Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

Abstract

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

The Effect of Recombinant Neurotoxins from the Sea Anemone Anthopleura sp. on Sodium Currents of Rat Cerebral Cortical Neurons

“…This action was inconsistent with single channel studies comparing the action of site-3 toxins in preparations expressing cardiac and neuronal channels (Benzinger et al 1999), which increased the rate of whole-cell recovery at all tested voltages.…”

“…So Thr 21 in Hk2a can also play an important role in the influence of Na+ current amplitude. However, whether Hk7a and Hk2a will equally reduce the current amplitude of the sodium channel in other kinds of cells is hard to say (Benzinger et al 1999). We thought that this difference might partly be due to the different methods of obtaining cells, different type of cells, and even different subtype of sodium channels on which the experiment was performed.…”

Domain IV voltage-sensor movement is both sufficient and rate limiting for fast inactivation in sodium channels

“…This suggests that movement of DIV is the rate-limiting step not only for development but also for recovery from fast inactivation. Consistent with this interpretation, toxins that inhibit activation of the DIV voltage sensor speed recovery from fast inactivated states and eliminate the lag preceding recovery onset (Richard Benzinger et al, 1999). All of the mutants slowed recovery from inactivation to some degree, although DIV-CN had the greatest effect.…”

Interaction between the transcriptional corepressor Sin3B and voltage-gated sodium channels modulates functional channel expression

“…Therefore, competitive binding of Sin3B 293 to REST could remove suppression and upregulate Na v -channel expression. However, although N1E-115 neuroblastoma cells express mRNA for several Na v -channel α-subunits2627, single cell RT-PCR experiments suggest that Na v 1.2 is the predominant isoform in this cell line27. Therefore, REST-dependent suppression of Scn2a expression may already be removed, preventing any further upregulation by overexpression of Sin3B 293.…”