Biophysical Properties, Pharmacology, and Modulation of Human, Neuronal L-Type ( alpha sub(1D), Ca sub(V)1.3) Voltage-Dependent Calcium Currents

• Published in: Journal of neurophysiology Vol. 85; no. 2; pp. 816 - 827
• Main Authors: Bell, D C, Butcher, A J, Berrow, N S, Page, K M, Brust, P F, Nesterova, A, Stauderman, KA, Seabrook, G R, Nuernberg, B, Dolphin, A C
• Format: Journal Article
• Language: English
• Published: 01.02.2001
• ISSN: 0022-3077

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha sub(1) subunit together with several accessory subunits, including alpha sub(2) Delta , beta , and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha sub(1S) (skeletal muscle), alpha sub(1C) (in heart and brain), alpha sub(1D) (mainly in brain, heart, and endocrine tissue), and alpha sub(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 sub(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha sub(1D) subunit, co-expressed with alpha sub(2) Delta -1 and beta sub(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha sub(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 super(2+)) and showed little inactivation in external Ba super(2+), while displaying rapid inactivation kinetics in external Ca super(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 sub(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 sub(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 sub(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha sub(1B)/ alpha sub(2) Delta -1/ beta sub(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha sub(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind G beta gamma , unlike the alpha sub(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha sub(1D) L-type currents are similar to alpha sub(1C) currents, and these currents are also resistant to modulation by G sub(i/o)-linked G-protein-coupled receptors.