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Biochemical, single-channel, whole-cell patch clamp, and pharmacological analyses of endogenous TRPM4 channels in HEK293 cells

Type of publication Peer-reviewed
Publikationsform Original article (peer-reviewed)
Publication date 2013
Author M.Y. Amarouch, N. Syam, H. Abriel,
Project Force-controlled patch clamp (pc-FluidFM)
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Original article (peer-reviewed)

Journal Neuroscience Letters
Volume (Issue) 541
Page(s) 105
Title of proceedings Neuroscience Letters
DOI 10.1016/j.neulet.2013.02.011

Abstract

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca2+-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22 pS conductance with a linear current–voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca2+. The channels were permeable to the monovalent cations Na+ and K+, but not to Ca2+. The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp “ruptured patch” configuration, the amplitude of the intracellular Ca2+-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca2+ sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9 ± 3.5 μM Ca2+ to be half-maximally activated versus 84.2 ± 1.5 μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca2+-activated channels.
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