Orientation of palmitoylated Ca(V)β2a relative to Ca(V)2.2 is critical for slow pathway modulation of N-type Ca(2+) current by tachykinin receptor activation

The G(q)-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca(2+) 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)β is coexpressed with Ca(V)2.2. When palmitoylated Ca(V)β2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca(V)β3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca(V)β2a 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)β2a 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)β2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the α interacting domain (AID) of Ca(V)2.2's I–II linker. Ca(V)βs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca(V)β's movements to Ca(V)2.2 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 Ca(V)2.2 coexpressed with Ca(V)β3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated Ca(V)β2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca(V)β2a's palmitoyl groups directly interact with an inhibitory site on Ca(V)2.2 to block N-current inhibition by SP.