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Suppression of human detrusor smooth muscle excitability and contractility via pharmacological activation of large conductance Ca2+-activated K+ channels

Sensitivity of TRPM7 channels to Mg2+ characterized in cell-free patches of Jurkat T lymphocytes

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Sensitivity of TRPM7 channels to Mg2+ characterized in cell-free patches of Jurkat T lymphocytes

Content Provider	PubMed Central
Author	Chokshi, Rikki Matsushita, Masayuki Kozak, J. Ashot
Copyright Year	2012
Abstract	Transient receptor potential melastatin 7 (TRPM7) channels were originally identified electrophysiologically when depletion of cytosolic Mg2+ resulted in the gradual development of an outwardly rectifying cation current. Conversely, inclusion of millimolar Mg2+ in internal solutions prevented activation of these channels in whole cell patch clamp. We recently demonstrated that the Jurkat T-cell whole cell TRPM7 channels are inhibited by internal Mg2+ in a biphasic manner, displaying high [IC50(1) ≈ 10 μM] and low [IC50(2) ≈ 165 μM] affinity inhibitor sites. In that study, we had characterized the dependence of the maximum cell current density on intracellular Mg2+ concentration. To characterize Mg2+ inhibition in Jurkat T cells in more detail and compare it to whole cell results, we recorded single TRPM7 channels in cell-free membrane patches and investigated the dependence of their activity on Mg2+ added on the cytoplasmic side. We systematically varied free Mg2+ from 265 nM to 407 μM and evaluated the extent of channel inhibition in inside-out patch for 58 patches. We found that the TRPM7 channel shows two conductance levels of 39.0 pS (γ1) and 18.6 pS (γ2) and that both are reversibly inhibited by internal Mg2+. The 39.0-pS conductance is the dominant state of the channel, observed most frequently in this recording configuration. The dose-response relation in inside-out patches shows a steeper Mg2+ dependence than in whole cell, yielding IC50(1) of 25.1 μM and IC50(2) of 91.2 μM.. Single-channel analysis shows that the primary effect of Mg2+ in multichannel patches is a reversible reduction of the number of conducting channels (No). Additionally, at high Mg2+ concentrations, we observed a saturating 20% reduction in unitary conductance (γ1). Thus Mg2+ inhibition in whole cell can be explained by a drop in individual participating channels and a modest reduction in conductance. We also found that TRPM7 channels in some patches were not sensitive to this ion at submaximal Mg2+ concentrations. Interestingly, Mg2+ inhibition showed the property of use dependence: with repeated applications, Mg2+ effect became gradually more potent, which suggests that Mg2+ sensitivity of the channel is a dynamic characteristic that depends on other membrane factors.
Related Links	http://dx.doi.org/10.1152/ajpcell.00037.2012
Starting Page	1642
File Format	PDF
ISSN	03636143
e-ISSN	15221563
Journal	American Journal of Physiology - Cell Physiology
Issue Number	11
Volume Number	302
Language	English
Publisher	American Physiological Society
Publisher Date	2012-06-01
Access Restriction	Open
Rights Holder	American Physiological Society
Subject Keyword	Cell Biology Physiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Cell Biology Physiology

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