CHARACTERISTICS OF SKELETAL-MUSCLE CHLORIDE CHANNEL CLC-1 AND POINT MUTANT R304E EXPRESSED IN SF-9 INSECT CELLS

Using the baculovirus system, the skeletal muscle chloride channel, CI C-1 (rat), and a point mutant replacing arginine 304 with glutamic aci d were expressed at high levels in cultured Sf-9 insect cells. Whole-c ell patch-clamping revealed large inwardly rectifying currents with ma xima up to 15 nA inward and 2.5 nA outward. Saturation was evident at voltage steps positive to +40 mV whilst steps negative to -60 mV produ ced inactivating currents made up of a steady state component and two exponentially decaying components with tau(1) = 6.14 +/- 0.92 ms, tau( 2) = 36.5 +/- 3.29 ms (S.D.) n = 7 for steps to -120 mV. Currents reco rded in the outside-out patch configuration were often unexpectedly la rge and up to 5% of whole-cell currents obtained in the same cell, sug gesting an uneven channel distribution in the plasmalemma of Sf-9 cell s. The pharmacology of a number of chloride channel blockers, includin g anthracene-9-carboxylate (A9C), niflumate, and perrhenate, was inves tigated and showed for the first time that perrhenate is an effective blocker of CIC-1 and that it has a complex mechanism of action. Furthe r, the potency of A9C was found to be dependent on external chloride c oncentration. As in studies on muscle cells themselves, blockade was r apidly effective and easily reversible, except when applying the indan yloxyacetate derivative, IAA94/95, which took up to 10 min to act, and , consistent with an intracellular site of action, was difficult to re verse by washing. Mutation of the highly conserved arginine at positio n 304 to a glutamic acid did not significantly alter the behaviour of the channel.