title: Calcium channels reconstituted from the skeletal muscle dihydropyridine receptor protein complex and its alpha 1 peptide subunit in lipid bilayers.
abstract: In the first part of this study, we show that sDHPR and pDHPR preparations reconstituted into lipid bilayers formed on the tips of patch pipettes exhibit two divalent cation-selective conductance levels of 9 and 20 pS, similar in single-channel conductance to VSCC reported in a variety of intact preparations (see Pelzer et al. and Tsien et al. for review). The larger conductance level is similar to the VSCC identified in intact rat t-tubule membranes and described in sDHPR and pDHPR preparations, and shares many properties in common with activity from L-type VSCC. It is sensitive to augmentation by the DHP agonist (+/-)-BAY K 8644 and cAMP-dependent phosphorylation, and to block by the phenylalkylamine (+/-)-D600 and the inorganic blocker CoCl2. Its open-state probability and open times are increased upon depolarization as expected for a voltage-dependent activation process. Upon depolarization beyond the reversal potential, however, open-state probability and open times decline again. A reasonable way to explain the bell-shaped dependence of open times and open-state probability on membrane potential is to assume voltage-dependent ion-pore interactions that produce closing of the channel at strong negative and positive membrane potentials. By contrast, the smaller conductance level may be similar to the 10.6-pS t-tubule VSCC described by Rosenberg et al. and may best be compared with T-type VSCC. It is largely resistant to augmentation by (+/-)-BAY K 8644 and cAMP-dependent phosphorylation or block by (+/-)-D600, but is sensitive to block by CoCl2. Its open times and open-state probability show a sole dependence on membrane potential where depolarization increases both parameters sigmoidally from close to zero up to a saturating level. Both elementary conductance levels do not exhibit significant inactivation over a wide potential range, which may suggest that skeletal muscle VSCC inactivation is either poorly or not voltage-dependent at all. This possibility seems in agreement with bilayer recordings on reconstituted intact t-tubule membranes and voltage-clamp recordings on intact fibers. It supports the idea that the decline of Ca2+ current in intact skeletal muscle fibers may be due to Ca2+ depletion from the t-tubule system and/or to inactivation induced by Ca2+ release from the sarcoplasmic reticulum. We consistently observe two conductance levels of 9 and 20 pS, either singly, or together in the same bilayer from solubilized DHPR samples and even highly purified DHPR preparations.(ABSTRACT TRUNCATED AT 400 WORDS)
authors: II. Physiologisches Institut, Medizinische Fakultät, Universität des Saarlandes, Homburg/Saar, Federal Republic of Germany.
authors: D
authors: D
authors: Pelzer
authors: A O
authors: AO
authors: Grant
authors: A
authors: A
authors: Cavalié
authors: S
authors: S
authors: Pelzer
authors: M
authors: M
authors: Sieber
authors: F
authors: F
authors: Hofmann
authors: W
authors: W
authors: Trautwein
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000502:physiology*; D002417:Cattle; D003035:Cobalt / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D004553:Electric Conductivity; D005711:Gallopamil / Q000494:pharmacology; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D009132:Muscles / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000502:physiology*
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D008051:Lipid Bilayers; D011978:Receptors, Nicotinic; D005711:Gallopamil; D003035:Cobalt; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D000242:Cyclic AMP; D011494:Protein Kinases; C018021:cobaltous chloride