Single Channel Properties and Regulated Expression of Ca2+ Release-Activated Ca2+ (Crac) Channels in Human T Cells

doi:10.1083/jcb.150.6.1435

Published online 18 September 2000. doi:10.1083/jcb.150.6.1435

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© The Rockefeller University Press, 0021-9525/2000//1435 $5.00
The Journal of Cell Biology, Volume 150, Number 6, , 2000 1435-1444

Original Article

Single Channel Properties and Regulated Expression of Ca²⁺ Release-Activated Ca²⁺ (Crac) Channels in Human T Cells

Alla F. Fomina^a, Christopher M. Fanger^a, J. Ashot Kozak^a, and Michael D. Cahalan^a

^a Department of Physiology and Biophysics, University of California Irvine, Irvine, California 92697-4561
Department of Physiology and Biophysics, University of California Irvine, Irvine, CA 92697-4561.(949) 824-3143(949) 824-7776

Although the crucial role of Ca²⁺ influx in lymphocyte activationhas been well documented, little is known about the propertiesor expression levels of Ca²⁺ channels in normal human T lymphocytes.The use of Na⁺ as the permeant ion in divalent-free solutionpermitted Ca²⁺ release-activated Ca²⁺ (CRAC) channel activation,kinetic properties, and functional expression levels to be investigatedwith single channel resolution in resting and phytohemagglutinin(PHA)-activated human T cells. Passive Ca²⁺ store depletionresulted in the opening of 41-pS CRAC channels characterizedby high open probabilities, voltage-dependent block by extracellularCa²⁺ in the micromolar range, selective Ca²⁺ permeation in themillimolar range, and inactivation that depended upon intracellularMg²⁺ ions. The number of CRAC channels per cell increased greatlyfrom $~$ 15 in resting T cells to $~$ 140 in activated T cells. Treatmentwith the phorbol ester PMA also increased CRAC channel expressionto $~$ 60 channels per cell, whereas the immunosuppressive drugcyclosporin A (1 µM) suppressed the PHA-induced increasein functional channel expression. Capacitative Ca²⁺ influx inducedby thapsigargin was also significantly enhanced in activatedT cells. We conclude that a surprisingly low number of CRACchannels are sufficient to mediate Ca²⁺ influx in human restingT cells, and that the expression of CRAC channels increases $~$ 10-fold during activation, resulting in enhanced Ca²⁺ signaling.

Key Words: T lymphocyte • Ca²⁺ channel • CRAC channel • T cell activation • Ca²⁺ signaling

Abbreviations used in this paper: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid; [Ca²⁺]_i, intracellular calcium concentration; CRAC, Ca²⁺release-activated Ca²⁺; CsA, cyclosporin A; HEDTA, N-hydroxyethyl-ethylenediamine-triaceticacid; K_Ca, Ca²⁺-activated K⁺; NF-AT, nuclear factor of activatedT cells; NMDG⁺, N-methyl D-glucamine; PHA, phytohemagglutinin;TCR, T cell receptor; Tg, thapsigargin.

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