Abstract
Cell membranes isolated from hamster insulinoma (HIT T15) cells at passages 65-74 contain high and low affinity receptors for a sulfonylurea derivative, 5-[125I]iodo,2-hydroxyglyburide (KD values of approximately 7 nM and 16 microM). Between passages 75 and 85, the estimated B(max) for the high affinity receptor decreases approximately 10-fold from approximately 1.6 to 0.16 pmol/mg membrane protein. By contrast, the density of low affinity binding sites, 800-1000 pmol/mg, is unaltered. The drop in high affinity receptors is paralleled by a decrease in the density of KATP channels assessed using patch-clamp and 86Rb(+)-efflux techniques. These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATP channel.
Highlights
The frequency of whole cellcurrents was scored as thenumber of cells which developed a current > 10 pA upon dialysis without ATP divided by the total number of trials
Mean no. of Frequency Of Passage no. chanNneol.so/pfatch channels open anism for closing is not critical, but two obvious candidates are either “rundown” in the absence of ATP, as has been described for these channels in membrane patches (Trube and Hescheler, 1984; Ohno-Shosaku et al, 1987),or reclosure following an increase in the ATP concentration
The results strongly support theidea that thehigh affinity receptor is an integral componentof the channel.We find a parallel drop in receptor number and channel activity as determbinye‘d‘jRb+ efflux, by estimates of channeldensity from patch-clamp experiments andby whole cell currents
Summary
K A ~ cphannels assessed using patch-clamp and “Rb+efflux techniques These results strongly support the idea that the high affinity sulfonylurea receptor is an integral part of the KATpchannel. Affinity P-cell receptors (Aguilar-Bryan et al, 1990; Nelson et al, 1992), strongly suggesting that the receptor is the 140kDa protein. In thispaper we have attempted to determine how “tight” the coupling is between the high affinity receptor and KATp channel activity by examining the density and behavior of the channels and receptors in the membranes of two sublines of HIT T15 cells at. We see a progressive parallel loss of the high affinity receptor and channel activity with increasing passage number in both sublines. A. B.), a Juvenile Diabetes Research grant To whom all correspondence should be addressed Dept. of Medicine, Baylor Collegeof Medicine, One Baylor Place, Houston, TX
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