Maitotoxin (MTX) activates a nonselective cation channel in Xenopus laevis oocytes

Abstract

 Maitotoxin (MTX) may exert its toxic effect by activating ion conductances and has been shown to elicit a fertilization-like response in Xenopus laevis oocytes. In the present study we investigated the electrophysiological response of stage V–VI Xenopus oocytes to MTX using the two-microelectrode voltage-clamp technique. Membrane voltage (V _m) measurements demonstrated that MTX (50 pM to 1 nM) depolarized the oocytes from –49±7 to –14±1 mV. Subsequent replacement of bath Na⁺ by the impermeant cation NMDG (N-methyl-d-glucamine) shifted V _m from –14±1 to –53±5 mV (n=29). This indicates that MTX activates a cation conductance. Indeed, current measurements at a holding potential of –60 or –100 mV showed that within 10 s of MTX application an inward current component developed which was largely abolished by extracellular Na⁺ removal. After a 1-min application of 1 nM MTX the NMDG-sensitive current increased more than 100-fold from 0.14±0.03 μA to a peak value of 21±3 μA (n=11). The effect of MTX was concentration dependent with an EC₅₀ of about 250 pM but only slowly reversible. Ion substitution experiments indicated that the stimulated conductance was nonselective for monovalent cations with a slight preference for NH₄ ⁺ (2.1) > K⁺ (1.5) > Na⁺ (1.0) > Li⁺ (0.7). Regarding divalent cations, a complex biphasic response to extracellular Na⁺ replacement by Ca²⁺ was observed, which suggests that the stimulated channels may have a small Ca²⁺ permeability but that exposure to high extracellular Ca²⁺ enhances recovery from MTX stimulation. No significant conductance for Mn²⁺ was observed. Application of 1 mM benzamil, 1 mM amiloride, or 100 μM 1-(β-[3-(4-Methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) reduced the MTX-stimulated inward current by 81%, 62%, or 65%, respectively. Gd³⁺ had an inhibitory effect of 29% and 38% at concentrations of 10 μM or 100 μM, respectively. Flufenamic acid, niflumic acid, (RS)-(3,4-dihydro-6,7-dimethoxyisoquinoline-1-γ1)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)-ethyl]-acetamide (LOE908), and 3′,5′-dichlorodiphenylamine-2-carboxylic acid (DCDPC), known blockers of other nonselective cation channels, had no significant effect. We conclude that MTX activates a nonselective cation conductance in Xenopus oocytes. The underlying channels may be involved in changes in V _m that occur during the early stages of fertilization.