Biophysical Properties, Pharmacology, and Modulation of Human, Neuronal L-Type (α1D, CaV1.3) Voltage-Dependent Calcium Currents

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Abstract
Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming α1 subunit together with several accessory subunits, including α2δ, β, and, in some cases, γ subunits. A family of VDCCs known as the L-type channels are formed specifically from α1S (skeletal muscle), α1C (in heart and brain), α1D (mainly in brain, heart, and endocrine tissue), and α1F (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 α1D calcium channels, we have examined calcium channel currents formed by the human neuronal L-type α1D subunit, co-expressed with α2δ-1 and β3a, stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The α1D-expressing 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 inactivation 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, α1D 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 (rD2long) 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 α1D currents. In contrast, in the same system, N-type currents, formed from transiently transfected α1B/α2δ-1/β3, showed strong G-protein–mediated inhibition. Furthermore, the I–II loop from the α1D clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gβγ, unlike the α1B I–II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human α1D L-type currents are similar to α1C currents, and these currents are also resistant to modulation by Gi/o-linked G-protein–coupled receptors.