Indanyloxyacetic acid-sensitive chloride channels from outer membranes of skeletal muscle

Abstract

In mature mammalian muscle, the chloride conductance of the membrane is an important factor in the regulation of excitability. Up to now, no ligand was available for the biochemical characterization of muscle chloride channels. In order to localize and characterize these channels, we have used indanyloxyacetic acid (IAA)-94, a ligand previously used for epithelial Cl- channels (Landry, D. W., Reitman, M., Cragoe, E. J., Jr., and Al-Awqati, Q. (1987) J. Gen. Physiol. 90, 779-798; Landry, D. W., Akabas, M. H., Redhead, C., Edelman, A., Cragoe, E. J., Jr., and Al-Awqati, Q. (1989) Science 244, 1469-1472). IAA induced myotonic responses when microinjected into mature mouse muscle fibers, indicating a blockade of Cl- channels from the cytoplasmic side. Membrane vesicles were prepared from rabbit skeletal muscle and separated by sucrose gradient centrifugation. Fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmatic reticulum (SR), and triads and mitochondria (TR/M). The fraction enriched for SL was characterized by high specific binding capacity for [3H]saxitoxin (Na+ channel), whereas TT-rich fractions bound [3H]PN 200-110 (dihydropyridine receptor) with high specific activity. Upon patch-clamping of lipid supplemented vesicles, IAA-sensitive Cl- channels were found in the SL fraction but not in the SR. Highest specific activities in electrical diffusion potential sensitive 36Cl transport and [3H]IAA-94 binding were found in the SL. SL vesicles were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate and subjected to IAA-Sepharose affinity chromatography. Specifically bound protein was eluted with 100 microM IAA-94 and either analyzed by SDS-gel electrophoresis or reconstituted into phospholipid vesicles. The eluate contained four polypeptides (specifically bound, mapp 110-120 and 60 kDa; unspecifically bound mapp 67 and 50 kDa) and was highly enriched for IAA-sensitive chloride channels as shown by patch-clamping after reconstitution. The IAA-sensitive 100/280-picosiemens chloride channels of the sarcolemma are likely to be responsible for its major chloride conductance and thereby for the stabilization of resting potential.