Inhibition of voltage-dependent calcium channels by omega-conotoxin MVIIC (omega-CTx-MVIIC) was studied in various types of rat neurons. When studied with 5 mM Ba2+ as charge carrier, omega-CTx-MVIIC block of N-type calcium channels in sympathetic neurons was potent, with half-block at 18 nM. Block of N-type channels had a rapid onset (tau approximately 1 sec at 1 microM omega-CTx-MVIIC) and quick reversibility (tau approximately 30 sec). The rate of block was proportional to toxin concentration, consistent with 1:1 binding of toxin to channels, with a rate constant (k on) of approximately 1 X 10(6) M-1. sec-1. Both potency and rate of block were reduced dramatically with increasing concentrations of extracellular Ba2+ omega-CTx-MVIIC also blocked P-type calcium channels in cerebellar Purkinje neurons, but both development and reversal of block were far slower than for N-type channels. The rate of block was proportional to toxin concentration, with k on -1.5 x 10(3) M-1. sec-1 at 5 mM Ba2+. From this value and an unblocking time constant of approximately 200 min, a dissociation constant of approximately 50 nM was estimated. Thus, block of P-type channels is potent but very slow. In hippocampal CA3 pyramidal neurons, omega-CTx-MVIIC blocked approximately 50% of the high-threshold calcium channel current; one component (approximately 20%) was blocked with the rapid kinetics expected for N-type channels, whereas the other component was blocked slowly. The component blocked slowly was reduced but not eliminated by preexposure to 200 nM or 1 microM omega-Aga-IVA.

L-Glutamate rapidly and reversibly suppressed Ca channel current in freshly dissociated pyramidal neurons from the CA3 region of the rat hippocampus. L-Glutamate inhibition of Ca channel current could be distinguished from activation of background conductance by appropriate ionic conditions and by distinct pharmacological profiles. Ca channel inhibition by glutamate was mimicked by quisqualate, ibotenate, racemïct-ACPD and 1S,3R-ACPD but not by kainate, AMPA, L-aspartate, NMDA, L-2-amino-4-phosphonobutyric acid, or 1R,3S-ACPD; 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the response. All agonists inhibited a similar fraction of high-voltage-activated Ca channel current, typically approximately 30%. Concentration-response relations for the agonists were consistent with mediation by a metabotropic glutamate receptor. The stereospecific agonist 1S,3R-ACPD was especially useful since it did not activate background conductances. The fraction of Ca channel current sensitive to 1S,3R-ACPD was partially blocked by omega-conotoxin GVIA but was not sensitive to dihydropyridine antagonists or agonists. The suppression of Ca channels by 1S,3R-ACPD became irreversible when cells were dialyzed with GTP-gamma-S. 1S,3R-ACPD suppressed Ca channel currents in outside-out membrane patches but not in cell-attached patches when applied outside the patch. These results suggest that metabotropic glutamate receptors suppress the activity of N-type Ca channels in CA3 neurons by a mechanism involving G-proteins but not readily diffusible second messengers.

BACKGROUND AND PURPOSEWe have developed a strategy to target the permanently charged lidocaine derivative lidocaine N-ethyl bromide (QX-314) selectively into nociceptive sensory neurons through the large-pore transient receptor potential cation channel subfamily V (TRPV1) noxious heat detector channel. This involves co-administration of QX-314 and a TRPV1 agonist to produce a long-lasting local analgesia. For potential clinical use we propose using lidocaine as the TRPV1 agonist, because it activates TRPV1 at clinical doses. EXPERIMENTAL APPROACHWe conducted experiments in rats to determine optimal concentrations and ratios of lidocaine and QX-314 that produce the greatest degree and duration of pain-selective block when administered nearby the sciatic nerve: reduction in the response to noxious mechanical (pinch) and to radiant heat stimuli, with minimal disruption in motor function (grip strength). KEY RESULTSA combination of 0.5% QX-314 and 2% lidocaine produced 1 h of non-selective sensory and motor block followed by >9 h of pain-selective block, where grip strength was unimpaired. QX-314 at this concentration had no effect by itself, while 2% lidocaine by itself produced 1 h of non-selective block. The combination of 0.5% QX-314 and 2% lidocaine was the best of the many tested, in terms of the duration and selectivity of local analgesia. CONCLUSIONS AND IMPLICATIONSTargeting charged sodium channel blockers into specific sets of axons via activation of differentially expressed large-pore channels provides an opportunity to produce prolonged local analgesia, and represents an example of how exploiting ion channels as a drug delivery port can be used to increase the specificity and efficacy of therapeutics.

Ionic currents activated by extracellular adenosine 5'-triphosphate (ATP) were studied in voltage-clamped dorsal root ganglion neurons from rats and bullfrogs. Under quasiphysiological ionic conditions, ATP-activated current reversed near 0 mV and showed strong inward rectification. Strong inward rectification was maintained even in symmetric solutions of divalent-free Cs glutamate. Examined with a resolution of 10s of microseconds, the rectification was instantaneous. Inward current was greatly reduced when N-methyl-D-glucamine was substituted for external Na. ATP-activated inward currents could be recorded with Ca as the only external cation; estimated from reversal potentials, the ratio of Ca to Na permeability is about 0.3. Unitary channel activity could be recorded when ATP was applied to outside-out patches. When activated, a single channel flickered rapidly, with a mean current of about 0.5 pA at -100 mV. Large concentrations of ATP put the channel in the activated, flickery condition virtually all the time, while at lower concentrations, periods of flickering were interspersed with closures. Analysis of whole-cell current fluctuations showed precisely the characteristics expected if such channels underlie the macroscopic currents.