Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Abstract

Voltage‐activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L‐type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage‐dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine‐type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.

Tissue-specific expression of calcium channels

“…The smooth muscle a, (CaChZb) subunit induces barium currents in CHO cells that are identical to those of native smooth muscle: the single-channel conductance was 26 psi in the presence of 80 mM Ba*+, the open probability increased with membrane depolarization, and the voltage dependence of activation and inactivation was similar to that of the native smooth muscle channel (Bosse et al 1992). Stable expression of the CaCh2b with the skeletal muscle p gene (CaBl) increased in parallel the number of DHPbinding sites and the amplitude of whole cell barium current, suggesting that the amplitude of the inward current is directly related to the number of expressed a, protein molecules (A.…”

“…Photoaffinity labeling of skeletal muscle a, subunit followed by limited proteolysis and immunoprecipitation indicates that the DHP-binding site is localized close to the SSl-SS2 region of repeat III (Striessnig et al 1991, Nakayama et al 1991 and to a sequence following the IVS6 segment (Regulla et al 1991), whereas the PAAbinding site has been located directly after the IVS6 segment (Striessnig et al 1990) (Figure 2). Binding studies with radiolabeled DHPs demonstrate that the stably expressed al subunits from skeletal and smooth muscle alone contain the allosterically coupled binding sites for the known CaCBs (Kim et al 1990, Bosse et al 1992). …”

“…A major difference between the cardiac and the smooth muscle a, subunits is the different IvS3 segment that results from alternative splicing of the primary transcript from the CaCh2 gene (Perez-Reyes et al 1990). The two alternative splice variants CaCh2a and CaCh2b have been expressed transiently and stably (Biel et al 199 1, Singer et al 1991, Bosse et al 1992. No major differences have been observed in basic electrophysiologic and pharmacologic properties of the two isoforms, including the amplitude of inward current, steady-state activation and inactivation, and DHP sensitivity (Welling et al 1992b).…”

The block of the expressed L‐type calcium channel is modulated by the β3 subunit

“…The contribution of the az/~ and the different fl subunits to the properties of the native channels is not clear at present, The expression of the alc subunit induces L-type calcium and barium currents (IBa) in CHO and HEK cells. These currents have many properties of the native current including the sensitivity against organic calcium channel blockers and agonists [2][3][4][5][6][7]. The binding site for dihydropyridines (DHP) and phenylalkylamines has been localized exclusively at the al subunit [8,9].…”

Application of Patch Clamp Methods to the Study of Calcium Currents and Calcium Channels

“…Detailed methodology regarding preparation of Ca 2+ channel subunit mRNA (or cRNA) for purposes of microinjection is found in Snutch and Mandel (1992). Methodology detailing the construction of mammalian cell lines (CHO cells, HEK293 cells, mouse L cells) that stably express L-and N-type Ca 2+ channel subunit cDNAs is found in the studies by Bosse et al (1992), Williams et al (1992), and Lacerda et al (1991). The construction of a Ca 2+ channel subunit cDNA expression vector for transfection of primary cell cultures of adrenal chromaffin cells is described by Ma et al (1992).…”