Ion channels and transporters in microglial function in physiology and brain diseases

Abstract

Microglial cells interact with all components of the central nervous system (CNS) and are increasingly recognized to play essential roles during brain development, homeostasis and disease pathologies. Functions of microglia include maintaining tissue integrity, clearing cellular debris and dead neurons through the process of phagocytosis, and providing tissue repair by releasing anti-inflammatory cytokines and neurotrophic factors. Changes of microglial ionic homeostasis (Na⁺, Ca²⁺, K⁺, H⁺, Cl⁻) are important for microglial activation, including proliferation, migration, cytokine release and reactive oxygen species production, etc. These are mediated by ion channels and ion transporters in microglial cells. Here, we review the current knowledge about the role of major microglial ion channels and transporters, including several types of Ca²⁺ channels (store-operated Ca²⁺ entry (SOCE) channels, transient receptor potential (TRP) channels and voltage-gated Ca²⁺ channels (VGCCs)) and Na⁺ channels (voltage-gated Na⁺ channels (Nav) and acid-sensing ion channels (ASICs)), K⁺ channels (inward rectifier K⁺ channels (K_ir), voltage-gated K⁺ channels (K_V) and calcium-activated K⁺ channels (K_Ca)), proton channels (voltage-gated proton channel (Hv1)), and Cl⁻ channels (volume (or swelling)-regulated Cl⁻ channels (VRCCs) and chloride intracellular channels (CLICs)). In addition, ion transporter proteins such as Na⁺/Ca²⁺ exchanger (NCX), Na⁺-K⁺-Cl⁻ cotransporter (NKCC1), and Na⁺/H⁺ exchanger (NHE1) are also involved in microglial function in physiology and brain diseases. We discussed microglial activation and neuroinflammation in relation to the ion channel/transporter stimulation under brain disease conditions and therapeutic aspects of targeting microglial ion channels/transporters for neurodegenerative disease, ischemic stroke, traumatic brain injury and neuropathic pain.

1. Introduction

Microglia, the resident immune cells of the central nervous system (CNS), originate from erythromyeloid progenitor cells in the embryonic yolk sac, migrate into the brain early in the development and then propagate, spread, and ramify throughout the brain parenchyma (Hansen et al., 2018a). Microglia constitute 5–10% of total brain cells in humans (Salter&Stevens, 2017; Li&Barres, 2018) and have been increasingly recognized as central players in CNS health and disease (Salter&Stevens, 2017). In healthy brains, microglia exhibit constant movement of processes to dynamically survey the brain environment for invading organisms, dying neurons, or synapses that need to be removed (Hansen et al., 2018a; Izquierdo et al., 2019). Microglia regulate neurogenesis by exerting trophic function, influencing programmed cell death, establishing and remodeling of neural circuits in the developing brain (Li&Barres, 2018). In response to pathogens or brain lesions, microglia directly extend processes to the regions of damage, engulf and phagocytose cellular debris, apoptotic neurons, or synapses, and generate immune-modulators, including reactive oxygen species (ROS) and cytokines that damage invading organisms, and alter neuronal and immune cell function, respectively (Izquierdo et al., 2019). Microglia also help prune developing synapses and regulate synaptic plasticity and function which provide new insights into how disruptions in microglia-synapse interactions could contribute to synapse loss and dysfunction, and consequently diseases (Hong et al., 2016). Lots of research have focused on changes of genes, such as Arg1, CD206 and iNOS, and changes of cytokines, such as TNF-α, IL-1β and IL-6 in microglial activation, especially during multi-dimensional activation transformation (Boche et al., 2013; Tang&Le, 2016); however, whether changes of intracellular ionic homeostasis and regulatory mechanisms play a role in microglial activation are less studied. Ion channels and transporters are involved in many microglial functions and their expression and function vary with different microglial morphological and functional states (Yu et al., 2015; Izquierdo et al., 2019). In this review, we focus on several major ion channels or transporters which regulate ionic changes, such as Ca²⁺, K⁺, Na⁺, H⁺ and Cl⁻, relating to microglial functions under physiological and pathophysiological conditions. (see Table 1)

Table 1.

