G-proteins in skeletal muscle. Evidence for a 40 kDa pertussis-toxin substrate in purified transverse tubules

Abstract

In muscle, it has been established that guanosine 5'-[gamma-thio]triphosphate (GTP[S]), a non-hydrolysable GTP analogue, elicits a rise in tension in chemically skinned fibres, and that pretreatment with Bordetella pertussis toxin (PTX) decreases GTP[S]-induced tension development [Di Virgilio, Salviati, Pozzan & Volpe (1986) EMBO J. 5, 259-262]. In the present study, G-proteins were analysed by PTX-catalysed ADP-ribosylation and by immunoblotting experiments at cellular and subcellular levels. First, the nature of the G-proteins present in neural and aneural zones of rat diaphragm muscle was investigated. PTX, known to catalyse the ADP-ribosylation of the alpha subunit of several G-proteins, was used to detect G-proteins. Three sequential extractions (low-salt-soluble, detergent-soluble and high-salt-soluble) were performed, and PTX was found to label two substrates of 41 and 40 kDa only in the detergent-soluble fraction. The addition of pure beta gamma subunits of G-proteins to the low-salt-soluble extract did not provide a way to detect PTX-catalysed ADP-ribosylation of G-protein alpha subunits in this hydrophilic fraction. In neural as well as in aneural zones, the 39 kDa PTX substrate, very abundant in the nervous system (Go alpha), was not observed. We then studied the nature of the G alpha subunits present in membranes from transverse tubules (T-tubules) purified from rabbit skeletal muscle. Only one 40 kDa PTX substrate was found in T-tubules, known to be the key element of excitation-contraction coupling. The presence of a G-protein in T-tubule membranes was further confirmed by the immunoreactivity detected with an anti-beta-subunit antiserum. A 40 kDa protein was also detected in T-tubule membranes with an antiserum raised against a purified bovine brain Go alpha. The presence of two PTX substrates (41 and 40 kDa) in equal amounts in total muscle extracts, compared with only one (40 kDa) found in purified T-tubule membranes, suggests that this 40 kDa PTX substrate might be involved in excitation-contraction coupling.

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

“…in T-tubule membranes was further confirmed by the detection of a 36 kd subunit by immunoblotting with an anti-: subunit antiserum (Toutant et al, 1988;Villaz, et al, 1990). In the present study, we investigated the cellular distribution of two subunits of G protein in comparison with the location of the dihydropyridine (DHP)-sensitive voltagedependent Ca2+ channels, by immunofluorescence using anti-Go3 and anti-Gooa antibodies.…”

“…The Goa immunoreactivity which was observed in T-tubule membranes (Toutant et al, 1988) is hardly detectable in HSR and LSR fractions (data not shown). The data obtained indicate that the 40 kd PTX substrate is in greater amounts in T-tubule membranes than in HSR, whereas in LSR, the labelling is very poor.…”

“…This important subsarcolemmal labelling is probably not due to soluble G proteins, since none of them could have been identified in the fraction obtained after low salt extraction (Toutant et al, 1988). A labelling with both G,B and Goca antibodies also appears very intense, in subsarcolemmal cytoplasm, particularly in slow muscle fibres.…”

“…Purified T-tubule membranes, LSR and HSR fractions (20 yg of protein for each fraction) were ADP-ribosylated with PTX (List Biological Laboratories, Campbell, CA, USA) as previously described (Toutant et al, 1987(Toutant et al, , 1988.…”

“…In a precedent study [Toutant et al, (1988), Biochem. In a precedent study [Toutant et al, (1988), Biochem.…”