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

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 K_ATP 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 K_ATP 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.