Biochemical characterization of ?M-RIIIJ, a Kv1.2 channel blocker: evaluation of cardioprotective effects of ?M-conotoxins

Conus snail (Conus) venoms are a valuable source of pharmacologically active compounds; some of the peptide toxin families from the snail venoms are known to interact with potassium channels. We report the purification, synthesis, and characterization of ?M-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family,?M-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, ?M-RIIIJ has an order of magnitude higher affinity (IC50=33 nM) to Kv1.2 than ?M-RIIIK (IC50=352 nM). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from ?M-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, ?M-RIIIK (100 ?g/kg of body weight), administered just before reperfusion, significantly reduces the infarct size in rat hearts in vivo without influencing hemodynamics, providing a potential compound for cardioprotective therapeutics. In contrast, ?M-RIIIJ does not exert any detectable cardioprotective effect. ?M-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than ?M-RIIIK, whereas the affinity of ?M-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC50=680 nM) than that of?M-RIIIJ (IC50=3.15 ?M). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 ?-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, ?M-RIIIK and ?M-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.