Ion channels and transporters in microglia.

Microglial ion channels/transporters	Type/Subunit	Ionic change	Inhibitor	Physiological Properties	Microglial Activation	Brian Diseases	References
Ca2+ channels and transporters	store-operated Ca2+ entry (SOCE) channels/Orai1, Stim1, Stim2	Ca2+ entry	SKF96365, 2-APB	SOCE, the main Ca2+ influx mechanism in non-excitable cells, typically occurs as a result of receptor-induced Ca2+ release from the endoplasmic reticulum (ER)	Migration/invasion, ramified morphology, microglial podosome formation	AD	(McLarnon et al., 2005; Kettenmann et al., 2011; Siddiqui et al., 2012; Heo et al., 2015; Michaelis et al., 2015; Gilbert et al., 2016)
	transient receptor potential (TRP) channels/TRPV1, TRPV2, TRPV4, TRPC3, TRPC6, TRPM2	Ca2+ entry	ruthenium red, capsazepine, AMG9810, 2-APB, SKF, RN1734	TRP channels mostly are nonselective Ca2+-permeable cation channels involved in cellular processes such as cytokine production, proliferation, and migration, all of which are important cellular activities in microglia	production of pro-inflammatory cytokines, an increase in TNF-α and IL-β expression and release	neuropathic pain, AD, vibroacoustic disease	(Venkatachalam&Montell, 2007; Haraguchi et al., 2012; Shi et al., 2013a; Echeverry et al., 2016; Mizoguchi&Monji, 2017b; Alawieyah Syed Mortadza et al., 2018; Hansen et al., 2018b; Shirakawa&Kaneko, 2018)
	voltage-gated Ca2+ channels (VGCCs)/Cav1.2, Cav2.2/NCX	Ca2+ entry	nifedipine, nimodipine, verapamil, diltiazem, tamoxifen	At physiologic or resting membrane potential, VGCCs are normally closed, while they are activated on membrane depolarization	phagocytosis, production of anti-inflammatory cytokines	AD, Creutzfeldt-Jakob disease, PD, neuropathic pain, ischemic stroke	(Valerio Silei, 1999; Venkatachalam&Montell, 2007; Boscia et al., 2009; Catterall, 2011; Saegusa&Tanabe, 2014; Wang et al., 2019)
K+ channels	inward rectifier K+ channels (Kir)/Kir 2.1, Kir6.1	inward current	Chloroethylclonidine	The Kir channels are responsible for stabilization of the resting membrane potential. Blockade of Kir channels depolarizes the cell and decreases the driving force for inwardly transported Ca2+ in microglial cells	migration	PD	(Tsai et al., 2013; Du et al., 2018)
	voltage-gated K+ channels (Kv)/Kv1.3	outward current	agitoxin-2	The Kv channels are important regulators for its role of regulating resting membrane potential and repolarization	amyloid-mediated microglial priming; ROS production; active state modulation	AD	(Maezawa et al., 2018; Zhang et al., 2020a)
	calcium-activated K+ channels (KCa)/KCa3.1	outward current	TRAM-34, triazole, cyclohexadiene	The activity of KCa channels depends on the intercellular Ca2+ concentration and thus constitutes a link between its important second messenger system and electrical activity of microglia cells	volume regulation; migration	traumatic brain injury	(Khanna et al., 2001; Mauler et al., 2004; Weaver et al., 2006; Kaushal et al., 2007; Schlichter et al., 2010; Skaper, 2011a; Ferreira et al., 2014; Sforna et al., 2018)
Cl− channels and transporters	volume (or swelling)-regulated Cl-channels (VRCCs)	outward current	flufenamic acid, NPPB, SITS, DIOA	VRCCs are of particular importance in regulating the cell volume and give rise to the swelling-activated Cl− current (ICl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling	phagocytosis	-	(Furtner et al., 2007; Kittl et al., 2019)
	chloride intracellular channels (CLICs)/CLIC1	outward current	IAA-94, NPPB, NS1652, NS3728	CLIC1 was widely expressed and found in several cell lines and was associated with activation of microglia	proliferation; production of pro-inflammatory cytokines; iNOS expression and NO production	AD	(Kjaer et al., 2009; Kettenmann et al., 2011; Skaper et al., 2013; Barbieri et al., 2019)
Na+ channels and transporters	voltage-gated Na+ channels (Nav)	inward current	tetrodotoxin (TTX)-sensitive, TTX-resistant	Microglia express Nav as the main sodium channels for Na+load, regulation of membrane polarization, migration, and phagocytosis	phagocytosis; release of proinflammatory cytokines and ROS	ischemic stroke, PD, neuropathic pain	(Black et al., 2009; Persson et al., 2014; Liu et al., 2016; Hossain et al., 2018; Park et al., 2020)
	acid-sensing ion channels (ASICs)	inward current	amiloride (unspecific), PcTx1 (ASIC1a antagonist)	ASICs are the voltage-insensitive Na+channels activated by extracellular protons and display high Na+ permeability, contributing to microglial Na+ load and migration	associated with microglial iNOS and COX-2 expressions	LPS, scrape wound	Yu et al. (2015)
H+ channels and transporters	voltage-gated proton channel (Hv1)	inward current	Zn2+	Regulation of proton current through Hv1 mediates pH regulation and membrane hyperpolarization	oxidative damage through NOX activation and ROS production, proinflammatory responses	ischemic stroke, chronic hypoperfusion (BCAS), cuprizone-induced demyelination	(Wu et al., 2012; Liu et al., 2015a; Yu et al., 2018, 2020)
	Na+/H+ exchanger-1 (NHE1)	outward H+ in exchange of inward Na+ current	HOE642 (cariporide)	NHE1 is activated upon intracellular acidosis or cell shrinkage, and is important inmaintaining the basal pHi, microglial migration, and triggers Ca2+ signaling via NCXrey	cell swelling, chemotaxis/migration, oxidative damage through NOX activation and ROS production, release of proinflammatory cytokines	ischemic stroke, glioma	(Liu et al., 2010; Lam et al., 2013; Shi et al., 2013b; Zhu et al., 2016; Song et al., 2018; Hasan et al., 2020)

