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

• Elevations in [Ca2+]i mediated by CRAC channel entry activates KCa3.1.
• Adequate Ca2+ entry for KCa3.1 activation requires a hyperpolarised potential.
• 2-Aminoethoxydiphenyl borate (2-APB) inhibits KCa3.1 resulting in depolarisation.
• 2-APB-induced depolarisation will reduce [Ca2+]i independent of CRAC block.
• Care must be taken when interpreting site of 2-APB block.

In the present experiments in HEL cells, we have investigated the requirement for a hyperpolarised resting membrane potential for the initial activation of the Ca2+ activated K+ channel, KCa3.1, following activation of the Ca2+ release activated Ca2+ (CRAC) entry pathway. In intact cells, fluorimetric measurements of [Ca2+]i following thapsigargin-mediated activation of CRAC entry revealed a sustained increase in [Ca2+]i. Block of KCa3.1 by application of charybdotoxin resulted in a 50% reduction in the steady-state [Ca2+]i, consistent with the well established role for KCa3.1-mediated hyperpolarisation in augmenting CRAC entry. Interestingly, subsequent depolarisation to 0 mV by application of gramicidin resulted in a fall in steady-state Ca2+ 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 0 mV 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 Ca2+ 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 [Ca2+]i changes, we investigated the hypothesis that inhibition of the CRAC-mediated changes in [Ca2+]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.

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