The patch-clamp method of single-channel recording was applied to K+ channels which are inhibited by intracellular adenosine 5'-triphosphate (ATP: K+-ATP channels) in membrane patches obtained from the insulin-secreting cloned cell line RINm5F. 2. The magnitude of K+ currents flowing outwards through these K+-ATP channels was reduced by internal Mg2+ ions in a dose-dependent manner. Currents flowing inwards through the channels were not affected by Mg2+. Internal Na+ ions had similar effects. 3. Divalent cations (Mg2+, Sr2+ and Ca2+) applied to the internal surface of the patch membrane inhibited the opening of K+-ATP channels in a dose-dependent manner. Internal Na+ ions had no effect. 4. K+-ATP channel activity was stimulated by adenosine 5'-diphosphate (ADP), guanosine 5'-triphosphate (GTP), guanosine 5'-diphosphate (GDP), guanosine 5'-o-(3-thiotriphosphate) (GTP gamma S) and guanosine 5'-o-(2-thiodiphosphate) (GDP beta S) when millimolar Mg2+ bathed the internal surface of the patch membrane. In the virtual absence of internal Mg2+ ions ADP, GTP, and GTP gamma S inhibited K+-ATP channels and GDP and GDP beta S were without effect. Adenosine 5'-o-(2-thiodiphosphate) (ADP beta S) inhibited K+-ATP channel activity in the presence and absence of Mg2+. 5. K+-ATP channel openings could be evoked by either ADP or GDP in the presence of an inhibitory concentration of ATP. These openings were abolished in the absence of internal Mg2+. 6. Run-down K+-ATP channels could be reactivated by ATP in the presence of internal Mg2+, but not in its absence. Analogues of ATP were unable to reactivate K+-ATP channels even in the presence of Mg2+. 7. It is concluded that internal Mg2+ ions (i) cause the rectification of the K+-ATP channel current-voltage relationship, (ii) are required for K+-ATP channel activity to be maintained by a phosphorylation process and (iii) are required for K+-ATP channel activity evoked by ADP, GTP and GDP.
