In this study we have identified multiple effects of ProTxII on macroscopic currents in NaV1.5. Previous reports have used measurements of peak current to track channel modification and measure toxin affinity. However, peak current, like most macroscopic current metrics, is a complex variable reflecting net changes in channel gating and pore conductance. Our data showed that ProTxII has multiple, distinct effects on NaV1.5 channels. This was evidenced by changes the current- and conductance-voltage relationships including a positive shift in the voltage range of activation, modification of gating transition rates along the activation pathway, and a decrease in Gmax. All three effects contribute to changes in peak current, making conventional assays of toxin affinity using dose-response relationships less useful. Data collected at various concentrations of ProTxII showed that the toxin-induced decrease in the voltage dependence of conductance and Gmax occurred at concentrations for which little or no positive shift in the range of activation was observed. This observation was most consistent with independent toxin binding events at multiple binding sites with different affinities.
Changes in tail current timecourse reflect the net contribution of channel deactivation (at very negative potentials) and reactivation (at more positive potentials) transition rates. Thus, analysis of tail current timecourse allowed us to further localize the effects of ProTxII to activation gating transitions. We observed faster tail current timecourse in the presence of very low concentrations of toxin which supported the idea that the decrease in the voltage dependence of conductance was indeed a result of activation gating modification.
Changes in tail current timecourse in the presence of ProTxII also indicated that at least some toxin-modified channels are able to activate. This is significant since channel drop-out could explain both the decrease in Gmax
and the shift in the range of activation. The fact that we are able to observe perturbed gating kinetics suggests that if such a population of permanently closed toxin-modified channels is responsible for those changes in the macroscopic current, it is distinct from a population of channels that are able to activate, albeit with altered gating properties. Similar changes in voltage dependent activation and Gmax
have been observed in voltage-gated K+
channels in the presence of Hanatoxin, another peptide toxin isolated from spider venom that shares extensive sequence homology with ProTxII (Priest et al., 2007
). Because Na+
channels are not four-fold symmetric, it is not suprising that ProTxII-induced changes in gating are more complex. Like Hanatoxin-bound K+
channels, at least some ProTxII-modified NaV
1.5 channels are able to activate as evidenced by toxin-induced changes in tail current timecourse. Hanatoxin binds to and impedes the movement of the voltage sensor in K+
channels (Swartz and MacKinnon, 1997a
; Li-Smerin et al., 2000
; Lee et al., 2005
; Phillips et al. 2005
), suggesting that interactions between ProTxII and the voltage sensors of voltage-gated Na+
channels are likely as well given the effects of the toxin on the voltage dependence of conductance and on tail current relaxation kinetics. Consistent with this idea, Sokolov et al. (2007) noted a decrease in total gating charge in the presence of ProTxII in the NaV
To further explore the possibility of interactions between ProTxII and NaV1.5 close to its voltage sensors, we checked for evidence of an electrostatic (surface charge-like) mechanism causing the shift in the range of activation we observed at concentrations above 200nM. We took advantage of the fact that by adding Ba2+ to the extracellular bath we would screen/bind surface charge, which would produce an apparent shift in gating without any direct changes to the gating transitions themselves. In these experiments we were able to distinguish between toxin effects that were sensitive to the presence of Ba2+, i.e. surface charge-like effects, and others that were insensitive and, therefore, surface charge-independent.
Tail current timecourse analysis revealed evidence of both types of effects. Tail currents at negative potentials responded to the surface charge agent, Ba2+ only in the absence of toxin. The simplest explanation for this finding is that Ba2+ and ProTxII “compete” for a site(s) that electrostatically affects the voltage sensors. The affinity of ProTxII is clearly much greater for such sites as toxin (5µM) speeds tail current relaxation to the same extent even in the presence of 40mM Ba2+. We also observed a persistent, toxin-induced, divergence in tail current timecourse at potentials positive to −90mV. Ba2+ alone did not produce such divergence indicating that this particular toxin effect is independent of its surface charge-like action. Changes in tail current time constants that cannot be accounted for by a shift in gating, reflect a perturbation of at least one activation gating transition. The fact that such changes were only apparent at more positive potentials indicates that the toxin is affecting gating transitions along the activation pathway distal to the final opening step.
