Ca(2+)-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers.
Oosawa Y., Ashcroft SJ., Ashcroft FM.
This study evaluates the use of the planar lipid bilayer as a functional assay of Ca(2+)-activated K+ channel activity for use in purification of the channel protein. Ca(2+)-activated K+ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K(+)-selective (PCl/PK = 0.046; PNa/PK = 0.027). Channels incorporated into the bilayer with two orientations. In 65% of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca2+ concentration of about 15 mumol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca2+ activated the channel. These results resemble those found for Ca(2+)-activated K+ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.