Nociceptive sensory neurons are unusual in expressing voltage-gated inward currents carried by sodium channels resistant to block by tetrodotoxin (TTX) as well as currents carried by conventional TTX-sensitive sodium channels and voltagedependent calcium channels. To examine how currents carried by each of these helps to shape the action potential in smalldiameter dorsal root ganglion cell bodies, we voltage clamped cells by using the action potential recorded from each cell as the command voltage. Using intracellular solutions of physiological ionic composition, we isolated individual components of current flowing during the action potential with the use of channel blockers (TTX for TTX-sensitive sodium currents and a mixture of calcium channel blockers for calcium currents) and ionic substitution (TTX-resistant current measured by the replacement of extracellular sodium by N-methyl-D-glucamine in the presence of TTX, with correction for altered driving force). TTX-resistant sodium channels activated quickly enough to carry the largest inward charge during the upstroke of the nociceptor action potential (ϳ58%), with TTX-sensitive sodium channels also contributing significantly (ϳ40%), especially near threshold, and high voltage-activated calcium currents much less (ϳ2%). Action potentials had a prominent shoulder during the falling phase, characteristic of nociceptive neurons. TTXresistant sodium channels did not inactivate completely during the action potential and carried the majority (58%) of inward current flowing during the shoulder, with high voltage-activated calcium current also contributing significantly (39%). Unlike calcium current, TTX-resistant sodium current is not accompanied by opposing calcium-activated potassium current and may provide an effective mechanism by which the duration of action potentials (and consequently calcium entry) can be regulated.
Camphor is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite its long history of common medical use, the underlying molecular mechanism of camphor action is not understood. Capsaicin and menthol, two other topically applied agents widely used for similar purposes, are known to excite and desensitize sensory nerves by acting on two members of transient receptor potential (TRP) channel superfamily: heat-sensitive TRP vanilloid subtype 1 (TRPV1) and cold-sensitive TRP channel M8, respectively. Camphor has recently been shown to activate TRPV3, and here we show that camphor also activates heterologously expressed TRPV1, requiring higher concentrations than capsaicin. Activation was enhanced by phospholipase C-coupled receptor stimulation mimicking inflamed conditions. Similar camphor-activated TRPV1-like currents were observed in isolated rat DRG neurons and were strongly potentiated after activation of protein kinase C with phorbol-12-myristate-13-acetate. Camphor activation of rat TRPV1 was mediated by distinct channel regions from capsaicin, as indicated by camphor activation in the presence of the competitive inhibitor capsazepine and in a capsaicin-insensitive point mutant. Camphor did not activate the capsaicin-insensitive chicken TRPV1. TRPV1 desensitization is believed to contribute to the analgesic actions of capsaicin. We found that, although camphor activates TRPV1 less effectively, camphor application desensitized TRPV1 more rapidly and completely than capsaicin. Conversely, TRPV3 current sensitized after repeated camphor applications, which is inconsistent with the analgesic role of camphor. We also found that camphor inhibited several other related TRP channels, including ankyrin-repeat TRP 1 (TRPA1). The camphor-induced desensitization of TRPV1 and block of TRPA1 may underlie the analgesic effects of camphor.
BackgroundForty million adults in the US suffer from anxiety disorders, making these the most common forms of mental illness. Transient receptor potential channel canonical subfamily (TRPC) members 4 and 5 are non-selective cation channels highly expressed in regions of the cortex and amygdala, areas thought to be important in regulating anxiety. Previous work with null mice suggests that inhibition of TRPC4 and TRPC5 may have anxiolytic effects.HC-070 in vitroTo assess the potential of TRPC4/5 inhibitors as an avenue for treatment, we invented a highly potent, small molecule antagonist of TRPC4 and TRPC5 which we call HC-070. HC-070 inhibits recombinant TRPC4 and TRPC5 homomultimers in heterologous expression systems with nanomolar potency. It also inhibits TRPC1/5 and TRPC1/4 heteromultimers with similar potency and reduces responses evoked by cholecystokinin tetrapeptide (CCK-4) in the amygdala. The compound is >400-fold selective over a wide range of molecular targets including ion channels, receptors, and kinases.HC-070 in vivoUpon oral dosing in mice, HC-070 achieves exposure levels in the brain and plasma deemed sufficient to test behavioral activity. Treatment with HC-070 attenuates the anxiogenic effect of CCK-4 in the elevated plus maze (EPM). The compound recapitulates the phenotype observed in both null TRPC4 and TRPC5 mice in a standard EPM. Anxiolytic and anti-depressant effects of HC-070 are also observed in pharmacological in vivo tests including marble burying, tail suspension and forced swim. Furthermore, HC-070 ameliorates the increased fear memory induced by chronic social stress. A careful evaluation of the pharmacokinetic-pharmacodynamic relationship reveals that substantial efficacy is observed at unbound brain levels similar to, or even lower than, the 50% inhibitory concentration (IC50) recorded in vitro, increasing confidence that the observed effects are indeed mediated by TRPC4 and/or TRPC5 inhibition. Together, this experimental data set introduces a novel, high quality, small molecule antagonist of TRPC4 and TRPC5 containing channels and supports the targeting of TRPC4 and TRPC5 channels as a new mechanism of action for the treatment of psychiatric symptoms.
When acutely dissociated small-diameter dorsal root ganglion (DRG) neurons were stimulated with repeated current injections or prolonged application of capsaicin, their action potential firing quickly adapted. Because TTX-resistant (TTX-R) sodium current in these presumptive nociceptors generates a large fraction of depolarizing current during the action potential, we examined the possible role of inactivation of TTX-R sodium channels in producing adaptation. Under voltage clamp, TTX-R current elicited by short depolarizations showed strong use dependence at frequencies as low as 1 Hz, although recovery from fast inactivation was complete in approximately 10-30 msec. This use-dependent reduction was the result of the entry of TTX-R sodium channels into slow inactivated states. Slow inactivation was more effectively produced by steady depolarization than by cycling channels through open states. Slow inactivation was steeply voltage dependent, with a Boltzmann slope factor of 5 mV, a midpoint near -45 mV (5 sec conditioning pulses), and completeness of approximately 93% positive to -20 mV. The time constant for entry (approximately 200 msec) was independent of voltage from -20 mV to +60 mV, whereas recovery kinetics were moderately voltage dependent (time constant, approximately 1.5 sec at -60 mV and approximately 0.5 sec at -100 mV). Using a prerecorded current-clamp response to capsaicin as a voltage-clamp command waveform, we found that adaptation of firing occurred with a time course similar to that of development of slow inactivation. Thus, slow inactivation of the TTX-R sodium current limits the duration of small DRG cell firing in response to maintained stimuli and may contribute to cross desensitization between chemical and electrical stimuli.
Voltage-dependent delayed rectifier K؉ channels regulate aspects of both stimulus-secretion and excitationcontraction coupling, but assigning specific roles to these channels has proved problematic. Using transgenically derived insulinoma cells (TC3-neo) and -cells purified from rodent pancreatic islets of Langerhans, we studied the expression and role of delayed rectifiers in glucose-stimulated insulin secretion.
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