ATP-sensitive K⁺ channels in an insulin-secreting cell line are inhibited byd-glyceraldehyde and activated by membrane permeabilization

Summary

The control of K⁺ channels in the insulin-secreting cell line RINm5F has been investigated by patch-clamp singlechannel current recording experiments. The unitary current events recorded from cell-attached patches are due to large and small inwardly rectifying ATP-sensitive K⁺ channels with conductance properties similar to the two channels previously identified in primary cultured rat islet cells (Findlay, I., Dunne, M.J., & Petersen, O. H.J. Membrane Biol. 88:165–172, 1985). Cell permeabilization through brief exposure to 10 μm digitonin or 0.05% saponin (outside the isolated membrane patch area) results in a dramatic increase in current through the cell-attached patch due to opening of many large and small K⁺-selective channels. These channels are inhibited in a dose-dependent manner by ATP applied to the bath (near-complete inhibition by 5mm ATP). During prolonged ATP exposure (1–5 min) the initial inhibition is followed by partial recovery of channel activity, although further activation does occur when ATP is subsequently removed. From the maximal number of coincident channel openings in the permeabilized cells (in the absence of ATP), it is estimated that there are on average 12 large ATP-sensitive K⁺ channels per membrane patch, but in the intact cells less than 5% of the membrane patches exhibited three or more coincident K⁺ channel openings, indicating the degree to which the channels are inhibited in the resting condition by endogenous ATP. Stimulation of RINm5F cells to secrete insulin was carried out by challenging intact cells with 10mm d-glyceraldehyde.d-glyceraldehyde induced depolarization of the membrane from about −70 to −20 mV and evoked a marked reduction in the open-state probability of both the large and small ATP-sensitive channels.d-glyceraldehyde also induced action potentials in a number of cases. All effects of stimulation were largely transient, lasting about 100 sec. The two ATP-sensitive K⁺ channels are probably responsible for the resting potential and play a crucial role in coupling metabolism to membrane depolarization.