Our homology molecular model of the open/inactivated state of the Na + channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below the selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level on permeation in hNa V 1.5 we replaced Phe-1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively charged residues as well as the neutral cysteine and alanine. These mutations eliminated use-dependent lidocaine block with no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel conductance (γ) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased γ by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations affected Na + /K + selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge. Local anaesthetic (LA) drugs such as lidocaine interfere with impulse conduction in nerve and muscle by binding to the inner pore of voltage-gated Na + channels and blocking current (Hille, 2001). The major drug mechanism of action is not resolved, with experimental evidence variously favouring steric block, stabilization of a closed state, or some combination of the two. Extensive site-directed mutagenesis experiments have provided strong evidence that lidocaine-like drugs (LA) bind in the inner pore. S6 segment residues in domains I, III and IV (but not II) have been shown to be important for use-dependent LA block (Ragsdale et al. 1994;Wright et al. 1998;Yarov-Yarovoy et al. 2001;Yarov-Yarovoy et al. 2002). Two residues in domain IV S6 are of particular importance -Phe-1759 (following the heart Na V 1.5 isoform numbering, corresponding to Phe-1579 in skeletal Na V 1.4 and Phe-1764 in brain Na V 1.2) and Tyr-1766 (Tyr-1586 in Na V 1.4; Tyr-1771 in Na V 1.2), because their alanine mutants exhibit the greatest changes in LA affinity. Open/inactivated state block of the brain isoform Na V 1.2 by etidocaine was reduced by 130-and 35-fold for the alanine substitutions of the phenylalanine and tyrosine, respectively (Ragsdale et al. 1994). Cysteine accessibility experiments with methanethiosulphonate (MTS) reagents confirm th...

Key points• Persistent firing can be triggered in a population of inhibitory interneurons found in the hippocampus and neocortex. Repeated stimulation eventually triggers an autonomous barrage of spikes that is generated and maintained in the axon, followed by antidromic propagation to the soma.• This barrage of spikes is generated and maintained in the axon, followed by antidromic propagation to the soma. The mechanisms underlying this 'retroaxonal barrage firing' are unknown.• We find that retroaxonal barrage firing is Ca 2+ dependent, is inhibited by the L-type Ca 2+ channel blockers cadmium, nifedipine and verapamil, and does not require synaptic transmission. Loading the stimulated interneuron with BAPTA did not block barrage firing, suggesting that the required Ca 2+ entry may occur in other cells.• Retroaxonal barrage firing was observed in mice lacking the Cx36 isoform (most common neuronal isoform), indicating that this particular isoform is not required. AbstractWe recently described a new form of neural integration and firing in a subset of interneurons, in which evoking hundreds of action potentials over tens of seconds to minutes produces a sudden barrage of action potentials lasting about a minute beyond the inciting stimulation. During this persistent firing, action potentials are generated in the distal axon and propagate retrogradely to the soma. To distinguish this from other forms of persistent firing, we refer to it here as 'retroaxonal barrage firing' , or 'barrage firing' for short. Its induction is blocked by chemical inhibitors of gap junctions and curiously, stimulation of one interneuron in some cases triggers barrage firing in a nearby, unstimulated interneuron. Beyond these clues, the mechanisms of barrage firing are unknown. Here we report new results related to these mechanisms. Induction of barrage firing was blocked by lowering extracellular calcium, as long as normal action potential threshold was maintained, and it was inhibited by blocking L-type voltage-gated calcium channels. Despite its calcium dependence, barrage firing was not prevented by inhibiting chemical synaptic transmission. Furthermore, loading the stimulated/recorded interneuron with BAPTA did not block barrage firing, suggesting that the required calcium entry occurs in other cells. Finally, barrage firing was normal in mice with deletion of the primary gene for neuronal gap junctions (connexin36), suggesting that non-neuronal gap junctions may be involved. Together, these findings suggest that barrage firing is probably triggered by a multicellular

Toxins have been used extensively to probe the gating mechanisms of voltage-gated ion channels. Relatively few such tools are available to study the low-voltage activated T-type Ca channels, which underlie thalamic neuron firing and affect sleep, resistance to seizures, and weight gain. Here we show that ProTxII, a peptide toxin recently isolated from the venom of the tarantula spider Thrixopelma pruriens, dose-dependently inhibited Ca V 3.1 causing a decrease in current (81.6% ± 3.1% at −30 mV in 5 μM toxin) and a positive shift in the voltage range of activation (+34.5 mV ± 4.4 mV). Toxin-modified currents were slower to activate and faster to deactivate and they displayed a longer lag in the onset of current, i.e. the Cole-Moore shift, consistent with the inhibition of gating transitions along the activation pathway, particularly the final opening transition. Single channel current amplitude and total gating charge were unaffected by toxin, ruling out a change in ion flux or channel drop-out as mechanisms for the decrease in macroscopic conductance. A positive shift in the voltage range of gating charge movement (+30.6 mV ± 2.6 mV shift in the voltage of half maximal charge movement in the presence of 5 μM toxin) confirmed that ProTxII-induced gating perturbations in this channel occur at the level of the voltage sensors, and kinetic modeling based on these findings suggested that reductions in current magnitude could be largely accounted for by kinetic perturbations of activation.

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na + and Ca 2+ channels. ProTxII is distinct from other known Na + channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage dependent activation, and current kinetics in Na V 1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G max appeared at relatively low concentrations (40-100nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (≥500nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba 2+ , known to bind and/or screen surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba 2+ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.

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