Photoaffinity labelling of the phenylalkylamine receptor of the skeletal muscle transverse‐tubule calcium channel

159

mentioning

confidence: 97%

Identification of a phenylalkylamine binding region within the alpha 1 subunit of skeletal muscle Ca2+ channels.

Striessnig

Glossmann

Catterall

1990

159

“…The L-type calcium channel is labeled by 1,4-dihydropyridine (DHP) and phenylalkylamine drugs (6)(7)(8), permitting its purification to homogeneity from skeletal muscle (9)(10)(11)(12) and heart (13,14). The purified receptor comprises four distinct subunits (a,, a2-6, f3, and y).…”

mentioning

confidence: 99%

Purified omega-conotoxin GVIA receptor of rat brain resembles a dihydropyridine-sensitive L-type calcium channel.

McEnery

Snowman

Sharp

et al. 1991

The w-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of '2SI-labeled CTX ('25I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-'2-I-CTX (HSA-'25I-CTX). Photoincorporation of HSA-'25I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of '25I-CTX binding to the purified CTX receptor closely resembles that ofthe native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (al) and four additional proteins with apparent molecular masses of 140 (a2), 110, 70 (P2), and 60 (pj) kDa. This subunit structure losely resembles that ofthe 1,4-dihydropyridine-sensitive L-type calcium channel.Biophysical studies have discriminated several types of voltage-gated calcium channels (designated L-, T-, P-, and N-type) in neurons (1-3) and other excitable tissues (4, 5). The L-type calcium channel is labeled by 1,4-dihydropyridine (DHP) and phenylalkylamine drugs (6-8), permitting its purification to homogeneity from skeletal muscle (9-12) and heart (13, 14). The purified receptor comprises four distinct subunits (a,, a2-6, f3, and y). Molecular cloning of the DHP-binding a1 subunit from several tissues (15-17) indicates a common protein structure. In the brain, molecular cloning reveals a heterologous family of calcium channels with homology to the a, subunit of the skeletal muscle DHP receptor (18)(19)(20)(21)(22).The N-type calcium channel, which regulates neurotransmitter release from synaptic endings, can be distinguished from the neuronal L-type calcium channel (23,24). The N-type channel is selectively inhibited by c-conotoxin GVIA (CTX) isolated from the venom of the snail Conus geographus (25,26) and can be labeled with 125I-labeled CTX (125I-CTX) (27). CTX receptors are more abundant than neuronal L-type channels (27, 28), but their instability has precluded purification. A photoaffinity derivative of CTX, N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX) labels a discrete 230-kDa band in brain membranes (29,30). As the DHP-binding protein represents only one of several L-type channel subunits, so the band labeled by HSA-1251-CTX may be only a portion of the CTX receptor.We now report purification to homogeneity of the CTX receptor with the isolated receptor protein retaining reversible ligand binding. The purified rat brain CTX receptor comprises a 230-kDa protein that is the CTX recognition site and four additional subunits. The subunit compositions of the CTX receptor and the L-type calcium channel are closely similar.MATERIALS AND METHODS Materials. Calpain inhibitors I and II and 3-1(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) used for CTX receptor solubilization were obtained from Calbiochem; CHAPS used in column buffers, heparinSepharose, ...

“…A recent report (33) shows that a verapamil derivative labels a,, but not a2, subunits. In contrast, another recent paper (34) reports only a single intact a2 subunit for calcium channels purified from frozen muscle, as found by Borsotto et al (4 …”

mentioning

confidence: 99%

Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.

Takahashi

Seagar

Jones

et al. 1987

410

215

Purified dihydropyridine-sensitive calcium channels from rabbit transverse-tubule membranes consist of three noncovalently associated classes of subunits: a (167 kDa), p (54 kDa), and y (30 kDa). Cleavage of disulfide bonds reveals two distinct a polypeptides and an additional component, 6.The a, subunit, a 175-kDa polypeptide that is not N-glycosylated, contains the dihydropyridine binding site, cAMP-dependent protein kinase phosphorylation site(s), and substantial hydrophobic domain(s). a2, a 143-kDa glycoprotein, has none of the properties characteristic of al but binds lectins and contains about 25% N-linked carbohydrate. a2 is disulfidelinked to 6, a 24-to 27-kDa glycopeptide. .3 (54 kDa) contains a cAMP-dependent phosphorylation site but is not N-glycosylated and does not have a hydrophobic domain. y (30 kDa) has a carbohydrate content of about 30% and extensive hydrophobic domain(s). Precipitation with affinity-purifiled anti-a, antibodies or a2-specific lentil lectin-agarose demonstrated that aja213yS behaves as a complex in the presence of digitonin or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, whereas the a26 complex dissociates from al.fy in the presence of Triton X-100. A model for subunit interaction and membrane insertion is proposed on the basis of these observations.