2. Calcium (Ca²⁺) channels and transporters in microglial function

2.1. Microglial Ca²⁺ channels and transporters in physiology

The microglial Ca²⁺ systems play a critical role in maintaining, handling and modifying the dynamic changes in the cellular Ca²⁺ levels (Giladi et al., 2016). Several evolutionary conserved molecular cascades are responsible for microglial Ca²⁺ transport across cellular membranes and intracellular Ca²⁺ buffering (Kettenmann et al., 2011). Microglial intracellular Ca²⁺ signals are shaped by electrochemically driven Ca²⁺ influx through membrane channels and receptors and Ca²⁺ efflux against the concentration gradient mediated by several families of Ca²⁺ pumps and exchangers (Kettenmann et al., 2011). These channels and transporters are differentially distributed within the cell, thus enabling different intracellular compartments to handle Ca²⁺ in a distinct way. Several Ca²⁺ channels and transporters have been identified on the surface of microglial cells, which act as doorways to help the Ca²⁺ enter or extrude from the cells and maintain intracellular Ca²⁺ homeostasis (Sharma&Ping, 2014). These microglial Ca²⁺ channels include store-operated Ca²⁺ entry (SOCE) channels, transient receptor potential (TRP) channels and voltage-gated Ca²⁺ channels (VGCCs), which mainly help Ca²⁺ enter the cell, while the Ca²⁺ extrusion from the cell is mainly through the Ca²⁺-ATPase and the Na⁺/Ca²⁺ exchanger (NCX) proteins (Table 1 and Fig. 1).

