The Ca2+ channel β subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

Abstract

In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AA's concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated CaVβ2a.