Albert T. Leung

The biochemistry and molecular biology of the dihydropyridine-sensitive calcium channel.

Abstract Calcium channels are known to exist in muscle, neuronal and secretory cells. The 1,4-dihydropyridines are potent blockers of L-type Ca 2+ channels, and have been used as specific probes in the study of dihydropyridine-sensitive Ca 2+ channels. The receptor for the 1,4-dihydropyridines has been purified from skeletal muscle in order to characterize the biochemistry and molecular biology of the dihydropyridine-sensitive Ca 2+ channel. This review summarizes recent findings on the subunit composition of the dihydropyridinesensitive Ca 2+ channel, and discusses the structure and possible function of the individual subunits.

32,000‐Dalton Subunit of the 1,4‐Dihydropyridine Receptor

Polyclonal antibodies to the 32,000-Da polypeptide of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel have been produced and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. The 32,000-Da polypeptide was found to copurify with alpha 1 and alpha 2 subunits at each step of the purification of the dihydropyridine receptor. Monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex that includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. Our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.

Monoclonal Antibody Characterization of the 1,4‐Dihydropyridine Receptor of Rabbit Skeletal Muscle

The 1,4-dihydropyridine receptor (DHPR) of the voltage-dependent Ca channel has been purified from transverse tubular membranes of skeletal muscle. Curtis and Catterall have shown that it consists of three polypeptides of 160,000 daltons, 50,000 Da, and 32,000 Da and that under reducing conditions the apparent molecular weight of the 160,000 Da subunit shifted to 130,000. Borsotto et al. have identified three polypeptides of 142,000 Da, 33,000 Da, and 32,000 Da in their preparation of the dihydropyridine receptor. Furthermore, they have shown by immunoblotting with polyclonal antibodies that the 142,000 Da and 32,000 Da subunits are produced by the reduction of a 170,000 Da polypeptide. We report here the identification and characterization with monoclonal antibodies (MAb) of an additional high molecular weight subunit of the DHPR which is distinct from that described by Curtis and Catterrall and Borsotto et al. Monoclonal antibodies capable of specifically immunoprecipitating the [H]PN200-110-labeled DHPR of rabbit skeletal muscle were produced by immunizing BALB/c mice initially with skeletal muscle triad vesicles followed by booster immunizations with purified DHPR from skeletal muscle triads. Hybridoma supernatants were screened for the production of anti-DHPR antibodies with an immunodot assay. Supernatants that reacted positively against the partially purified DHPR were then tested for their ability to immunoprecipitate the [H]PN200-110-labeled receptor from digitonin-solubilized triads (see TABLE 1). All three MAbs (IIC12, IIF7, IIID5) that were capable of immunoprecipitating the DHPR recognized a protein with an Mr of 170,000 on nitrocellulose transfers of skeletal muscle triads and transverse tubular membranes separated on sodium dodecyi sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions (FIGURE la). Wheat-germ agglutinin (WGA) peroxidase stained a 175,000 Da protein on similar nitrocellulose transfers. Neither the 170,000 Da polypeptide nor the 175,000 Da polypeptide was detected in light sarcoplasmic reticulum vesicles, a preparation devoid of DHPR. Under reducing conditions, the Mr of the 170,000 Da

Ca2+ Channel Antibodies: Subunit-Specific Antibodies as Probes for Structure and Function

Voltage–dependent Ca2+ channels are known to exist in cardiac, skeletal, and smooth muscle cells as well as in neuronal and secretory cells [1, 2]. 1, 4–Dihydropyridines are potent blockers of voltage–dependent Ca2+ channels [3], and the receptor for 1, 4– dihydropyridines has been found to be highly enriched in the transverse tubular system of skeletal muscle [4]. Curtis and Catterall [5] were the first to purify the dihydropyridine receptor from rabbit skeletal muscle T–system membranes. Analysis of their preparation of receptor by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) suggested that the dihydropyridine receptor consisted of three subunits: an a subunit of 160000 Da, a β subunit of 50000 Da, and γ y subunit of 32000 Da. The apparent molecular weight of the a subunit in their preparation shifted from 160000 to 130000 upon reduction, whereas the molecular weight of the α and y subunits did not change upon reduction. The dihydropyridine receptor has also been purified from skeletal muscle membranes by Borsotto et al. [6] and Flockerzi et al. [7]. These groups also identified three subunits in their preparations of dihydropyridine receptor but the exact composition of subunits and molecular weight of the subunits differ from the original report of Curtis and Catterall [5]. Our laboratory has shown that the purified l, 4–dihydropyridine receptor from rabbit skeletal muscle triads contains four protein components of 175 000 Da (α2), 170000 Da (α1), 52000Da (β) and 32000 Da (γ) and that the 170000 Da and 175000 Da components are distinct polypeptides [8]. The 170000 Da polypeptide (a subunit) has been shown by photoaffinity labeling with [3H]azidopine and [3H]PN200–110 to contain the dihydropyridine binding site of the receptor [9,10], and the 170000 Da (α1 subunit) polypeptide and 52000 Da polypeptide (β subunit) have been shown to be substrates for various protein kinases [11–15]. Finally, the primary structure of the α1 subunit shows considerable sequence and structural similarities to the α subunit of the sodium channel [16].

Structural characterization of the nitrendipine receptor of the voltage-dependent Ca2+ channel: evidence for a 52,000 dalton subunit.

The nitrendipine receptor of the voltage-dependent Ca2+ channel purified from rabbit skeletal muscle has been shown to contain four polypeptide components of 175,000, 170,000, 52,000, and 32,000 daltons. Despite the existence of a substantial amount of data on the composition of the nitrendipine receptor, little is known about the relationship between the 175,000 and 170,000 dalton subunits of the receptor and the lower molecular weight components of the receptor. A monoclonal antibody specific to the 52,000 dalton component of the receptor has now been produced. The monoclonal antibody is capable of specifically immunoprecipitating the [3H]dihydropyridine-labeled nitrendipine receptor from detergent-solubilized membranes. Immunoprecipitation experiments with 32P-labeled nitrendipine receptor have demonstrated a tight association between the 170,000 dalton nitrendipine binding subunit and the 52,000 dalton polypeptide of the receptor. Immunoblotting experiments have shown that the 52,000 dalton polypeptide copurifies with the 175,000 and 170,000 dalton subunits of the nitrendipine receptor at all stages of the purification. In addition, the higher molecular weight subunits of the receptor were not labeled by the antibody. Densitometric scanning of Coomassie blue stained SDS-polyacrylamide gels of the purified nitrendipine receptor has shown that the 175,000, 170,000, 52,000, and 32,000 dalton subunits of the nitrendipine receptor exists in a 1:1:1:1 stoichiometric ratio. In conclusion, we have demonstrated that the 52,000 dalton polypeptide is an integral and distinct subunit of the purified nitrendipine receptor of the voltage-dependent Ca2+ channel.