Orientation of palmitoylated CaVβ2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

Abstract

The Gq-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca2+ channel (CaV2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which CaVβ is coexpressed with CaV2.2. When palmitoylated CaVβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when CaVβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of CaVβ2a may occupy an inhibitory site on CaV2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of CaVβ2a relative to CaV2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of CaVβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of CaV2.2's I–II linker. CaVβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple CaVβ's movements to CaV2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from CaV2.2 coexpressed with CaVβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated CaVβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that CaVβ2a's palmitoyl groups directly interact with an inhibitory site on CaV2.2 to block N-current inhibition by SP.