Fig. 1.

Ion channels and transporters in microglia. Dynamic microglial ionic homeostasis is important for microglial proliferation, migration, cytokine release, and reactive oxygen species (ROS) production. Dysregulation of microglial ion channels and transporters plays a role in neurodegenerative diseases and brain disorders. Especially, stimulation of Ca²⁺ channels (CRAC, TRP, VGCCs) and transporters (NCX) increases Ca²⁺ concentration, leading to microglial migration/invasion, proliferation, morphological changes, podosome formation, and production of TNF-α and IL-β pro-inflammatory cytokines. Other channels such as K⁺ channels (K_ir, K_ca, K_V), Na⁺ channels (ASICs, Na_V), and Cl⁻ channels (CLICs, VRCCs) also contribute to microglial migration, phagocytosis, production and release of ROS and cytokines. Activation of Na⁺/H⁺ exchanger 1 (NHE1) extrudes H⁺ in exchange of Na⁺ influx, leading to cell swelling, NCX reversal, chemotaxis migration, and oxidative damage.

2.1.1. SOCE channels

Intracellular Ca²⁺ signaling is important for microglial functions including regulation of microglial activation state, chemotaxis function (targeting processes to damaged areas), phagocytosis, and release of ROS and cytokines (Mizoguchi&Monji, 2017a; Tvrdik&Kalani, 2017; Izquierdo et al., 2019). The most important signaling factor in the differentiation of immune-active cells, such as microglial cells after stimulation, is the sustained elevation of Ca²⁺ concentration in the cytosol, which is needed for phagocytic receptors to respond and regulate subsequent steps that are involved in the maturation of phagosomes (Heo et al., 2015). SOCE is comprised of calcium release-activated Ca²⁺ channel (CRAC; Orai, highly Ca²⁺-selective channels) and stromal interaction molecule (Stim, the Ca²⁺ sensor in the endoplasmic reticulum (ER)) (Heo et al., 2015) (Fig. 1). Michaelis et al. (2015) reported that microglial SOCE is mediated by Stim1, Stim2 and Orai1 using experimental mice deficient for the above genes (Michaelis et al., 2015). Cyclopiazonic acid (CPA) is a blocker of ER Ca²⁺ pumps which could induce efficient store depletion and cause Ca²⁺ release followed by SOCE after Ca²⁺ re-addition. CPA-induced SOCE was nearly abolished in Stim1^−/− cells compared with wild-type. Stim2^−/− microglia showed a less pronounced but significantly decreased SOCE. In addition, SOCE was substantially decreased in Orai1^−/− microglia cells (Michaelis et al., 2015). Purines such as ATP, ADP and UDP released by damaged brain cells promote microglia activation, such as motility towards injury sites and phagocytosis which are mediated by calcium-dependent purinergic signaling, either via P2Y-induced Ca²⁺ release from internal stores or P2X-induced plasmalemmal Ca²⁺ entry (Gilbert et al., 2016). However, recent studies also showed that SOCE was also identified as an integral part of purinergic activation in primary mouse microglia (Heo et al., 2015; Michaelis et al., 2015). Pre-treatment with the pharmacological inhibitors of SOC (SKF96365 or 2APB) resulted in the absence of the UDP-induced increase in phagocytosis. Knockdown of Orai1 or Stim1 also showed the conclusive effect of SOCE on the phagocytic activity of microglia (Heo et al., 2015; Michaelis et al., 2015). In addition, intercellular Ca²⁺ elevation after stimulation is a central event in microglial cytokine secretion (Anja Hoffmann, 2003a), such as TNF-α and IL-6 which has been shown to contribute to nerve injury pain and neurodegenerative diseases (Tang&Le, 2016). Lipopolysaccharide (LPS), the stimuli of microglia, treatment for microglia cells induced TNF-α and IL-6 accumulation which were reduced by cotreatment with SOC inhibitor SKF96365 (Heo et al., 2015). Besides, CRAC/Orai1 channel is also involved in microglial podosome formation, migration and/or invasion (Siddiqui et al., 2012). Podosomes identified in several cell types have the ability to adhere to and degrade extracellular matrix (ECM) molecules (Murphy&Courtneidge, 2011). Siddiqui et al. (2012) found that microglial podosomes are enriched in CRAC/Orai1 channel and closely associated with Stim1 (Siddiqui et al., 2012). 50 μM of 2-APB and a more selective CRAC channel blocker BTP2 nearly abolished podosomes in microglia (Siddiqui et al., 2012). However, the detailed information of how CRAC/Orai1 regulate microglial podosomes formation needs further study.

