Intracellular calcium andpH regulate basolateral potassium and chloride channels in colonic epithelial cells.

Abstract

Intracellular calcium and pH are cellular regulators that play a major role in the modulation of basolateral ion channels in absorptive epithelial cells. To define the mechanisms underlying the actions of these cellular messengers, a digitonin-permeabilized epithelial cell layer from turtle colon was developed. Exposure of apical membranes to digitonin provided access to and control over the cell interior by chemically removing the apical membranes of the cells as a permeability barrier. The degree of apical membrane permeabilization induced by digitonin treatment permitted the introduction of organic buffer systems to clamp cell calcium and pH at specified values. Initial studies employed the chelator EGTA at a pH of 6.6 in the mucosal bathing solutions to optimize the calcium-buffering properties of EGTA. Raising cytosolic calcium from the nanomolar to the micromolar range activated basolateral conductances for K$\sp+$ and Cl$\sp-$. Differences in ion selectivity, blocker specificity, calcium activation kinetics and divalent cation activation selectivity indicated that these calcium-activated conductances represent two separate populations of ion channels. This permeabilized preparation offers a means for the identification of macroscopic currents that are due to presumed Ca$\sp{2+}$-activated single channels and provides a model system for the functional reconstitution of channel regulatory mechanisms. To examine the role of intracellular pH as a modulator of basolateral K$\sp+$ and Cl$\sp-$ conductances, the pH-insensitive chelator BAPTA was used to buffer internal calcium in digitonin-permeabilized cells. Increasing the intracellular pH from 6.6 to 8.0 enhanced the sensitivity of both ionic conductances to activation by intracellular calcium, but changing extracellular pH had no effect. Maximal K$\sp+$ and Cl$\sp-$ currents activated by calcium were not affected by changes in intracellular pH. Hill analysis of the calcium activation process revealed that raising the cytosolic pH from 6.6 to 8.0 reduced the K$\sb{1/2}$ for calcium activation and increased the Hill coefficient. In the absence of calcium, changes in intracellular pH did not have a significant effect on the K$\sp+$ and Cl$\sp-$ conductances. These results are consistent with the notion that changes in pH modify the action of calcium perhaps by acting at the activation site to provide a "variable gain control" mechanism. Protons do not appear to alter the conduction properties of the channels.