MECHANISM OF ACTION OF K CHANNEL OPENERS ON SKELETAL-MUSCLE K-ATP CHANNELS - INTERACTIONS WITH NUCLEOTIDES AND PROTONS

The molecular mechanisms underlying the actions of K channel openers ( KCOs) on K-ATP channels were studied with the patch clamp technique in excised inside-out patches from frog skeletal muscle fibers. Benzopyr an KCOs (levcromakalim and SR 47063) opened channels partially blocked by ATP, ADP, or ATP gamma s, with and without Mg2+, but they had no e ffects in the absence of internal nucleotides, even after channel acti vity had significantly declined because of rundown. The effects of KCO s could therefore be attributed solely to a competitive inter-action b etween KCOs and nucleotides, as confirmed by observations that ATP dec reased the apparent affinity for KCOs and that, conversely, KCOs decre ased ATP or ADP sensitivity. Protons antagonized the action of the non -benzopyran KCOs, pinacidil and aprikalim, by enhancing their dissocia tion rate. This effect resembled the effect of acidification on benzop yran KCOs (Forestier, C., Y. Depresle, and M. Vivaudou. FEBS Lett. 325 :276-280, 1993), suggesting that, in spite of their structural diversi ty, KCOs could act through the same binding sites. Detailed analysis o f the inhibitory effects of protons on channel activity induced by lev cromakalim or SR 47063 revealed that, in the presence of 100 mu M ATP, this effect developed steeply between pH 7 and 6 and was half maximal at pH 6.6. These results are in quantitative agreement with an allost eric model of the K-ATP channel possessing four protonation sites, two nucleotidic sites accessible preferentially to Mg2+-free nucleotides, and one benzopyran KCO site. The structural implications of this mode l are discussed.