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

Abstract

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 kappaM-conotoxin RIIIJ from the venom of a fish-hunting species, Conus radiatus. This conopeptide, like a previously characterized peptide in the same family, kappaM-RIIIK, inhibits the homotetrameric human Kv1.2 channels. When tested in Xenopus oocytes, kappaM-RIIIJ has an order of magnitude higher affinity (IC(50) = 33 nm) to Kv1.2 than kappaM-RIIIK (IC(50) = 352 nm). Chimeras of RIIIK and RIIIJ tested on the human Kv1.2 channels revealed that Lys-9 from kappaM-RIIIJ is a determinant of its higher potency against hKv1.2. However, when compared in a model of ischemia/reperfusion, kappaM-RIIIK (100 mug/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, kappaM-RIIIJ does not exert any detectable cardioprotective effect. kappaM-RIIIJ shows more potency for Kv1.2-Kv1.5 and Kv1.2-Kv1.6 heterodimers than kappaM-RIIIK, whereas the affinity of kappaM-RIIIK to Kv1.2-Kv1.7 heterodimeric channels is higher (IC(50) = 680 nm) than that of kappaM-RIIIJ (IC(50) = 3.15 mum). Thus, the cardioprotection seems to correlate to antagonism to heteromultimeric channels, involving the Kv1.2 alpha-subunit rather than antagonism to Kv1.2 homotetramers. Furthermore, kappaM-RIIIK and kappaM-RIIIJ provide a valuable set of probes for understanding the underlying mechanism of cardioprotection.

FIGURE 1.

Isolation of κM-RIIIJ from C. radiatus venom. The asterisk in each HPLC chromatogram indicates the location of the fraction containing the peptide κM-RIIIJ. All of the elution buffers had 0.1% trifluoroacetic acid. A, the venom extract was chromatographed in a preparative Vydac C18 column eluted with 2–100% B90. B, the peak containing κM-RIIIJ in A was subfractionated using an analytical Vydac C18 column and an elution gradient of 10–15% B90 at a flow rate of 0.2%B90/min. C, the peak containing κM-RIIIJ in B was subfractionated using the same gradient as in B. D, the peak containing κM-RIIIJ in C was subfractionated using the gradient of 7–15% B90 at a flow rate of 0.2% B90/min.

SEQUENCE 1.

FIGURE 2.

The electrophysiological effect of κM-RIIIJ. Whole cell currents recorded from an oocyte expressing human Kv1.2 channel evoked by test potentials to −20, 0, and 20 mV (top panel) from a holding potential of −90 mV. The addition of 50 and 150 nm of κM-RIIIJ results in the block of the currents with a dose-dependent effect (two middle panels), which are reversible (bottom panel).

FIGURE 3.

Dose-response curves for the block by κM-RIIIJ (▲) and κM-RIIIK (■). Whole cell currents were recorded from oocytes expressing human Kv1.2 channel evoked by test potentials to 20 mV (n = 3–5). The error bars represent S.E.M.

FIGURE 4.

The IC₅₀ of κM-RIIIJ, κM-RIIIK and chimeras on the human Kv1.2 channel expressed in Xenopus oocytes. Whole cell currents were recorded from oocytes expressing human Kv1.2 channel evoked by test potentials to 20 mV (n = 3–5). The error bars represent S.E.M.

FIGURE 5.

A, blood pressure development at the time points of conotoxin application (25 min) and reperfusion (30 min) after treatment with κM-RIIIK or κM-RIIIJ. The average values of mean blood pressures of five to seven experiments are depicted. Application of κM-RIIIJ appeared to cause a slight acute increase in blood pressure that was not statistically verified. However, κM-RIIIJ enhanced the drop of blood pressure upon reperfusion, whereas the blood pressure development after κM-RIIIK was equivalent to that of the control experiments. B and C, infarct size expressed as a percentage of the area at risk in rats treated with κM-RIIIK (B) or with κM-RIIIJ (C) administration 5 min before reperfusion. *, significant difference versus vehicle (p < 0.05). The values represent the means ± S.E.M.

FIGURE 6.

The IC₅₀ of κM-RIIIJ and κM-RIIIK on concatenated human Kv1.2 channel heterodimers expressed in Xenopus oocytes. Whole cell currents were recorded from oocytes expressing a given channel heterodimer evoked by test potentials to 40 mV from a holding potential of −80 mV. The IC₅₀ values were obtained from the fractional currents (Fc) in the presence of the toxins (IC₅₀ = Fc/1 − Fc * [Tx]) (n = 4–5). The error bars represent S.E.M.