Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage‐dependent calcium channels

• Published in: The EMBO journal 1990-02, Vol.9 (2), p.363-369
• Main Authors: Toutant, M., Gabrion, J., Vandaele, S., Peraldi‐Roux, S., Barhanin, J., Bockaert, J., Rouot, B.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies - isolation & purification; Binding and carrier proteins; Biological and medical sciences; calcium; Calcium Channels - physiology; Calcium Channels - ultrastructure; Diaphragm; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; GTP-Binding Proteins - analysis; guanine nucleotide-binding protein; Immunohistochemistry; Macromolecular Substances; Microtubules - ultrastructure; Muscles - cytology; Muscles - ultrastructure; Pertussis Toxin; Proteins; Rabbits; T-tubules; Virulence Factors, Bordetella - metabolism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1990.tb08119.x

GTP binding proteins have been proposed to play a role in excitation‐‐contraction coupling. In a precedent study [Toutant et al., (1988), Biochem. J., 405‐409], we determined that Bordetella pertussis toxin is able to catalyse ADP‐ribosylation of two substrates in the detergent soluble fraction of total muscle extracts. Purified fractions of transverse tubule membranes (T‐tubule membranes), a key element of the excitation‐‐contraction coupling, were shown to exhibit a major ADP‐ribosylated substrate at 40 kd and an immunoreactivity with antisera raised against purified bovine brain Go alpha or G beta. In the present study, we have investigated the cellular distribution of G protein subunits in comparison with that of the voltage‐dependent Ca2+ channels by immunofluorescence on transverse and longitudinal sections of fast and slow muscles. With affinity‐purified antibodies against G beta subunits, a fluorescent labelling underlined the myofibrils and sarcolemma, whereas a strong immunoreaction in a dotted pattern evoked the presence of the subunit in repetitive triadic structures. With anti‐Go alpha antibodies, the immunofluorescence was more clearly focussed on a dotted pattern and the co‐location with the voltage‐dependent Ca2+ channel immunoreactivity indicates that both proteins were located in very close subcellular structures. Immunoblot analysis and PTX ADP‐ribosylation of the purified light sarcoplasmic reticulum (LSR), heavy sarcoplasmic reticulum (HSR) and T‐tubule subcellular fractions indicate the discrete presence of G proteins in LSR, an unambiguous labelling of the HSR fraction, while T‐tubule membranes clearly appear very rich in a Go‐like protein, confirming the observed preferential immunocytochemical distribution of G protein subunits.