GLYCOSPHINGOLIPIDS OF SKELETAL-MUSCLE - II - MODULATION OF CA2-FLUX IN TRIAD MEMBRANES BY GANGLIOSIDES()

Membrane vesicles of rabbit skeletal muscle were prepared and separate d by sucrose density gradient centrifugation. The fractions obtained ( in the order of increasing density) were sarcolemma (SL), T-tubules (T T), sarcoplasmic reticulum (SR1 and SR2) and triads/mitochondria (Tr/M ) as characterized by their specific marker enzymes, ligand binding, a nd ion flux activities. The distribution of neutral glycosphingolipids and gangliosides In these membrane preparations has been documented i n the preceding paper (J. Muthing, U. Maurer, U. Neumann, B. Kniep, an d S. Weber-Schurholz, Carbohydr. Res., (1988) 135-145). G(M3)(Neu5Ac) is the dominant ganglioside, neolacto-series gangliosides are moderate ly expressed and ganglio-series gangliosides were found in minor quant ities, however, all showing different qualitative and quantitative mem brane-type specific patterns. The voltage dependent Ca2+ -channels of skeletal muscle reside prevalently in the triad enriched membrane frac tions deduced from highest binding capacity of 1,4-dihydropyridines. C alcium channel complexes of triads were reconstituted into unilamellar phospholipid vesicles of 400 nm defined size and the active Ca-45(2+) -uptake into intravesicular space was measured after incorporation of muscle specific gangliosides into the outer vesicle lipid bilayer in p arallel to control liposomes without gangliosides. G(M3)(Neu5Ac) stron gly increased the uptake of Ca-45(2+) (+ 285%) whereas G(M3)(Neu5Gc) s everely inhibited the ion flux (-61%). Neolacto-series gangliosides ev oked miscellaneous effects upon Ca-45(2+)-flux depending on isomeric s ialic acid configuration, oligosaccharide size and fatty acid chain le ngth of the ceramide portion. VI(3)Neu5Ac-nLcOse(6)Cer (C-24-fatty aci d), IV(3)Neu5Ac-nLcOse(4)Cer (C-16-fatty acid) and IV(6)Neu5Ac-nLcOse( 4). Cer (C-16-fatty acid) strongly enhanced the Ca-45(2+)-flux (+ 208, + 162, and + 120%, respectively), whereas IV(3)Neu5Ac-nLcOse(4)Cer (C -24-fatty acid), VI(3)Neu5Ac-nLcOse(6)Cer (C-16-fatty acid) and IV(6)N eu5Ac-nLcOse(4)Cer (C-24-fatty acid) slightly reduced Ca-45(2+)-flux ( -3, -6, and -17%, respectively). Out of all gangliosides tested in thi s 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, whic h regulates muscle contraction. (C) 1998 Elsevier Science Ltd. All rig hts reserved.