title: Inhibition of KCa3.1 by depolarisation and 2-aminoethoxydiphenyl borate (2-APB) during Ca²⁺ release activated Ca²⁺ (CRAC) entry in human erythroleukemia (HEL) cells: Implications for the interpretation of 2-APB inhibition of CRAC entry.
abstract: In the present experiments in HEL cells, we have investigated the requirement for a hyperpolarised resting membrane potential for the initial activation of the Ca(2+) activated K(+) channel, KCa3.1, following activation of the Ca(2+) release activated Ca(2+) (CRAC) entry pathway. In intact cells, fluorimetric measurements of [Ca(2+)]i following thapsigargin-mediated activation of CRAC entry revealed a sustained increase in [Ca(2+)]i. Block of KCa3.1 by application of charybdotoxin resulted in a 50% reduction in the steady-state [Ca(2+)]i, consistent with the well established role for KCa3.1-mediated hyperpolarisation in augmenting CRAC entry. Interestingly, subsequent depolarisation to 0mV by application of gramicidin resulted in a fall in steady-state Ca(2+) levels to values theoretically below that required for activation of KCa3.1. Whole cell patch clamp experiments confirmed the lack of KCa3.1 activation at 0mV following activation of the CRAC entry pathway, indicating an absolute requirement for a hyperpolarised resting membrane potential for the initial activation of KCa3.1 leading to hyperpolarisation and augmented Ca(2+) entry. Current clamp experiments confirmed the requirement for a hyperpolarised resting membrane potential in KCa3.1 activation by CRAC entry. Given the critical role played by KCa3.1 and membrane potential in general in the control of CRAC-mediated [Ca(2+)]i changes, we investigated the hypothesis that inhibition of the CRAC-mediated changes in [Ca(2+)]i observed following 2-APB addition may in part arise from direct inhibition of KCa3.1 by 2-APB. Under whole cell patch clamp, 2-APB, at concentrations typically used to block the CRAC channel, potently inhibited KCa3.1 in a reversible manner (half maximal inhibition 14.2 μM). This block was accompanied by a marked shift in the reversal potential to depolarised values approaching that set by endogenous membrane conductances. At the single channel level, 2-APB applied to the cytosolic face resulted in a significant reduction in open channel probability and a fall in the mean open time of the residual channel activity. Our data highlight the absolute requirement for a hyperpolarising resting membrane conductance for the initial activation of KCa3.1 by CRAC entry. Additionally, our results document direct inhibition of KCa3.1 by 2-APB, thus highlighting the need for caution when ascribing the site of inhibition of 2-APB exclusively to the CRAC entry pathway in experiments where membrane potential is not controlled.
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK.
authors.forename: Robert
authors.initials: R
authors.lastname: Littlechild
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Zaidman
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Khodaverdi
authors.affiliation: Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK. Electronic address: mjm39@cam.ac.uk.
authors.forename: Michael James
authors.initials: MJ
authors.lastname: Mason
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry; D001896:Boron Compounds / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects*; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism / Q000473:pathology; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; D001896:Boron Compounds; D015220:Calcium Channels; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496923:KCNN4 protein, human; D014966:Xanthenes; C059715:Fluo-3; D019284:Thapsigargin; C109986:2-aminoethoxydiphenyl borate; D002118:Calcium
keywords: 2-APB; CRAC channel; Current clamp; HEL cells; Intracellular Ca(2+); KCa3.1; Membrane potential; Patch clamp