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.
Highlights
Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception
ProTx Protoxin I and II (II) is a preferential blocker of hCav3.2 ProTx II was originally identified as potent inhibitor of sodium channels with fifteen to hundred- fold selectivity for hNav1.7 versus other sodium channels [18,19]
Our observations indicate that ProTx II blocks hCav3.2 more potently than the other T-type calcium channels
Summary
Low-voltage-activated (T-type) calcium channels play a crucial role in a number of physiological processes, including neuronal and cardiac pacemaker activity and nociception. Low-voltage-activated (LVA) or “T-type” calcium channels are encoded by one of three different types of Cav α1 subunits (Cav3.1, Cav3.2 and Cav3.3, known as α1G, α1H, and α1I, respectively) whose membrane topology is similar to those of sodium channels [1] They are activated by small membrane depolarizations and display rapid activation and inactivation kinetics [2] and they are responsible for triggering low-threshold depolarizations that in turn lead to the initiation of action potentials. Identification of drugs and molecules that selectively interact with T-type calcium channels has, so far proven difficult, recently novel small organic scaffolds for T-type channel inhibitors have been derived from blockers of other calcium channel subtypes, such as L-type channels [12,13,14] Another class of molecules that are known to be effective blockers of voltage gated ion channels are polypeptide toxins. Given the structural similarities between these two classes of ion channels, we tested to what extent these two toxins inhibited T-type calcium channels and identified channel structural determinants of toxin block
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