REGULATION OF AN INWARDLY RECTIFYING K-CHANNEL IN THE T-84 EPITHELIAL-CELL LINE BY CALCIUM, NUCLEOTIDES AND KINASES

Agonists that elevate calcium in T-84 cells stimulate chloride secreti on by activating K-BIC an inwardly rectifying K channel in the basolat eral membrane. We have studied the regulation of this channel by calci um, nucleotides and phosphorylation using patch clamp and shore-circui t current (I-SC) techniques. Open probability (P-o) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 mu M-5 mM), ATP (0.1 mM) + protein kin ase A (PKA; 180 nM), or is obutylmethylxanthine (IBMX; 1 mM). Analysis of event durations suggest ed that the channel has at least two open and two closed states, and t hat rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or A DP. Activity was further enhanced when PKA was added in the presence o f MgATP, but only if free calcium concentration was elevated (400 nM). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical memb rane using alpha-toxin, Finally, protein kinase C (PKC) inhibited sing le K-BIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phospho rylation by PKA and PI(C modulate the responsiveness of the inwardly r ectifying K channel to Ca-mediated secretagogues.