Charge-dependent And Isoform-specific Interactions Between ProTxii And T-type Calcium Channels

ProTxII, peptide toxin isolated from the venom of the tarantula Thrixopelma puriens, modifies voltage-dependent activation of both T-type calcium (Ca) channels and voltage-gated sodium (Na) channels. In the presence of ProTxII (5μM) the voltage at half maximal activation (V1/2) of the CaV3.1 isoform is shifted positive (>25mV) and maximum conductance (Gmax) decreases (∼50%). Interestingly, the toxin's effects on this channel were completely precluded in the presence of high extracellular divalent concentrations indicating a role for surface charge-like, electrostatic interactions with the channel. Several mutant toxins in which individual basic residues were neutralized were tested for activity on CaV3.1. Three of these mutants, R13Q, R22A, and K28A, significantly disrupted the ability of the toxin to both shift channel activation and decrease Gmax. Two other mutations: K4Q and K14A, showed minimal or no effect, thus indicating an important yet specific role of charge in ProTxII's interaction with CaV3.1. The gating kinetics of T-type Ca channels varies among the three known isoforms suggesting there might be differences in the gating structures and, therefore, potential gating modifier toxin interaction surfaces as well. In CaV3.3, 5μM ProTxII reduced Gmax by approximately 60%, similar to what was seen for CaV3.1. However, unlike in CaV3.1, this concentration of toxin produced only a minimal shift in voltage dependent activation (2mV). These results suggest significant differences in the extracellular surface of T-type Ca channels across isoforms, particularly in terms of surface charge distribution close to one or more of the channels’ voltage sensors.