Biophysical properties, pharmacology, and modulation of human, neuronal L-type (α1D, Cav1.3) voltage-dependent calcium currents

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha (1) subunit together with several accessory subunits, 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 alpha (1S) (skeletal muscle), alpha (1C) (in heart and brain), alpha (1D) (mainly in brain, heart, and endocrine tissue), and alpha (1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha (1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha (a1)D 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)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba(2+)) and showed little inactivation in external Ba(2+), while displaying rapid inactivation kinetics in external Ca(2+). 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 currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha (1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha (1B)/alpha (2)delta -1/beta (3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha (1D) clone, expressed as a glutathione-S-transferase (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 pharmacological properties of recombinant human alpha (1D) L-type currents are similar to alpha (1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.