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Methylene Blue Inhibits Cromakalim-Activated K+ Currents in Follicle-Enclosed Oocytes
Dmytro Isaev1, Keun-Hang Susan Yang2, Georg Petroianu3
1Department of Cellular Membranology, Bogomoletz Institute of Physiology, 01024 Kiev, Ukraine.
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.
Insights
Methylene blue (MB) directly inhibits potassium (K+) currents activated by cromakalim in Xenopus oocytes. This action on KATP channels occurs independently of calcium pathways and guanylate cyclase signaling.
Area of Science:
- Pharmacology
- Molecular Biology
- Oocyte Physiology
Background:
- Potassium channels, particularly ATP-sensitive potassium (KATP) channels, play crucial roles in cellular function.
- Cromakalim is a known activator of KATP channels.
- Methylene blue (MB) is a compound with various biological effects, but its precise interaction with ion channels requires elucidation.
Purpose of the Study:
- To investigate the inhibitory effects of methylene blue (MB) on cromakalim-induced potassium (K+) currents.
- To determine the mechanism of MB's action on KATP channels in Xenopus oocytes.
- To explore potential signaling pathways involved in MB's inhibitory effect.
Main Methods:
- Electrophysiological recordings of K+ currents in follicle-enclosed Xenopus oocytes.
- Application of varying concentrations of methylene blue (MB) and cromakalim.
- Utilizing calcium chelator BAPTA and a C-terminally deleted Kir6.2 subunit (KirΔC26).
- Radioligand binding assays with [3H]glibenclamide and experiments with cGMP analogues and guanylate cyclase activators.
Main Results:
- Methylene blue (MB) noncompetitively and voltage-independently inhibited cromakalim-activated K+ currents with an IC50 of 22.4 μM.
- MB's inhibitory effect was not altered by BAPTA, indicating no involvement of Ca2+-activated pathways.
- MB inhibited K+ currents through KirΔC26, suggesting direct interaction with the Kir6.2 subunit.
- MB did not affect [3H]glibenclamide binding or interfere with cGMP-mediated signaling.
Conclusions:
- Methylene blue (MB) directly inhibits cromakalim-activated K+ currents in Xenopus oocytes.
- The mechanism of inhibition involves direct interaction with the Kir6.2 subunit of KATP channels.
- MB's action is independent of calcium signaling and guanylate cyclase pathways.
