T Imagawa

Role of the Ryanodine Receptor of Skeletal Muscle in Excitation‐Contraction Coupling

In skeletal muscle, contraction is initiated by a depolarization of the transverse tubular membrane (t-tubule), which in turn signals the release of Ca from the sarcoplasmic reticulum (SR). A key protein involved in this process is the ryanodine receptor, an SR membrane protein of MW 450,000 that binds the alkaloid ryanodine with nanomolar affinity and is present exclusively at the junction between t-tubule and SR membranes. The ryanodine receptor plays a dual role: Functionally, it is the putative Ca-release channel of the SR, and structurally, it is the major protein responsible for forming “bridges” or “feet” that anatomically connect t-tubule and SR. Here we demonstrate that the ryanodine receptor is steeply gated by both voltage and protons, and for the first time in vitro, we measured nonlinear capacitance (charge movement) that may be involved in the gating of this channel protein. We recently identified the 450,000-Da ryanodine receptor-feet protein (FIG. 1A) as the Ca-release channel of native SR. This was achieved using the planar bilayer recording technique and by comparing ligand-dependent gating, ionic selectivity, and pharmacology of purified ryanodine receptors to that of native Ca-release channels. Voltage dependence was a gating property notoriously absent in our study and in studies by others that followed. Its inconspicuousness in our earlier work is related to the effect of protons. At pH 7.4 (FIG. 1B) the channel dwells in a fast gating mode (p = 0.38). A drop to pH 7.2 drives the channel into an almost closed condition (p = 0.08) and at pH 7.0 the channel never opens (p < 0.01). Reversibility is shown in the last record of FIGURE 1 where alkalinization from pH 7.0 to pH 7.6 reopens the channel, resulting in a higher level of activity (p = 0.72) than seen at pH 7.4. Over this narrow range of pH, slope conductance is not affected (FIG. 2C, inset), and the kinetics remain fast, with a mean open event duration of approximately 100 μsec. Thus for all practical purposes, a change of 0.6 unit in solution pH from 7.6 to 7.0 units is sufficient to make the channel switch from an almost all-open to an almost all-closed conformation. The fitted Hill coefficient for data in FIGURE 1 was n = 6.8 and the apparent pK a was 7.5. FIGURE 2 describes the ensuing changes in voltage

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.