Basolateral K channel activated by carbachol in the epithelial cell line T84.
Cholinergic stimulation of chloride secretion involves the activation of a basolateral membrane potassium conductance, which maintains the electrical gradient favoring apical Cl efflux and allows K to recycle at the basolateral membrane. We have used transepithelial short-circuit current (Isc), fluorescence imaging, and patch clamp studies to identify and characterize the K channel that mediates this response in T84 cells. Carbachol had little effect on Isc when added alone but produced large, transient currents if added to monolayers prestimulated with cAMP. cAMP also enhanced the subsequent Isc response to calcium ionophores. Carbachol (100 microM) transiently elevated intracellular free calcium ([Ca2+]i) by approximately 3-fold in confluent cells cultured on glass coverslips with a time course resembling the Isc response of confluent monolayers that had been grown on porous supports. In parallel patch clamp experiments, carbachol activated an inwardly rectifying potassium channel on the basolateral aspect of polarized monolayers which had been dissected from porous culture supports. The same channel was transiently activated on the surface of subconfluent monolayers during stimulation by carbachol. Activation was more prolonged when cells were exposed to calcium ionophores. The conductance of the inward rectifier in cell-attached patches was 55 pS near the resting membrane potential (-54 mV) with pipette solution containing 150 mM KCl (37 degrees C). This rectification persisted when patches were bathed in symmetrical 150 mM KCl solutions. The selectivity sequence was 1 K > 0.88 Rb > 0.18 Na >> Cs based on permeability ratios under bi-ionic conditions. The channel exhibited fast block by external sodium ions, was weakly inhibited by external TEA, was relatively insensitive to charybdotoxin, kaliotoxin, 4-aminopyridine and quinidine, and was unaffected by external 10 mM barium. It is referred to as the KBIC channel based on its most distinctive properties (Ba-insensitive, inwardly rectifying, Ca-activated). Like single KBIC channels, the carbachol-stimulated Isc was relatively insensitive to several blockers on the basolateral side and was unaffected by barium. These comparisons between the properties of the macroscopic current and single channels suggest that the KBIC channel mediates basolateral membrane K conductance in T84 cell monolayers during stimulation by cholinergic secretagogues.
Tabcharani
J A
JA
Department of Physiology, McGill University, Montreal, Quebec, Canada.
Harris
R A
RA
Boucher
A
A
Eng
J W
JW
Hanrahan
J W
JW
D015761:4-Aminopyridine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D018999:Charybdotoxin; D003110:Colonic Neoplasms / Q000473:pathology* / Q000503:physiopathology* / Q000648:ultrastructure; D000242:Cyclic AMP / Q000494:pharmacology; D004848:Epithelium / Q000737:chemistry / Q000473:pathology / Q000648:ultrastructure; D005453:Fluorescence; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000032:analysis / Q000187:drug effects / Q000502:physiology*; D011802:Quinidine / Q000494:pharmacology; D012604:Scorpion Venoms / Q000494:pharmacology; D013696:Temperature; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured
D015221:Potassium Channels; D012604:Scorpion Venoms; D013757:Tetraethylammonium Compounds; C072276:kaliotoxin; D018999:Charybdotoxin; D000001:Calcimycin; D015759:Ionomycin; D019789:Tetraethylammonium; D002217:Carbachol; D015761:4-Aminopyridine; D000242:Cyclic AMP; D011802:Quinidine