Abstract
Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning.
Highlights
Voltage-gated calcium (Cav) channels are membrane proteins that facilitate communication via electrical and chemical signaling in a wide variety of cells and organisms
It has been proposed that Cav channels, like their Kv and Nav relatives, have functional paddle motif substructures within each voltage sensor[34,35]
We investigated the molecular interactions of tarantula toxins with the Cav3.1 channel and lipid vesicles
Summary
Voltage-gated calcium (Cav) channels are membrane proteins that facilitate communication via electrical and chemical signaling in a wide variety of cells and organisms. The four channel domains (DI–DIV) have similar sequences, there is significant variability within each channel and across channel types[2] This variability may render the four voltage sensors functionally and pharmacologically distinct. Like other gating-modifier toxins, ProTx-II slows activation kinetics, accelerates deactivation, decreases the macroscopic current, and shifts the activation curve (I–V) to more positive potentials in both channel types, with no significant effect on steady-state availability or recovery from inactivation[29,32]. Our results demonstrate that sequence differences across the four voltage-sensors cause functional heterogeneity in the voltage-sensing and pharmacological properties of LVA channels. Only a few differences in the amino acid sequence of the toxins studied here can cause differences in channel inhibition and lipid partitioning. These two aspects, channel inhibition and lipid partitioning, seem to be positively correlated
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