimulate d with cAMP. cAMP also enhanced the subsequent I-sc response to calciu m ionophores. Carbachol (100 mu M) transiently elevated intracellular free calcium ([Ca2+](i)) by similar to 3-fold in confluent cells cultu red on glass coverslips with a time course resembling the I-sc respons e of confluent monolayers that had been grown on porous supports. In p arallel patch clamp experiments, carbachol activated an inwardly recti fying potassium channel on the basolateral aspect of polarized monolay ers which had been dissected from porous culture supports. The same ch annel was transiently activated on the surface of subconfluent monolay ers during stimulation by carbachol. Activation was more prolonged whe n cells were exposed to calcium ionophores. The conductance of the inw ard rectifier in cell-attached patches was 55 pS near the resting memb rane potential (-54 mV) with pipette solution containing 150 mM KCI (3 7 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCI solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na much greater than Cs based on permeability ratios un der bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively inse nsitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, a nd was unaffected by external 10 mM barium. It is referred to as the K -BIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single K-BIC channels, the c arbachol-stimulated I-sc was relatively insensitive to several blocker s on the basolateral side and was unaffected by barium. These comparis ons between the properties of the macroscopic current and single chann els suggest that the K-BIC channel mediates basolateral membrane K con ductance in T-84 cell monolayers during stimulation by cholinergic sec retagogues.