DNAs for other putative channel subunits

Other recent and very compelling evidence that the P subunit may also be necessary for full activity [83] comes from the study of a high threshold but DHP-insen-sitive brain channel that has an a 1-like channel core. The cDNA for this brain channel was recently isolated and the mRNA was expressed in Xenopus oocytes. The current corresponding to this channel was increased when the mRNA for either the skeletal muscle 0.2 or P subunits were co-injected. However, when the brain ai-like mRNA was co-expressed with the combination of the skeletal muscle 2 and p mRNAs, the current was dramatically increased from 31 nA for the brain ai alone to 6 500nA for the combination [83]. These striking results are the best evidence so far obtained that the P subunit has a functional role in channel activity. Although these data were obtained with a DHP-insensitive channel, they pro- 

Reconstitution of Ca channels Since L-type Ca channels were purified as high affinity DHP receptors, it was important to demonstrate that the purified preparations could act as functional DHP-sensitive Ca channels. Purified DHP receptor preparations from skeletal 

The recent studies with the skeletal muscle channels reconstituted in bilayers also established that an intrinsic property of the skeletal muscle Ca channels is that they possess a very low probability of opening. Even under optimal conditions of voltage, etc., these channels open only 8% of the time [95]. This previously unappreciated characteristic of these channels provides an alternative explanation to an earlier study in which it was proposed that only 5% of the DHP receptors in skeletal muscle can form functional channels [91]. In that study a probability of opening of 1.0 was assumed. However, if one adjusts this to 0.08, then the very different conclusion is that all the DHP receptors can make functional channels, but that they open only a very small percentage of the time. 

Biochemical studies with purified preparations incorporated into liposomes have also been performed [32,33,96-98]. Reconstituted receptors from skeletal muscle bound DHPs, PAAs and diltiazem with high affinity and in a 1 1 1 stoichiometry [97], In general, the reconstituted proteins exhibit the characteristic pharmacological properties expected for these channels. In recent studies, our laboratory has reconstituted partially purified channels into liposomes containing the Ca -sensitive fluorescent dye, fluo-3 [33,96]. These channels exhibit Ca influx that is sensitive to activation by Ca channel activators and inhibitors with affinities similar to those observed in intact cells, and the Ca influx is dependent on the establishment of a gradient in the presence of valinomycin [132]. This assay provides a convenient and rapid approach to obtaining a macroscopic picture of the activity of the channels under different conditions, while the more complex studies in lipid bilayers provide a more complete analysis of the single channel behavior. 

L-type Ca channels are primarily regulated by voltage and are thus opened and closed in response to changes in membrane potential, but in addition, they are regulated by receptor-dependent processes involving protein phosphorylation and G-proteins (Fig. 2). Many lines of evidence support the hypothesis that Ca channels are regulated by phosphorylation by several protein kinases, in particular by 

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