The human CC chemokine receptor 5 (CCR5) is a G protein–coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

BACKGROUND AND PURPOSE The µ‐conopeptide family is defined by its ability to block voltage‐gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ‐conopeptide (µ‐CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant. EXPERIMENTAL APPROACH µ‐CnIIIC was sequenced, synthesized and characterized by its direct block of elicited twitch tension in mouse skeletal muscle and action potentials in mouse sciatic and pike olfactory nerves. µ‐CnIIIC was also studied on HEK‐293 cells expressing various rodent VGSCs and also on voltage‐gated potassium channels and nicotinic acetylcholine receptors (nAChRs) to assess cross‐interactions. Nuclear magnetic resonance (NMR) experiments were carried out for structural data. KEY RESULTS Synthetic µ‐CnIIIC decreased twitch tension in mouse hemidiaphragms (IC50= 150 nM), and displayed a higher blocking effect in mouse extensor digitorum longus muscles (IC = 46 nM), compared with µ‐SIIIA, µ‐SmIIIA and µ‐PIIIA. µ‐CnIIIC blocked NaV1.4 (IC50= 1.3 nM) and NaV1.2 channels in a long‐lasting manner. Cardiac NaV1.5 and DRG‐specific NaV1.8 channels were not blocked at 1 µM. µ‐CnIIIC also blocked the α3β2 nAChR subtype (IC50= 450 nM) and, to a lesser extent, on the α7 and α4β2 subtypes. Structure determination of µ‐CnIIIC revealed some similarities to α‐conotoxins acting on nAChRs. CONCLUSION AND IMPLICATIONS µ‐CnIIIC potently blocked VGSCs in skeletal muscle and nerve, and hence is applicable to myorelaxation. Its atypical pharmacological profile suggests some common structural features between VGSCs and nAChR channels.

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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.