Biochemical and molecular characterization

L-type Ca channels are the only Ca channels that have been extensively characterized at the biochemical level. The success in characterizing L-type channels was largely due to their rich pharmacology, and in particular to the availability of radio-labelled DHPs. However, a second major contributor to this successful characterization came from the seminal findings of Glossmann and colleagues [45] and Fosset, Lazdunski and colleagues [43] who identified skeletal muscle transverse tubule membranes as a relatively rich source of this rare membrane protein. 

Isolation and purification of the multisubunit dihydropyridine-sensitive Ca channels from skeletal muscle 

For reasons that are not yet clear, skeletal muscle transverse (T)-tubule membranes contain 50-100-fold more high affinity DHP receptors than any other source yet identified [43,45]. Transverse tubule membranes contain 30-70 pmol/mg protein of DHP receptors that bind [ H]PN 200-100 with a of 0.1-0.2nM. The strategy utilized for the purification of L-type channels was similar to that used for the purification of other high affinity ligand binding proteins, and its success was predicted from the prior use of such an approach for the purification of other ion channels [54,55]. Thus the L-type channels were purified as high affinity DHP receptors, with the anticipation that the purified component(s) would constitute functional Ca channels. 

The purified L-type channels from skeletal muscle appear to be a multisubunit protein containing the products of four different and unrelated genes. A cartoon depicting some of the features of the purified protein is shown in Fig. 1. The subunits of the channel are commonly referred to as the ai, a2, ]8, y, and 8. These have been observed to migrate in a variety of SDS-polyacrylamide gels with apparent 

The ai polypeptide contains the receptors for the Ca channel effectors as it can be photolabelled by DHPs and PAAs [30,39-41,56,57,63] and various other Ca channel effectors. The other subunits do not appear to be required for proper binding of Ca channel effectors [64], although there is some evidence from biochemical studies that the other subunits may modulate binding [65]. The oti and y subunits are very hydrophobic, while the a.2jd and jS subunits are very hydrophilic [57]. The a [30], 2, 6 and y [57] peptides are glycosylated, but oc is much less so than 2 [30]. The of both a peptides varies slightly on different types of SDS gels 

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