2.1.2. TRP channels

TRP channels are also the main source for Ca²⁺ entry and play critical roles in responses to all major classes of external stimuli, including light, sound, chemicals, temperature, and touch (Venkatachalam&Montell, 2007). TRP channels mostly are nonselective Ca²⁺-permeable cation channels involved in cellular processes such as cytokine production, proliferation, and migration, all of which are important cellular activities in microglia (Shirakawa&Kaneko, 2018). The TRP family currently comprises 28 mammalian cation channels which can be divided into six subfamilies: TRPC (canonical), TRPM (melastatin), TRPV (vanilloid), TRPA (ankyrin), TRPP (polycystin), and TRPML (mucolipin) according to their sequence homology (Echeverry et al., 2016; Shirakawa&Kaneko, 2018). TRPV, TRPM, and TRPC have been shown to be expressed in microglia involved in osmotic regulation, cytokine production, proliferation, activation, cell death, and oxidative stress responses (Echeverry et al., 2016). TRPV1 activity in microglia has mainly been associated with neurotoxicity, through the production of proinflammatory cytokine and oxidative stress (Echeverry et al., 2016). TRPV1localized primarily in intracellular organelles such as mitochondrial plays an important role in inducing microglial migration (Miyake et al., 2015). Activation of TRPV1 triggers an increase in intra-mitochondrial Ca²⁺ concentration and depolarization of mitochondria, which stimulates ROS production, MAPK activation, and enhancement of chemotactic activity in microglia (Miyake et al., 2015). Hassan et al. (2014) also revealed that cannabidiol increased the expression of TRPV2 and TRPV1 proteins and caused a translocation of TRPV2 to the microglial BV-2 cell membrane which produced a sustained increase in intracellular Ca²⁺ concentration upregulated and was abolished by TRP channel blocker ruthenium red and the TRPV1 antagonists capsazepine and AMG9810 leading to enhanced phagocytosis of BV-2 cells (Hansen et al., 2018a). Mizoguchi et al. (2014) found that brain-derived neurotrophic factor (BDNF), which is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders, induces sustained intracellular Ca²⁺ elevation through the up-regulation of microglial surface TRPC3 channels and it could be important for the BDNF-induced suppression of the NO production in activated microglia (Mizoguchi et al., 2014).

2.1.3. VGCCs

Microglia cells were also reported to express VGCCs that mediates Ca²⁺ entry. VGCCs belong to several families of VGCCs, including Cav1, Cav2, and Cav3, with several subtypes within each family. The specific channels that have been detected in microglia are the Cav1.2 and Cav2.2 subtypes (Wang et al., 2019). Structurally, all VGCC family members are formed as complex of several different subunits: α1, α2, β, δ, and γ and the α1 subunit is the pore forming component and functions as the voltage sensor (Espinosa-Parrilla et al., 2015). The heterogeneity of each subunit gives rise to different VGCC pharmacological and electrophysiological properties and to the classification of the P-, Q-, N-, L-, R-, and T-type channels (Espinosa-Parrilla et al., 2015). In many different cell types, Ca²⁺ entering the cytosol via VGCCs regulates enzyme activity, gene expression, and other biochemical processes (Catterall, 2011). At physiologic or resting membrane potential, VGCCs are normally closed, while they are activated on membrane depolarization (Catterall, 2011). Many papers have shown that VGCCs modulate microglial pro-inflammatory activity and play an important role in several neurodegenerative diseases which will be discussed in detail in the following section.

