Glycosphingolipids of skeletal muscle: II. Modulation of Ca 2+-flux in triad membranes by gangliosides

Abstract

Membrane vesicles of rabbit skeletal muscle were prepared and separated by sucrose density gradient centrifugation. The fractions obtained (in the order of increasing density) were sarcolemma (SL), T-tubules (TT), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M) as characterized by their specific marker enzymes, ligand binding, and ion flux activities. The distribution of neutral glycosphingolipids and gangliosides in these membrane preparations has been documented in the preceding paper (J. Müthing, U. Maurer, U. Neumann, B. Kniep, and S. Weber-Schürholz, Carbohydr. Res., (1988) 135–145). G M3(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderately expressed and ganglio-series gangliosides were found in minor quantities, however, all showing different qualitative and quantitative membrane-type specific patterns. The voltage dependent Ca 2+-channels of skeletal muscle reside prevalently in the triad enriched membrane fractions deduced from highest binding capacity of 1,4-dihydropyridines. Calcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active 45Ca 2+-uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in parallel to control liposomes without gangliosides. G M3(Neu5Ac) strongly increased the uptake of 45Ca 2+ (+285%) whereas G M3(Neu5Gc) severely inhibited the ion flux (−61%). Neolacto-series gangliosides evoked miscellaneous effects upon 45Ca 2+-flux depending on isomeric sialic acid configuration, oligosaccharide size and fatty acid chain length of the ceramide portion. VI 3Neu5Ac– nLcOse 6Cer (C 24-fatty acid), IV 3Neu5Ac– nLcOse 4Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 16-fatty acid) strongly enhanced the 45Ca 2+-flux (+208, +162, and +120%, respectively), whereas IV 3Neu5Ac– nLcOse 4Cer (C 24-fatty acid), VI 3Neu5Ac– nLcOse 6Cer (C 16-fatty acid) and IV 6Neu5Ac– nLcOse 4Cer (C 24-fatty acid) slightly reduced 45Ca 2+-flux (−3, −6, and −17%, respectively). Out of all gangliosides tested in this study, G M1 showed the strongest stimulatory effect (+327%). G D1a and G T1b gave rise to remarkable flux-stimulation of +283 and +255%, respectively, whereas G D1b exhibited only a slightly positive effect (+38%). This data suggest a functional role of gangliosides in subcellular muscle membranes giving strong evidence that gangliosides are capable of modulating the cytosolic calcium level of muscle, which regulates muscle contraction.