CRAC intermediate 1

Inhibition of KCa3.1 by depolarisation and 2-aminoethoxydiphenyl borate (2-APB) during Ca?? release activated Ca?? (CRAC) entry in human erythroleukemia (HEL) cells: Implications for the interpretation of 2-APB inhibition of CRAC entry

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.

Selective activation of KCa3.1 and CRAC channels by P2Y2 receptors promotes Ca(2+) signaling, store refilling and migration of rat microglial cells

Microglial activation involves Ca(2+) signaling, and numerous receptors can evoke elevation of intracellular Ca(2+). ATP released from damaged brain cells can activate ionotropic and metabotropic purinergic receptors, and act as a chemoattractant for microglia. Metabotropic P2Y receptors evoke a Ca(2+) rise through release from intracellular Ca(2+) stores and store-operated Ca(2+) entry, and some have been implicated in microglial migration. This Ca(2+) rise is expected to activate small-conductance Ca(2+)-dependent K(+) (SK) channels, if present. We previously found that SK3 (KCa2.3) and KCa3.1 (SK4/IK1) are expressed in rat microglia and contribute to LPS-mediated activation and neurotoxicity. However, neither current has been studied by elevating Ca(2+) during whole-cell recordings. We hypothesized that, rather than responding only to Ca(2+), each channel type might be coupled to different receptor-mediated pathways. Here, our objective was to determine whether the channels are differentially activated by P2Y receptors, and, if so, whether they play differing roles. We used primary rat microglia and a rat microglial cell line (MLS-9) in which riluzole robustly activates both SK3 and KCa3.1 currents. Using electrophysiological, Ca(2+) imaging and pharmacological approaches, we show selective functional coupling of KCa3.1 to UTP-mediated P2Y2 receptor activation. KCa3.1 current is activated by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC/Orai1) channels, and both CRAC/Orai1 and KCa3.1 channels facilitate refilling of Ca(2+) stores. The Ca(2+) dependence of KCa3.1 channel activation was skewed to abnormally high concentrations, and we present evidence for a close physical association of the two channel types. Finally, migration of primary rat microglia was stimulated by UTP and inhibited by blocking either KCa3.1 or CRAC/Orai1 channels. This is the first report of selective coupling of one type of SK channel to purinergic stimulation of microglia, transactivation of KCa3.1 channels by CRAC/Orai1, and coordinated roles for both channels in store refilling, Ca(2+) signaling and microglial migration.

Ca2+-Activated K+ Channel KCa3.1 as a Therapeutic Target for Immune Disorders

In lymphoid and myeloid cells, membrane hyperpolarization by the opening of K⁺ channels increases the activity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels and transient receptor potential (TRP) Ca²⁺ channels. The intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 plays an important role in cell proliferation, differentiation, migration, and cytokine production in innate and adaptive immune systems. K_Ca3.1 is therefore an attractive therapeutic target for allergic, inflammatory, and autoimmune disorders. In the past several years, studies have provided new insights into 1) K_Ca3.1 pharmacology and its auxiliary regulators; 2) post-transcriptional and proteasomal regulation of K_Ca3.1; 3) K_Ca3.1 as a regulator of immune cell migration, cytokine production, and phenotypic polarization; 4) the role of K_Ca3.1 in the phosphorylation and nuclear translocation of Smad2/3; and 5) K_Ca3.1 as a therapeutic target for cancer immunotherapy. In this review, we have assembled a comprehensive overview of current research on the physiological and pathophysiological significance of K_Ca3.1 in the immune system.

Role of KCa3.1 Channels in Modulating Ca2+ Oscillations during Glioblastoma Cell Migration and Invasion

Cell migration and invasion in glioblastoma (GBM), the most lethal form of primary brain tumors, are critically dependent on Ca²⁺ signaling. Increases of [Ca²⁺]_i in GBM cells often result from Ca²⁺ release from the endoplasmic reticulum (ER), promoted by a variety of agents present in the tumor microenvironment and able to activate the phospholipase C/inositol 1,4,5-trisphosphate PLC/IP? pathway. The Ca²⁺ signaling is further strengthened by the Ca²⁺ influx from the extracellular space through Ca²⁺ release-activated Ca²⁺ (CRAC) currents sustained by Orai/STIM channels, meant to replenish the partially depleted ER. Notably, the elevated cytosolic [Ca²⁺]_i activates the intermediate conductance Ca²⁺-activated K (KCa3.1) channels highly expressed in the plasma membrane of GBM cells, and the resulting K? efflux hyperpolarizes the cell membrane. This translates to an enhancement of Ca²⁺ entry through Orai/STIM channels as a result of the increased electromotive (driving) force on Ca²⁺ influx, ending with the establishment of a recurrent cycle reinforcing the Ca²⁺ signal. Ca²⁺ signaling in migrating GBM cells often emerges in the form of intracellular Ca²⁺ oscillations, instrumental to promote key processes in the migratory cycle. This has suggested that KCa3.1 channels may promote GBM cell migration by inducing or modulating the shape of Ca²⁺ oscillations. In accordance, we recently built a theoretical model of Ca²⁺ oscillations incorporating the KCa3.1 channel-dependent dynamics of the membrane potential, and found that the KCa3.1 channel activity could significantly affect the IP? driven Ca²⁺ oscillations. Here we review our new theoretical model of Ca²⁺ oscillations in GBM, upgraded in the light of better knowledge of the KCa3.1 channel kinetics and Ca²⁺ sensitivity, the dynamics of the Orai/STIM channel modulation, the migration and invasion mechanisms of GBM cells, and their regulation by Ca²⁺ signals.

Immune Checkpoint Inhibitors Regulate K+ Channel Activity in Cytotoxic T Lymphocytes of Head and Neck Cancer Patients

Programmed death receptor-1 (PD-1) and its ligand (PD-L1) interaction negatively regulates T cell function in head and neck squamous cell carcinoma (HNSCC). Overexpression of PD-1 reduces intracellular Ca²⁺ fluxes, and thereby T cell effector functions. In HNSCC patients, PD-1 blockade increases KCa3.1 and Kv1.3 activity along with Ca²⁺ signaling and mobility in CD8⁺ peripheral blood T cells (PBTs). The mechanism by which PD-L1/PD-1 interaction regulates ion channel function is not known. We investigated the effects of blocking PD-1 and PD-L1 on ion channel functions and intracellular Ca²⁺ signaling in CD8⁺ PBTs of HNSCC patients and healthy donors (HDs) using single-cell electrophysiology and live microscopy. Anti-PD-1 and anti-PD-L1 antibodies increase KCa3.1 and Kv1.3 function in CD8⁺ PBTs of HNSCC patients. Anti-PD-1 treatment increases Ca²⁺ fluxes in a subset of HSNCC patients. In CD8⁺ PBTs of HDs, exposure to PD-L1 reduces KCa3.1 activity and Ca²⁺ signaling, which were restored by anti-PD-1 treatment. The PD-L1-induced inhibition of KCa3.1 channels was rescued by the intracellular application of the PI3 kinase modulator phosphatidylinositol 3-phosphate (PI3P) in patch-clamp experiments. In HNSCC CD8⁺ PBTs, anti-PD-1 treatment did not affect the expression of KCa3.1, Kv1.3, Ca²⁺ release activated Ca²⁺ (CRAC) channels, and markers of cell activation (CD69) and exhaustion (LAG-3 and TIM-3). Our data show that immune checkpoint blockade improves T cell function by increasing KCa3.1 and Kv1.3 channel activity in HNSCC patients.