Changes in activation due to surface charge effects and gating perturbations will all influence the voltage dependence of conductance. This explains the more complicated effect adding Ba2+ had on this relationship. If the shift of the conductance voltage relationship were due entirely to the surface charge-like effects of ProTxII we would expect data in the presence of toxin to overlay regardless of Ba2+ concentration, as was the case for tail current timecourse. However, this is not what we observed. The shift in the V1/2 of activation in the presence of both Ba2+ and ProTxII was largely additive reflecting the contribution of surface charge-like and surface charge-independent toxin actions. The extent to which the effects of Ba2+ and toxin were not additive most likely reflects the toxin’s surface charge-like effect.
ProTxII’s effect on Gmax
was also sensitive to the presence of Ba2+
indicating competition between the two agents. However, in contrast to the toxin’s effect on tail current timecourse, Ba2+
precluded any further decrease in Gmax
upon addition of ProTxII. The ability of Ba2+
to directly out-compete toxin in this case is unlikely since the effective concentration of toxin is nearly two orders of magnitude lower than the effective concentration of Ba2+
. Also consistent with a high affinity toxin effect on Gmax
, two-thirds of the toxin-induced decrease in Gmax
was recovered within 250ms of depolarization. Ba2+
binds to and voltage dependently blocks ion conduction through the pore in NaV
1.5. However, it seems unlikely that Ba2+
and ProTxII would be directly competing for a binding site within the pore, as toxin effects on activation gating and the relief of toxin modification with depolarization suggested an interaction site at the voltage sensors, which are far from the central pore (Jiang et al., 2003
; Lee et al., 2005
; Long et al., 2005
; Long et al., 2007
). Furthermore, experiments conducted in NaV
1.2 in the presence of TTX, a known pore blocker, showed a ProTxII-induced decrease in gating charge, which indicates ProTxII is able to interact with that channel in the presence of TTX (Sokolov et al., 2007). The effect of ProTxII on that particular isoform appeared to be limited to a decrease in Gmax
, lending further support to the idea that whatever the mechanisms of this effect, it is independent of TTX binding to the pore. As an alternative to direct competition between ProTxII and Ba2+
for a biding site within the permeation pathway, Ba2+
may indirectly disrupt a toxin binding site(s) via its interactions with the channel, thus precluding yet another distinct toxin action. These data also support the idea of a gating mechanism underlying the toxin-induced decrease in Gmax
and more strongly suggest that the loss of current reflects stabilization of one or more native closed states or the introduction of an additional absorbing non-conducting state that dose-dependently sequestered channels outside of the activation pathway.
Our data showed multiple effects on NaV
1.5 activation gating, including an effect on Gmax
, which differed in their affinity and mechanism. In contrast, modification of the NaV
1.2 isoform by ProTxII resulted only in a decrease in Gmax
(Sokolov et al., 2007). This difference in ProTxII’s effects between channel isoforms also are consistent with multiple binding sites. Interestingly, studies of β-scorpion toxins have revealed evidence of multiple toxin-channel interaction sites that together determine the characteristics of gating modification and toxin affinity that vary among isoforms (Leipold et al., 2006
). Perhaps there is a similar basis for the complexity of ProTxII action on NaV
1.5 and the differences in toxin effects among Na+
channel isoforms (Middleton et al., 2002
; Sokolov et al., 2007). Because the domains of these channels are non-identical, even analogous binding sites could vary in their toxin affinities and would be expected to produce very different consequences for channel gating. Mutagenesis experiments in NaV
1.2 suggested that ProTxII action in that channel is controlled by binding close to the domain II voltage sensor (Sokolov et al., 2007) making it likely that at least one interaction site for ProTxII on NaV
1.5 is in this domain. Interactions with the domain IV voltage sensor seem highly unlikely since movement of that voltage sensor is closely linked to inactivation from the open state (Cha et al., 1999
; Sheets and Hanck, 2005
), and there is no evidence for toxin interference with that transition. If indeed ProTxII binds to multiple domains, then the remaining candidates (assuming one interaction site on domain II) would be domain I and domain III, both of which have been closely linked to activation (Cha et al., 1999
). Because of its exclusive effects on channel activation, determining the binding site(s) and mechanism(s) of action of ProTxII promises to help elucidate differences in gating between channel isoforms and channel types that underlie characteristics matching channel activity to physiological function.