Neurokinin 1 Receptors Trigger Overlapping Stimulation and Inhibition of Ca_V2.3 (R-Type) Calcium Channels

Abstract

Neurokinin (NK) 1 receptors and Ca V 2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of Ca V 2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of Ca V 2.3. In particular, weak activation of these receptors evokes mainly stimulation of Ca V 2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by Ca V 2.3, L-type (Ca V 1.3), Ntype (Ca V 2.2), and P/Q-type (Ca V 2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of Ca V 2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either G␣ q/11 (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-␤1) or G␤␥ subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering G␤␥, whereas a slow component of inhibition was reduced by buffering G␣ q/11 . When both G␤␥ and G␣ q/11 were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of Ca V 2.3. We also report that NK1 receptors accelerate the inactivation kinetics of Ca V 2.3 currents. Altogether, our results indicate that NK1 receptors modulate Ca V 2.3 using three different signaling mechanisms: a fast inhibition mediated by G␤␥, a slow inhibition mediated by G␣ q/11 , and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.

Protein Kinase C-Mediated Inhibition of Recombinant T-Type CaV3.2 Channels by Neurokinin 1 Receptors

“…1B). No evidence for receptor desensitization was observed during NKA applications lasting up to 3 min, in agreement with our previous report (Meza et al, 2007). As shown in Fig.…”

Double Mutant Cycle Analysis Identified a Critical Leucine Residue in the IIS4S5 Linker for the Activation of the CaV2.3 Calcium Channel

“…Oocytes were injected with 46 or 4.6 nl of a solution containing cRNA coding for the Ca V ␣1, Ca V ␣2b␦, and Ca V ␤3 subunits in a 6:2:3 weight ratio. Coexpression with the ancillary Ca V ␤3, which is predominant in the brain (30), promotes strong functional expression (31,32) and emphasizes closed-state inactivation (33) of Ca V 2.3. Functional expression of mutants was deemed significant with whole cell Ba 2ϩ currents larger than Ϫ300 nA.…”

Reactive oxygen species are second messengers of neurokinin signaling in peripheral sensory neurons

“…These results were unexpected, given that in other tissues and expression systems NKR were shown to couple to G q/11 (5,6). Thus, we expressed NK1-3 receptors in CHO cells and tested major components of the G q/11 pathway by (i) confocal imaging of PIP 2 hydrolysis and IP 3 release using PLCδ-PH-GFP; (ii) confocal imaging of DAG release with the PKCγ-C1-GFP probe; (iii) fura-2 Ca 2+ imaging to monitor Ca 2+ release; and (iv) patchclamp recording of recombinant M-channel inhibition by NKR.…”

“…However, the nature of such actions is controversial, and the molecular events triggered by NKR in peripheral nociceptors are not well established. The NKR traditionally are classed as G q/11 -coupled G protein-coupled receptors (GPCR) that activate phospholipase C (PLC), with subsequent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and release of inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) (5,6). Common downstream steps of G q/11 signaling include IP 3 -mediated release of Ca 2+ from intracellular stores and DAG-mediated activation of PKC.…”