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

Abstract

BackgroundLow-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Therefore, finding specific modulators and/or blockers of T-type channels has become an important field of drug discovery. One characteristic of T-type calcium channels is that they share several structural similarities with voltage-gated sodium channels (VGSCs). We therefore hypothesized that binding sites for certain sodium channel blocking peptide toxins may be present in T-type calcium channels.FindingsThe sodium channel blocker ProTx I tonically blocked native and transiently expressed T-type channels in the sub- to low micro molar range with at least a ten-fold selectivity for the T-type calcium channel hCav3.1 over hCav3.3, and more than one hundred fold selectivity over hCav3.2. Using chimeras of hCav3.1 and hCav3.3, we determined that the domain IV region of hCav3.1 is a major determinant of toxin affinity, with a minor contribution from domain II. Further analysis revealed several residues in a highly conserved region between T-type and sodium channels that may correspond to toxin binding sites. Mutagenesis of several of these residues on an individual basis, however, did not alter the blocking effects of the toxin. ProTx II on the other hand preferentially blocked hCav3.2 and significantly shifted the steady state inactivation of this channel.ConclusionsProTx I blocks hCav3.1 both selectively and with high affinity. Domain IV appears to play a major role in this selectivity with some contribution from domain II. Given the structural similarities between sodium and T-type calcium channels and the apparent conservation in toxin binding sites, these data could provide insights into the development and synthesis of novel T-type channel antagonists.

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 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).…”

“…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).…”

Cochlear protein biomarkers as potential sites for targeted inner ear drug delivery

“…L-and T-type calcium channels are the two major forms of calcium channels within the cell [67]. Several natural peptides have been identified that target these calcium channels [68,74]. In addition to these natural peptides, there is also a range of synthetic calcium channel binding compounds that have been developed by the pharmaceutical industry that may be used as targeting compounds.…”

Toxins as tools: Fingerprinting neuronal pharmacology

“…Off-target activity may occur within channels or receptors of the same class due to high sequence homology or it may occur within different ion channel or receptor classes due to structural homology. Promiscuous activity is particularly evident amongst venom-derived peptides that target the voltage-sensing domain of voltage-gated ion channels, with venom-derived peptides from spiders often reported to have activity at NaV, KV and CaV channels [16,65,132,164]. However, it should be noted that compared to most other probes, including synthetic small molecules and endogenous ligands, toxins in general remain to be the most selective modulators available for voltage-gated and ligand-gated ion channels.…”