Identification and Subcellular Localization of the Subunits of L-type Calcium Channels and Adenylyl Cyclase in Cardiac Myocytes

Tianyan Gao,Tipu S Puri,Brian L Gerhardstein,Andy J Chien,Richard D Green,M Marlene Hosey

doi:10.1074/jbc.272.31.19401

Tianyan Gao, Tipu S Puri + Show 4 more

Open Access

https://doi.org/10.1074/jbc.272.31.19401

Copy DOI

Abstract

The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming alpha1 subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the alpha1C and beta2 subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the beta2 and the full-length alpha1C subunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the alpha1C subunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.

Highlights

The regulation of ion channels by protein phosphorylation and dephosphorylation is a common theme in neurobiology
It is not known if the channel subunits and adenylyl cyclase are spatially localized in cardiac myocytes in such a way that the increased Ca2ϩ resulting from ylrhodamine isothiocyanate; PAGE, polyacrylamide gel electrophoresis; AC, adenylyl cyclase; PBS, phosphate-buffered saline; WGA, wheat germ agglutinin
The results shown are the raw data from a single experiment and are representative of four separate experiments in which antibody binding was performed under identical conditions to paired groups of transfected human embryonic kidney (HEK) cells and cardiac myocytes

Summary

EXPERIMENTAL PROCEDURES

Materials—Adult male rabbits were purchased from commercial suppliers. The human embryonic kidney (HEK) 293 cells were a gift from Dr Ron Kopito (Stanford University). Membranes were solubilized in solubilization buffer (50 mM Tris, pH 7.4, 5 mM EDTA, 5 mM EGTA, 0.1%SDS, 1% Triton X-100 and protease inhibitors [16]) and incubated with the ␤gen antibody coupled to protein G. To test the specificity of the AC antibodies, HEK/ACV cells were sonicated in homogenization buffer (20 mM HEPES, 1 mM EDTA, pH 7.4, and protease inhibitors [16]) and crude membrane fractions were obtained by centrifugation. Membranes were solubilized in immunoprecipitation buffer (40 mM sodium phosphate, pH 7.4, 50 mM sodium fluoride, 5 mM EDTA, 5 mM EGTA, 0.5 M NaCl, 1% Nonidet P-40, 0.5% deoxylcholate, 0.1% SDS, and protease inhibitors) and solubilized proteins were incubated with the indicated AC antibodies immobilized to protein G (Pierce). Nonspecific binding from primary antibodies was determined after preabsorbing antibodies with their immunizing antigens

RESULTS

Since the method relies on quantifying the signal from Card

Rabbit myocytes

DISCUSSION