Modulation of Cav2.2 Channels via Activation of Human GABAB Receptors Expressed in HEK293 Cells by Analgesic α-Conotoxins

Abstract

Numerous G protein-coupled receptors, including the GABAB receptor (GABABR), provide negative feedback to modulate the activity of neuronal (N)-type voltage-gated calcium channels (Cav2.2), which play a critical role in nociception. We have previously shown that analgesic α-conotoxins indirectly inhibit Cav2.2 channels via GABABR activation in mammalian dorsal root ganglion neurons (1). We reconstituted GABABR-mediated modulation of stably expressed CaV2.2 channels (α1, α2δ and β3) in HEK293 cells, co-transfected with cDNAs of cloned human GABABR subunits. GABABR expression was demonstrated using receptors labelled with fluorescent antibodies against the epitope tags of GABAB1 and GABAB2 subunits. Modulation of Cav2.2 by the agonists GABA and baclofen, and the α-conotoxins Vc1.1 and RgIA was studied using the whole-cell recording configuration of the patch clamp technique. Voltage-dependent Ba2+ currents were inhibited by baclofen, GABA and α-conotoxins Vc1.1 and RgIA in HEK293 cells transfected with Cav2.2 and GABABRs but not in cells transfected with Cav2.2 alone. In the presence of the GABABR, the biophysical properties of the Cav2.2 channels, including the current-voltage relationship, and activation and steady-state inactivation curves, were unchanged. The concentration-response relationships obtained for inhibition of Cav2.2 by baclofen, GABA, RgIA, Vc1.1, and cyclized Vc1.1 resulted in half-maximal inhibitory concentrations (IC50) of 3 μM, 68 nM, 130 nM, 120 nM and 10 nM, respectively. The inhibition by baclofen and GABA could be reversed by a depolarizing prepulse to +80 mV, whereas the effect of the α-conotoxins was slower and unaffected by a prepulse, suggesting the involvement of a voltage-independent pathway. Taken together, HEK293 cells provide a suitable expression system to study GABABR modulation of Cav2.2 channels and confirm the role of GABABRs in mediating the effects of analgesic α-conotoxins.1. Callaghan et al. (2008) J. Neurosci. 28:10943-51.