Studies on the structural requirements for the activity of the skeletal muscle dihydropyridine receptor/slow Ca2+ channel. Allosteric regulation of dihydropyridine binding in the absence of alpha 2 and beta components of the purified protein complex.
A rabbit skeletal muscle dihydropyridine (DHP) receptor can be purified as an alpha 1-alpha 2-delta-beta-gamma complex, of which alpha 2 and delta are disulfide bonded. This complex has Ca2+ channel activity when incorporated into lipid bilayers. We reported recently that expression of alpha 1 in murine L cells (LCa cells) leads to appearance of both DHP binding and Ca2+ currents, and that we failed to detect alpha 2 by immunoblotting. LCa cell Ca2+ channel currents resembled those in rabbit skeletal muscle in their sensitivity to both voltage and the DHP agonist Bay K 8644, but differed in that they responded to depolarization much more slowly. We now report details of the molecular cloning of the cDNA encoding the 1857-amino acid long alpha 1 transfected into the L cells and results from studies on expression of beta, as well as, on allosteric regulation of DHP binding to these cells. The alpha 1 cDNA was cloned by a combination of cDNA library screening (5355 base pairs) and chemical synthesis (508 base pairs). Using rabbit labeled beta cDNA, which cross-reacts with murine beta mRNA, we failed to observe cross-hybridizing beta mRNA in LCa cells. Using a labeled single stranded 200-base long rabbit alpha 2 cDNA that cross-reacts with mouse alpha 2 mRNA, we likewise failed to observe cross-hybridizing alpha 2 mRNA in LCa cells and hence confirmed the absence of an endogenous murine alpha 2 in these cells. Using LCa cell membranes as DHP receptor source we found the binding of the DHP antagonist (+)-[3H]PN200-110 to be regulated by both verapamil and diltiazem as it is in rabbit skeletal muscle membranes. However, we noted a difference; at concentrations above 10(-6) M, verapamil inhibited residual DHP binding in LCa but not in skeletal muscle membranes. We conclude that neither alpha 2 nor beta are essential for expression of alpha 1 on the cell surface, or for its functioning as a voltage-gated Ca2+ channel, or for its allosteric regulation of DHP binding by Ca2+ channel antagonists. The studies neither exclude roles for gamma and delta, nor for alpha 2 or beta in determining more subtle properties of this channel.
Kim
H S
HS
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Wei
X Y
XY
Ruth
P
P
Perez-Reyes
E
E
Flockerzi
V
V
Hofmann
F
F
Birnbaumer
L
L
D000494:Allosteric Regulation; D000818:Animals; D015152:Blotting, Northern; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D007700:Kinetics; D007739:L Cells / Q000378:metabolism; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009132:Muscles / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014162:Transfection
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D004247:DNA