TY - JOUR
T1 - Digitonin-permeabilized colonic cell layers
T2 - Demonstration of Calcium-activated Basolateral K+ and Cl− Conductances
AU - Chang, Dean
AU - Dawson, David C.
PY - 1988/9/1
Y1 - 1988/9/1
N2 - Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl− currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+ buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl−. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+ increases in the K+ and Cl− conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl−. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+ channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.
AB - Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl− currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+ buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl−. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+ increases in the K+ and Cl− conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl−. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+ channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.
UR - http://www.scopus.com/inward/record.url?scp=0023689357&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0023689357&partnerID=8YFLogxK
U2 - 10.1085/jgp.92.3.281
DO - 10.1085/jgp.92.3.281
M3 - Article
C2 - 2465372
AN - SCOPUS:0023689357
SN - 0022-1295
VL - 92
SP - 281
EP - 306
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 3
ER -