Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(v)1.3) voltage-dependent calcium currents

Voltage-dependent calcium channels (VDCCs) are multimeric complexes compose d of a pore-forming alpha (1) subunit together with several accessory subun its, including alpha (2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from a lpha (1S) (skeletal muscle), alpha (1C) (in heart and brain), alpha (1D) (m ainly in brain, heart, and endocrine tissue), and alpha (1F) (retina). Neur oendocrine L-type currents have a significant role in the control of neuros ecretion and can be inhibited by GTP-binding (G-) proteins. However, the su bunit composition of the VDCCs underlying these G-protein-regulated neuroen docrine L-type currents is unknown. To investigate the biophysical and phar macological properties and role of G-protein modulation of alpha (1D) calci um channels, we have examined calcium channel currents formed by the human neuronal L-type alpha D-a1 subunit, coexpressed with alpha (2)delta -1 and beta (3a), stably expressed in a human embryonic kidney (HEK) 293 cell line , using whole cell and perforated patch-clamp techniques. The alpha (1D)-ex pressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inacti vation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha (1D) L-type curr ents were not modulated by activation of a number of G-protein pathways. Ac tivation of endogenous somatostatin receptor subtype 2 (sst2) by somatostat in-14 or activation of transiently transfected rat D2 dopamine receptors (r D2(long)) by quinpirole had no effect. Direct activation of G-proteins by t he nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also ha d no effect on the alpha (1D) currents. In contrast, in the same system, N- type currents, formed from transiently transfected alpha (1B)/alpha (2)delt a -1/beta (3), showed strong G-protein-mediated inhibition. Furthermore, th e I-II loop from the alpha (1D) clone, expressed as a glutathione-S-transfe rase (GST) fusion protein, did not bind G beta gamma, unlike the alpha (1B) I-II loop fusion protein. These data show that the biophysical and pharmac ological properties of recombinant human alpha (1D) L-type currents are sim ilar to alpha (1C) currents, and these currents are also resistant to modul ation by G(i/o)-linked G-protein-coupled receptors.