Related Experiment Videos
Calcium channel activity in a purified dihydropyridine-receptor preparation of skeletal muscle
J S Smith1, E J McKenna, J J Ma
1Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Abstract:
A purified dihydropyridine-receptor complex (DHPR) of skeletal muscle consisting of a major polypeptide of Mr 150K under reducing conditions induces divalent cation selective channels when incorporated into planar lipid bilayers. Channels were inserted into preformed planar bilayers by two techniques: (i) direct dilution of detergent-solubilized DHPR into the aqueous chambers adjacent to the bilayer membrane or (ii) reconstitution of DHPR into phospholipid vesicles followed by fusion of the preformed vesicles to the planar bilayer membrane. Unlike native membrane preparations of t-tubules, which only have one major Ca channel type of slope conductance of 12 pS in symmetrical 100 mM Ba, the purified DHPR complex induced at least two channel types with conductances of 12-14 and 22 pS. Some recordings suggest that these two channels are statistically coupled in time, i.e., that they may correspond to substrates of the same DHPR channel. Activity was found to occur spontaneously in the absence of the Ca channel agonist Bay k 8644. The 12-14-pS channel from DHPR exhibits voltage-dependent kinetics, is highly selective for barium ions, and was inhibited by micromolar nitrendipine. The 12-14-pS DHPR channel appears to be identical with functional Ca channels previously described in native t-tubules.
Insights
Purified skeletal muscle dihydropyridine-receptor (DHPR) complexes form cation channels in lipid bilayers. These channels exhibit distinct conductances, suggesting DHPR may regulate multiple calcium channel types.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Physiology
Background:
- Skeletal muscle dihydropyridine-receptor (DHPR) is crucial for excitation-contraction coupling.
- Native t-tubule membranes contain a single major calcium channel type with a 12 pS conductance.
- The precise functional properties and diversity of purified DHPR channels remain incompletely understood.
Purpose of the Study:
- To investigate the ion channel properties of purified skeletal muscle DHPR.
- To determine if purified DHPR can reconstitute functional cation channels in artificial membranes.
- To compare the characteristics of channels formed by purified DHPR with those in native t-tubules.
Main Methods:
- Purification of the skeletal muscle dihydropyridine-receptor (DHPR) complex.
- Incorporation of purified DHPR into planar lipid bilayers via detergent dilution or vesicle fusion.
- Electrophysiological recording of ion channel activity in reconstituted systems.
Main Results:
- Purified DHPR reconstituted into lipid bilayers formed cation-selective channels.
- At least two distinct channel types were observed, with conductances of 12-14 pS and 22 pS.
- The 12-14 pS channel displayed voltage-dependent kinetics, high Ba selectivity, and inhibition by nitrendipine, resembling native t-tubule Ca channels.
- Spontaneous channel activity occurred without Bay k 8644, and some channels showed statistical coupling.
Conclusions:
- Purified skeletal muscle DHPR complex reconstitutes functional cation channels in planar lipid bilayers.
- The observed channel diversity suggests DHPR may be associated with multiple calcium channel subtypes or exhibit complex gating.
- The 12-14 pS DHPR channel is functionally similar to native t-tubule calcium channels, validating its role in excitation-contraction coupling.