Mechanism and molecular basis for the sodium channel subtype specificity of µ‐conopeptide CnIIIC

Abstract

BACKGROUND AND PURPOSEVoltage-gated sodium channels (NaV channels) are key players in the generation and propagation of action potentials, and selective blockade of these channels is a promising strategy for clinically useful suppression of electrical activity. The conotoxin m-CnIIIC from the cone snail Conus consors exhibits myorelaxing activity in rodents through specific blockade of skeletal muscle (NaV1.4) NaV channels. EXPERIMENTAL APPROACHWe investigated the activity of m-CnIIIC on human NaV channels and characterized its inhibitory mechanism, as well as the molecular basis, for its channel specificity. KEY RESULTSSimilar to rat paralogs, human NaV1.4 and NaV1.2 were potently blocked by m-CnIIIC, the sensitivity of NaV1.7 was intermediate, and NaV1.5 and NaV1.8 were insensitive. Half-channel chimeras revealed that determinants for the insensitivity of NaV1.8 must reside in both the first and second halves of the channel, while those for NaV1.5 are restricted to domains I and II. Furthermore, domain I pore loop affected the total block and therefore harbours the major determinants for the subtype specificity. Domain II pore loop only affected the kinetics of toxin binding and dissociation. Blockade by m-CnIIIC of NaV1.4 was virtually irreversible but left a residual current of about 5%, reflecting a 'leaky' block; therefore, Na + ions still passed through m-CnIIIC-occupied NaV1.4 to some extent. TTX was excluded from this binding site but was trapped inside the pore by m-CnIIIC. CONCLUSION AND IMPLICATIONSOf clinical significance, m-CnIIIC is a potent and persistent blocker of human skeletal muscle NaV1.4 that does not affect activity of cardiac NaV1.5.

Mammalian Neuronal Sodium Channel Blocker μ-Conotoxin BuIIIB Has a Structured N-Terminus That Influences Potency

“…At least two possible explanations may account for such a residual current: A) there are two populations of channels, with the majority being sensitive to the peptide and a small minority being insensitive; B) the blocking efficacy of the peptide is less than 100%, such as that observed when μ-KIIIA and its derivatives were tested on Na V 1.2 and Na V 1.4,(29-31) and when μ-CnIIIA was tested on Na V 1.4. (32) Further tests, which are beyond the scope of the present study, are necessary to pinpoint the mechanism responsible for the small (∼5%) residual current observed with [ d -Ala2]BuIIIB and Na V 1.3.…”

Where cone snails and spiders meet: design of small cyclic sodium‐channel inhibitors

“…Considerable effort has been made in elucidating the molecular determinants for the subtype specificity of peptide toxins toward particular Na V channel subtypes. More precisely, many studies have focused on the key residues responsible for subtype selectivity among m-conotoxins (15,(39)(40)(41)(42)(43). These studies paved the way for the rational design of selective Na V channel antagonists and will assist in further peptide engineering of the PnCS scaffold with the aim of designing small, cyclic, selective, and potent Na V channel inhibitors for therapeutically relevant Na V subtypes.…”

µ-Conotoxins Modulating Sodium Currents in Pain Perception and Transmission: A Therapeutic Potential

“…The µ-CTX CnIIIC through the potent and selective antagonism of Nav channel 1.4 has been shown to elicit a block in rodents’ sciatic nerves and muscles emerging as a promising pharmacological tool in the development of myorelaxants and analgesics [ 56 , 86 ]. The recent findings of alternative modes by which µ-PIIIA binds Nav 1.4 channel also suggested a novel role of the binding properties for combating pain-associated diseases [ 43 ].…”