Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes

Abstract

The effects of methylene blue (MB) on cromakalim-induced K+ currents were investigated in follicle-enclosed Xenopus oocytes. In concentrations ranging from 3–300 μM, MB inhibited K+ currents (IC50: 22.4 μM) activated by cromakalim, which activates KATP channels. MB inhibited cromakalim-activated K+ currents in a noncompetitive and voltage-independent manner. The respective EC50 and slope values for cromakalim-activation of K+ currents were 194 ± 21 µM and 0.91 for controls, and 206 ± 24 µM and 0.87 in the presence of 30 μM MB. The inhibition of cromakalim-induced K+ currents by MB was not altered by pretreatment with the Ca2+ chelator BAPTA, which suggests that MB does not influence Ca2+-activated second messenger pathways. K+ currents mediated through a C-terminally deleted form of Kir6.2 (KirΔC26), which does not contain the sulfonylurea receptor, were still inhibited by MB, indicating direct interaction of MB with the channel-forming Kir6.2 subunit. The binding characteristics of the KATP ligand [3H]glibenclamide are not altered by MB in a concentration range between 1 μM-1 mM, as suggested by radioligand binding assay. The presence of a membrane permeable cGMP analogue (8-Br-cGMP, 100 µM) and a guanylate cyclase activator (BAY 58-2667, 3 µM) did not affect the inhibitory effects of MB, suggesting that MB does not inhibit cromakalim-activated K+ currents through guanylate cyclase. Collectively, these results suggest that MB directly inhibits cromakalim-activated K+ currents in follicular cells of Xenopus oocytes.

The Xenopus Oocyte: A Tool for Membrane Biology

“…Numerous membrane receptors can be expressed in the Xenopus oocyte to characterize their biophysical or pharmacological properties using the two-electrode voltage clamp technique. In a study by Isaev et al [9], the antagonistic effect of methylene blue on K ATP channels was demonstrated by measuring the intensity of a current induced by cromakalim. And Cav2.1 channel variants causing ataxic channelopathies revealed electrophysiological gains or losses of function in the study by Folacci et al [10]; the molecular modelling of these mutations or of the associated neuronal excitability confirms the electrophysiological findings.…”