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

The G(q)-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca2+ channel (Ca(V)2.2 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 Ca-V beta is coexpressed with Ca(V)2.2. When palmitoylated Ca-V beta 2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca-V beta 3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca-V beta 2a may occupy an inhibitory site on Ca(V)2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca-V beta 2a relative to Ca(V)2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca-V beta 2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the alpha interacting domain (AID) of Ca(V)2.2's I-II linker. Ca-V beta s bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca-V beta's movements to Ca(V)2.2 gating. Although Bdel1/beta 2a currents exhibited more variable enhancement by SP, Bdel2/beta 2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca(V)2.2 coexpressed with Ca-V beta 3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/beta 2a currents, suggesting that when palmitoylated Ca-V beta 2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca-V beta 2a's palmitoyl groups directly interact with an inhibitory site on Ca(V)2.2 to block N-current inhibition by SP.