Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers.

Abstract

The purified 1,4-dihydropyridine receptor from skeletal muscle has been incorporated into planar bilayers, and its channel characteristics have been investigated.Conductances showed the characteristics of an L-type Ca2+ channel: divalent cation selectivity (PBa/PNa 30), blockage of Na+ conductance by micromolar Ca2e, and blockage of the Ca2" channel by D890 and by Cd2+. The a, subunit of the receptor must be phosphorylated by the cAMP-dependent protein kinase to give channel activity. BAY K 8644 did not activate nonphosphorylated channels, and (+ )-PN200-110 caused dramatic prolongation of mean open times when applied after phosphorylation. Channel properties were found to be dependent on association of receptor molecules in the bilayer. Single receptor molecules form channels of 0.9 pS (100 mM Ba2") and show no voltage-dependent gating. Upon association, both voltage-dependent gating and higher conductance events are recovered; stabilized conductance levels assume values of even multiples of 0.9 pS, predominately 7.5 and 15 pS and multiples of these values up to 60 pS. Thus, individual channels become functionally coupled (synchronous opening and closing) with association, reinstating the characteristics of one larger unitary channel. It is concluded that the L-type Ca2+ channel represents an oligomer of 1,4-dihydropyridine-receptor protein complexes, each of which constitutes a channel, where the array of channels (oligochannel) opens and closes in concerted action.Ca2+ channels play a central role in the physiology and pharmacology of excitable cells. The L-type Ca2" channels have different classes of allosterically interacting drugreceptor sites-e.g., for 1,4-dihydropyridines (DHPs), the phenylalkylamines, and the benzothiazepines. These drug receptors have been purified from the richest mammalian source, the skeletal muscle transverse (T)-tubule membrane (1-5). Upon reconstitution of the purified Ca2" antagonist receptor in planar membranes, L-type Ca2" channel activity was observed (5-7). However, in intact skeletal muscle fibers, a large apparent discrepancy is observed between the number of DHP binding sites and the apparent number of T-tubule Ca2+ channels (8). An involvement of the receptor protein in charge movement, relevant to excitation-contraction coupling (9), has been suggested (10-12). The "feet structures," which bridge the sarcoplasmic reticulum (SR) and T-tubule membranes, are now known to be identical with the ryanodine receptor (13, 14), which recently has been shown to form the Ca2 +-release channel when incorporated into planar bilayers (15). The "feet structures" consist of an oligomeric associate of 12-16 single polypeptides (Mr = 360,000) (14), and the purified ryanodine receptor forms arrays of synchronized Ca2" channels in the bilayer (15). We now find oligomer formation of the purified DHP receptor in planar bilayers to be essential for establishing channel properties expected for the L-type Ca2" channel of T-tubule (16), suggesting a model of directly coupled SR and T-tubu...

Calcium Channel α2δ Subunits: Structure, Functions and Target Site for Drugs

“…However, we did notice an additional small conductance mode of several calcium channel α1 subunits expressed in the absence of accessory subunits, which may relate to a different conformation of the α1 subunit expressed alone [22]. Furthermore, others have shown that purified L type calcium channels require oligomerisation to exhibit native single channel properties in planar lipid bilayers [23].…”

Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands.

“…However Hymel et al (1988) found that Bay K 8644 did not activate channels unless they were pre-phosphorylated.…”

“…In early studies it was concluded that cardiac calcium channel activation still occurs in the absence of phosphorylation (Kameyama et al 1986), and this is supported by findings that purified skeletal muscle Ca2+ channels reconstituted into lipid bilayers show single-channel activity in the absence of phosphorylating conditions ). However, evidence has been presented that Ca2+ channels in GH3 cells need to be phosphorylated to open (Armstrong & Eckert, 1987), and the conclusion reached by Hymel, Striessnig, Glossmann & Schindler (1988) and by Nunoki, Florio & Catterall (1989) was that purified skeletal muscle calcium channels are activated in a stoichiometric manner by phosphorylation, and that the basal activity may be due to residual phosphorylation of the purified channels. The lack of effect of forskolin on control IBa in the present experiments may indicate that the calcium channels, or associated proteins are fully phosphorylated.…”

Multiple Genes, Tissue Specificity, and Expression-Dependent Modulation Contribute to the Functional Diversity of Potassium Channels in Arabidopsis thaliana

“…Channel molecules may interact cooperatively at high packing density. Intermolecular cooperativity in biological membranes has been suggested for many animal receptors and ion channels (Hymel et al, 1988;Taleb and Betz, 1994), including voltage-gated K+ channels (Honore et al, 1992;Moran et al, 1992), and has been recently suggested for a plant anion channel (Schmidt and Schroeder, 1994). In addition, clustering of high densities of channel proteins is well known in animal cells and has been shown to require cytoskeletal elements for localization and stabilization (Cooper, 1987;Grimminger et al, 1991;Peter et al, 1991).…”