ISOLATION AND CHARACTERIZATION OF DISTINCT DOMAINS OF SARCOLEMMA AND T-TUBULES FROM RAT SKELETAL-MUSCLE

Several cell-surface domains of sarcolemma and T-tubule from skeletal- muscle fibre were isolated and characterized. 2. A protocol of subcell ular fractionation was set up that involved the sequential low- and hi gh-speed homogenization of rat skeletal muscle followed by KCl washing , Ca2+ loading and sucrose-density-gradient centrifugation. This proto col led to the separation of cell-surface membranes from membranes enr iched in sarcoplasmic reticulum and intracellular GLUT4-containing ves icles. 3. Agglutination of cell-surface membranes using wheat-germ agg lutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) an d a T-tubule fraction enriched in protein tt28 and the alpha(2)-compon ent of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions o f biochemical markers: SM2 was characterized by high levels of beta(1) -integrin and dystrophin, and SM1 was enriched in beta(1)-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was v ery abundant in SM1, SM2 and T-tubules, suggesting the presence of cav eolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-t ubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existen ce of subcompartments in sarcolemma from muscle fibre, featuring selec tive mutually exclusive components. T-tubules contain caveolin and cla thrin suggesting that they contain caveolin- and clathrin-rich domains . Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.