Privileged crosstalk between TRPV1 channels and mitochondrial calcium shuttling machinery controls nociception

Abstract

The nociceptive noxious heat-activated receptor - TRPV1, conducts calcium and sodium, thus producing a depolarizing receptor potential, leading to activation of nociceptive neurons. TRPV1-mediated calcium and sodium influx is negatively modulated by calcium, via calcium-dependent desensitization of TRPV1 channels. A mitochondrial Ca uniporter - MCU, controls mitochondrial Ca entry while a sodium/calcium transporter - NCLX shapes calcium and sodium transients by mediating sodium entry into and removing calcium from the mitochondria. The functional interplay between TRPV1, MCU and NCLX, in controlling the cytosolic and mitochondrial calcium and sodium transients and subsequently the nociceptive excitability, is poorly understood. Here, we used cytosolic and mitochondrial fluorescent calcium and sodium imaging together with electrophysiological recordings of TRPV1-induced currents in HEK293T cells and nociceptor-like dissociated rat dorsal root ganglion neurons, while modulating NCLX or MCU expression using specific small interfering RNA (siNCLX). We show that the propagation of the TRPV1-induced cytosolic calcium and sodium fluxes into mitochondria is dependent on coordinated activity of NCLX and MCU. Thus, knocking-down of NCLX triggers down regulation of MCU dependent mitochondrial Ca uptake. This in turn decreases rate and amplitude of TRPV1-mediated cytosolic calcium, which inhibits capsaicin-induced inward current and neuronal firing. TRPV1-mediated currents were fully rescued by intracellular inclusion of the fast calcium chelator BAPTA. Finally, NCLX controls capsaicin-induced cell death, by supporting massive mitochondrial Ca shuttling. Altogether, our results suggest that NCLX, by regulating cytosolic and mitochondrial ionic transients, modulates calcium-dependent desensitization of TRPV1 channels, thereby, controlling nociceptive signaling.

The Role of Kv7/M Potassium Channels in Controlling Ectopic Firing in Nociceptors

“…To mimic the capsaicin-induced current, we first introduced an inward current with the activation and inactivation kinetics resembling TRPV1-induced inward current evoked by short (5s) puff application of capsaicin (1 μM) into our single-compartment model (see Methods). The stimulation of the soma with a modeled 5 s application of capsaicin (capsaicin-like stimulation) resulted in an inward current and action potential firing, which resembled the experimental data (Figures 3C,D, compare to Supplementary Figure 2A,B respectively, see also Nita et al, 2016). …”

“…To that end, we simulated a current induced by application of capsaicin, an activator of the noxious heat-sensitive TRPV1 channel, expressed by nociceptive neurons. We and others have previously demonstrated that application of capsaicin produces depolarization of nociceptive neurons, followed by a stereotypic pattern of action potential firing (Nita et al, 2016; Blair and Bean, 2003; see also Supplementary Figure 2, here). To mimic the capsaicin-induced current, we first introduced an inward current with the activation and inactivation kinetics resembling TRPV1-induced inward current evoked by short (5s) puff application of capsaicin (1 μM) into our single-compartment model (see Methods).…”

“…Neurons were isolated from lumbar dorsal ganglions of 6–9 week old Sprague Dawley male rat as previously described (Binshtok et al, 2008; Nita et al, 2016). In short: lumbar DRGs (L1-L6) were removed and placed into DMEM with 1% penicillin-streptomycin, then digested in 5 mg ml −1 collagenase, 1 mg ml −1 Dispase II (Roche) and 0.25% trypsin, followed by addition of 0.25% trypsin inhibitor.…”

“…A capsaicin-like induced current was introduced into a single simplified voltage clamp point-process with a fast exponential activation and slow exponential inactivation mimicking the experimental kinetics of puff-applied 1 μM capsaicin induced current (Nita et al, 2016):…”

“…The current was first tuned to fit experimental results (Supplementary Figure 2A, see also Nita et al, 2016) and then it was applied to the model neuron to verify that the induced voltage response for the experimental capsaicin-induced firing (Supplementary Figure 2A, see also Nita et al, 2016). Since our experimental recordings were obtained in DRG culture neurons which are considered to consist of only DRG soma, both adjustment and verification were performed in a soma-like model.…”