Inhibition of the activation pathway of the T-type calcium channel CaV3.1 by ProTxII

Abstract

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.

Block of T-type calcium channels by protoxins I and II

“…Contrary to previous findings [18], ProTx II appeared to preferentially block hCav3.2. This toxin block caused a significant negative shift in half inactivation voltage of hCav3.2, but in contrast with previous studies on sodium and calcium channels, no significant change in half activation potential [18,30,31]. The apparent differences between some of our results and those of previous studies, may be in part be due to the different expression systems, recording methods and clones used.…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…Both peptides were subsequently shown to block Ca V 3 channels in a subtype-dependent manner (Edgerton et al, 2010). Protoxin I preferentially blocks Ca V 3.1 channels over Ca V 3.3 and even more so over Ca V 3.2 (Ohkubo et al, 2010;Bladen et al, 2014b).…”

“…Protoxin I preferentially blocks Ca V 3.1 channels over Ca V 3.3 and even more so over Ca V 3.2 (Ohkubo et al, 2010;Bladen et al, 2014b). Protoxin II appears to act as a gating modifier, with the highest affinity for Ca V 3.2 channels (Edgerton et al, 2010;Bladen et al, 2014b). Another spider toxin that blocks T-type calcium channels is PsPTx3, a peptide isolated from Theraphosidae tarantula that has apparent selectivity for Ca V 3.2 calcium channels (French patent application FR2940973).…”

From Foe to Friend: Using Animal Toxins to Investigate Ion Channel Function

“…Recent studies identified other toxins ( e.g. , ProTx-I and ProTx-II) that inhibit T-type channels 153; 154 . However, their affinity and effects on channel gating are different compared with those of kurtoxin.…”