EFFECTS OF THIOL-MODIFYING AGENTS ON K-ATP CHANNELS IN GUINEA-PIG VENTRICULAR CELLS

ATP-sensitive K+ (K-ATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of K-ATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in K-ATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (0 mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups (p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal) were tested. The membrane-impermeable compound, pCMPS ( greater than or equal to 5 mu M), caused a quick and irreversible inhibition of K-ATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 mu M) caused a rapid, but spontaneously reversible, block of K-ATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block K-ATP channel activity. Thimerosal (100-500 mu M) induced a DTT-reversible block of partially rundown K-ATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block K-ATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (K-i) was increased from 47 +/- 12 to 221 +/- 35 mu M (n = 6, P < 0.05). This was not due to a spontaneous change with time. After patch excision, the K-i for ATP changed from 211 mu M within 60 s after patch excision to 46 mu M within 420 s after patch excision. Thus K-ATP channels in situ may be more sensitive to small changes in ATP levels (even in the physiological range) than those observed in excised patches. We conclude that SH-group modifying agents may have diverse effects on cardiac K-ATP channels. Some substances inhibited K-ATP channel activity, whereas others led to a decrease in ATP sensitivity and thus an increased likelihood of K-ATP channel opening for a given concentration of ATP.