Bell, Damian C.; (2000) Electrophysiological investigations into the G-protein modulation of the neuronal L-type (alpha1D, Cav1.3) and N-type (alpha1B Cav2.2) voltage dependent calcium channels. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this thesis two aspects of the G-protein modulation of voltage-dependent calcium channels were investigated using the whole-cell and perforated patch clamp techniques. 1). Neuroendocrine L-type calcium currents play a prominent role in neurosecretion, a process that can be modulated by G-proteins. This G-protein modulatory pathway was investigated using two clonal cell lines: a GH4C1 (derived from rat pituitary tumour tissue) cell line which express predominantly L-type currents; and, an HEK 293 cell line stably expressing a neuronal L-type calcium channel subunit complex (α1D, α2δ, β3). The channel biophysics and pharmacology exhibited by each cell line was shown to be typical of L-type currents. For example, currents in each cell line were shown to be long lasting (displaying little time dependent inactivation) and sensitive to the L-type specific dihydropyridine compounds (e.g. antagonised by nifedipine, and enhanced by the agonist S(-)-BayK8644). Using G-protein coupled receptors expressed in these cells (e.g. endogenous somatostatin type 2 or exogenously expressed dopamine D2 receptors) the G-protein modulation of the L-type calcium currents was investigated by the perfusion of the respective receptor agonists. No G-protein modulation was observed. A more direct method of G-protein activation was attempted, employing the use of the non-hydrolysable GTP and GDP analogues (GTP-γS and GDP-βS): again, no modulation was observed. In contrast, a positive control of HEK 293 cells transfected with α1D, α2δ and β3a (a calcium channel composition known to be G-protein modulated) displayed obvious G-protein modulation in both of these G-protein activating conditions. 2). The role of auxiliary calcium channel β subunits in the G-protein pathway was investigated by transfecting am alone or am co-expressed with β2a channel subunits in COS-7 cells. Cells expressing α1B/β2a subunits displayed typical G-protein modulation; however, in cells expressing α1B alone G-protein modulation was less apparent and atypical.

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