Electrophysiological Properties of Endogenous Single Ca2+ Activated Cl- Channels Induced by Local Ca2+ Entry in HEK293.

Dmitrii Kolesnikov,Anastasiia Perevoznikova,Alexey Shalygin,Lyubov Glushankova,Elena Kaznacheyeva,Konstantin Gusev

doi:10.3390/ijms22094767

Dmitrii Kolesnikov, Anastasiia Perevoznikova + Show 4 more

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https://doi.org/10.3390/ijms22094767

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Abstract

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca2+ entry (SOCE). Ca2+ release through inositol 1,4,5-trisphosphate receptor (IP3R) and subsequent Ca2+ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca2+, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca2+ entry through TRPC1 channels activated endogenous Ca2+-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca2+ concentration and/or transmembrane potential, conversely lowering of intracellular Ca2+ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca2+ concentration. Experiments with Ca2+ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca2+-activated chloride channels are involved in Ca2+ entry microdomains.

Highlights

Published: 30 April 2021In non-excitable cells, activation of phospholipase C (PLC) mediates the calcium (Ca2+ )release from the inositol 1,4,5-trisphosphate (IP3 )-sensitive intracellular Ca2+ stores, activation of STIM proteins which are sensors of intraluminalCa2+ concentration, and Ca2+ influx through the plasma membrane store-operated Orai channels [1,2,3]
We studied the functional interaction between Ca2+ -activated chloride channels (CaCCs) and TRPC1 channels by means of “slow” (EGTA) and “fast” (BAPTA)
The application of 2.5 μM IP3 in the inside-out patches of HEK293 cells resulted in the activation of inward calcium channels followed by activation of outward currents pronounced at positive potentials (Figure 1A)

Summary

Introduction

Ca2+ concentration, and Ca2+ influx through the plasma membrane store-operated Orai channels [1,2,3]. Store depletion and Orai channel activation can lead to the activation of TRPC (transient receptor potential-canonical) channels, which amplify and modulate downstream Ca2+ signaling [4,5]. On the one hand TRPC1 can be directly regulated by STIM proteins in overexpressed systems [6,7], on the other hand in some cells TRPC1 activation by store depletion requires Ca2+ entry through Orai channels [8]. It was shown that in T-lymphocytes store-operated channels have impact on the activity of Ca2+ -activated chloride channels (CaCCs) and, by this way, cell proliferation [9]

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