Heteromeric Assembly of GABABR1 and GABABR2 Receptor Subunits Inhibits Ca2+Current in Sympathetic Neurons

Abstract

Neuronal GABA_B receptors regulate calcium and potassium currents via G-protein-coupled mechanisms and play a critical role in long-term inhibition of synaptic transmission in the CNS. Recent studies have demonstrated that assembly of GABA_B receptor GABA_BR1 and GABA_BR2 subunits into functional heterodimers is required for coupling to potassium channels in heterologous systems. However whether heterodimerization is required for the coupling of GABA_B receptors to effector systems in neurons remains to be established. To address this issue, we have studied the coupling of recombinant GABA_B receptors to endogenous Ca²⁺ channels in superior cervical ganglion (SCG) neurons using nuclear microinjection to introduce both sense and antisense expression constructs. Patch-clamp recording from neurons injected with both GABA_BR1a/1b and GABA_BR2 cDNAs or with GABA_BR2 alone produced marked baclofen-mediated inhibition of Ca²⁺ channel currents via a pertussis toxin-sensitive mechanism. The actions of baclofen were blocked by CGP62349, a specific GABA_B antagonist, and were voltage dependent. Interestingly, SCGs were found to express abundantly GABA_BR1 but not GABA_BR2 at the protein level. To determine whether heterodimerization of GABA_BR1 and GABA_BR2 subunits was required for Ca²⁺inhibition, the GABA_BR2 expression construct was microinjected with a GABA_BR1 antisense construct. This resulted in a dramatic decrease in the levels of the endogenous GABA_BR1 protein and a marked reduction in the inhibitory effects of baclofen on Ca²⁺ currents. Therefore our results suggest that in neurons heteromeric assemblies of GABA_BR1 and GABA_BR2 are essential to mediate GABAergic inhibition of Ca²⁺ channel currents.