Cellular distribution and biochemical characterization of G proteins in skeletal muscle: comparative location with voltage-dependent calcium channels.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Toutant
M
M
Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France.
Gabrion
J
J
Vandaele
S
S
Peraldi-Roux
S
S
Barhanin
J
J
Bockaert
J
J
Rouot
B
B
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D015220:Calcium Channels / Q000502:physiology / Q000648:ultrastructure*; D003964:Diaphragm; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis*; D007150:Immunohistochemistry; D046911:Macromolecular Substances; D008870:Microtubules / Q000648:ultrastructure; D009132:Muscles / Q000166:cytology* / Q000648:ultrastructure; D037342:Pertussis Toxin; D011817:Rabbits; D010566:Virulence Factors, Bordetella / Q000378:metabolism
D000906:Antibodies; D015220:Calcium Channels; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins