title: Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents.
abstract: 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(1D) subunit, co-expressed 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 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, 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 Gbetagamma, 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.
authors.affiliation: Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
authors.forename: D C
authors.initials: DC
authors.lastname: Bell
authors.forename: A J
authors.initials: AJ
authors.lastname: Butcher
authors.forename: N S
authors.initials: NS
authors.lastname: Berrow
authors.forename: K M
authors.initials: KM
authors.lastname: Page
authors.forename: P F
authors.initials: PF
authors.lastname: Brust
authors.forename: A
authors.initials: A
authors.lastname: Nesterova
authors.forename: K A
authors.initials: KA
authors.lastname: Stauderman
authors.forename: G R
authors.initials: GR
authors.lastname: Seabrook
authors.forename: B
authors.initials: B
authors.lastname: Nürnberg
authors.forename: A C
authors.initials: AC
authors.lastname: Dolphin
mesh_terms: D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D002120:Calcium Channel Agonists / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D004095:Dihydropyridines / Q000819:agonists / Q000037:antagonists & inhibitors / Q000494:pharmacology; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D002120:Calcium Channel Agonists; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C038806:1,4-dihydropyridine; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins