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

Highlights

• 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.

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 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.

Introduction

Ca²⁺ entry mediated by depletion of endosomal Ca²⁺ stores in non-excitable cells is a ubiquitous mechanism leading to Ca²⁺ mediated down-stream signalling events. The role of intracellular stores in regulation of the Ca²⁺ permeability of the plasma membrane was first highlighted by Putney and co-workers in 1986 [1]. Store operated Ca²⁺ entry (SOCE) as it has subsequently come to be known has since been under extensive investigation and has moved from the observation of augmented Ca²⁺ entry following depletion of endosomal Ca²⁺ stores [1], to detection of an inward Ca²⁺ current accompanying store depletion [2], to the more recent identification of several components of the transduction and pore-forming elements including Orai1, 2 and 3 and Stim1 and 2 which signal the Ca²⁺ loss from endosomal compartments to the Orai components in the plasma membrane [as reviewed in 3]. The best electrophysiologically studied variant of the current is the calcium release activated Ca²⁺ (CRAC) channel, first isolated by Hoth and Penner [2] in rat basophilic leukemia cells and later by Zwiefach and Lewis [4] in Jurkat cells. This current displays marked inward rectification thus making net Ca²⁺ entry particularly susceptible to depolarisation.

SOCE pathways and CRAC entry in particular, play important roles in health and disease [as reviewed in 5]. As such, Ca²⁺ entry pathways controlled by the Ca²⁺ status of intracellular Ca²⁺ stores are important targets for therapeutic modulation. To date, high affinity, selective blockers of the signalling cascade leading to SOCE have remained elusive. Numerous organic compounds are known to block CRAC currents and inhibit elevations in [Ca²⁺]_i ascribed to Ca²⁺ entry by these pathways [as reviewed in 5]. 2-Aminoethoxyphenyl borate (2-APB) is a well established blocker of CRAC currents [6], [7], [8]. Its ability to modulate CRAC currents is complex. At low concentrations it has been shown to augment channel conductance while at high concentrations it is inhibitory [7]. As this compound became more widely used as a SOC/CRAC entry blocker its off target effects began to mount up. These include block of endosomal Ca²⁺ pumps [9], voltage-gated K⁺ channels [10], the non-selective cation channel TRPM7 [11], an Mg²⁺-inhibited K⁺ conductance described in human erythroleukemia (HEL) cells [12] and mitochondrial Ca²⁺ release [7]. Although it is well established to block CRAC currents at higher concentrations, its additional inhibitory influences make for cautious interpretation of effects observed during 2-APB application. This is particularly important when ascribing a site of action of this agent.

In the present experiments we have undertaken experiments to investigate the requirement for a hyperpolarised resting potential in ensuring adequate CRAC-mediated changes in [Ca²⁺]_i for activation of KCa3.1 in HEL cells. Our results highlight the absolute requirement for a hyperpolarised potential while confirming a critical role for the hyperpolarisation mediated by the Ca²⁺-activated K⁺ channel, KCa3.1 in maximising Ca²⁺ entry. Importantly, we demonstrate that 2-APB potently inhibits KCa3.1, independent of its effects on CRAC channel function and propose that inhibition of KCa3.1 may underlie, in part, the inhibitory influence of 2-APB on Ca²⁺ elevations mediated by CRAC entry in experiments in which membrane potential is not controlled.