Extracellular interaction of the voltage-dependent Ca2+ channel alpha2delta and alpha1 subunits.

Christina A Gurnett,Kevin P Campbell,Ricardo Felix

doi:10.1074/jbc.272.29.18508

Christina A Gurnett, Kevin P Campbell + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.29.18508

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Abstract

The role of the extracellular domain of the voltage-dependent Ca2+ channel alpha2delta subunit in assembly with the alpha1C subunit was investigated. Transiently transfected tsA201 cells processed the alpha2delta subunit properly as disulfide linkages and cleavage sites between the alpha2 and delta subunits were shown to be similar to native channel protein. Coimmunoprecipitation experiments demonstrated that in the absence of delta subunits, alpha2 subunits do not assemble with alpha1 subunits. Furthermore, the transmembrane and cytoplasmic sequences in delta can be exchanged with those of an unrelated protein without any effect on the association between the alpha2delta and alpha1 proteins. Extracellular domains of the alpha2delta subunit are also shown to be responsible for increasing the binding affinity of [3H]PN200-110 (isopropyl-4-(2,1, 3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-([3H]methoxycarbonyl)-pyridine-3-carboxylate) for the alpha1C subunit. Investigation of the corresponding interaction site on the alpha1 subunit revealed that although tryptic peptides containing repeat III of native alpha1S subunit remain in association with the alpha2delta subunit during wheat germ agglutinin chromatography, repeat III by itself is not sufficient for assembly with the alpha2delta subunit. Our results suggest that the alpha2delta subunit likely interacts with more than one extracellular loop of the alpha1 subunit.

Highlights

The ␣2␦ subunit has been identified in every voltage-dependent Ca2ϩ channel purified to date from various mammalian tissues, including skeletal muscle [1, 2], brain [3, 4], and heart [5, 6]
An antibody directed against the ␣2 subunit recognized a protein of 175 kDa in both skeletal muscle triads and in membranes prepared from tsA201 cells transfected with the full-length ␣2␦ subunit (Fig. 1)
There was a noticeable shift in molecular mass of the transfected ␣2␦ protein, suggesting that the cleavage site and disulfide linkages are similar to native protein

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—tsA201 cells (SV40 large T antigen transformed HEK 293 cells) (Cell Genesis, Foster City, CA) were maintained at 5% CO2 in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 2 mM L-glutamine, 50 units/ml penicillin, and 50 ␮g/ml streptomycin. Cell Membrane Preparation—tsA201 cells were harvested 48 h after transfection by washing two times with 10 ml of phosphate-buffered saline and collected by centrifugation at 3,000 rpm for 5 min. Cell membranes were prepared immediately by resuspending cell pellet from one 150-mm plate in 20 ml of ice-cold hypotonic lysis buffer (10 mM Tris, pH 7.4 with 0.64 mM benzamidine, and 0.23 mM PMSF).. Solubilized protein was isolated by centrifugation at 50,000 rpm for 15 min at 4 °C and subsequently diluted 2-fold with ice-cold double distilled H20. Inserts were amplified from pure phage positives by polymerase chain reaction using primers directed to ␭gt phage arms. These were directly inserted into a T-vector (made from Bluescript SkϪ plasmid) for sequencing.

RESULTS

NaCl and incubated with protein

DISCUSSION