2.1.4. NCX

Calcium extrusion following intracellular Ca²⁺ elevation is accomplished by plasmalemmal Ca²⁺ pumps (Ca²⁺-ATPase) and NCX (Kettenmann et al., 2011). The microglial Ca²⁺ pumps have not been yet characterized in detail which need further investigation. NCX is a bidirectional ion transporter that conducts Ca²⁺ efflux in exchange to Na⁺ influx. The gene family of mammalian NCX proteins consists of three gene isoforms (NCX1–3) which generate at least 17 splice variants (Giladi et al., 2016). Under physiological conditions, NCX operates in forward mode, transporting Na⁺ down their concentration gradient into cells and exporting Ca²⁺ in return (Takuma et al., 2013). However, the reverse mode of NCX becomes predominant in pathological settings, which can significantly alter Ca²⁺ homeostasis with output affecting numerous Ca²⁺-dependent events that take place at the cellular or systemic levels (Giladi et al., 2016). Newell et al. (2007) provided the direct evidence that Ca²⁺ entry by reversed NCX can contribute to microglia phagocytosis (Newell et al., 2007). Matsuda et al. (2006) found that NCX is involved in NO-induced depletion of Ca²⁺ in the endoplasmic reticulum (ER), leading to ER stress (Matsuda et al., 2006). Ifuka et al. (2007) also showed that blocking the reverse mode of NCX completely inhibited bradykinin-induced microglial migration (Ifuku et al., 2007).

2.2. Microglial Ca²⁺ channels and transporters in brain diseases

2.2.1. VGCCs

Microglial VGCCs have been shown to play a role in several neurodegenerative diseases (Saegusa&Tanabe, 2014; Wang et al., 2019). In microglia, VGCCs contribute to the increase in intracellular Ca²⁺ that occurs when the cell is in the presence of inflammatory stimuli (Valerio Silei, 1999). Microglia can exist in two activation states: the neuroinflammatory M1 state and the neuroprotective M2 state (Boche et al., 2013). The presence of acute inflammatory stimuli in microglia initially activates the neuroinflammatory state, but eventually the cell transitions to its neuroprotective state to eliminate the hazard and return the brain to homeostasis (Hopp, 2020). However, long-term stimuli, such as those associated with neurodegenerative diseases. e.g. Lewy bodies, Aβ and tau aggregates, can chronically activate the M1 state, causing inflammation and overall neurotoxicity. Once microglia reach this toxic state, they become less sensitive to additional stimuli, and neuroprotective effects are suppressed. The transition between these two states is very important and is regulated by multiple components, with evidence suggesting that levels of intracellular Ca²⁺ plays a role (Hopp, 2020). Anti-inflammatory stimuli, such as IL-4 or IL-10, reduce intracellular Ca²⁺ levels and promote the transition to the M2 state (Turovskaya et al., 2012). This activated state induces the phagocytotic abilities and production of anti-inflammatory cytokines by microglia for threat removal (Chhor et al., 2013). Acute inflammatory stimuli, such as liposaccharide (LPS), TNF-α, IL-1β, or IFNγ promote activation of the M1 state, causing microglia to release pro-inflammatory cytokines (Chhor et al., 2013). M1 activation by LPS leads to an increase in intracellular Ca²⁺ (Anja Hoffmann, 2003b), as evidenced by an increase in expression of the Cacna1c gene encoding the α−1 subunit of Cav1.2 channels in LPS-activated microglia (Wang et al., 2019).

Treatment of IL-4-activated microglia with nifedipine, a Cav1.2 antagonist, was shown to decrease expression of arginase 1, indicating a decline in M2 activation (Wang et al., 2019). However, treatment of LPS-activated microglia with nifedipine brought about conflicting results: with low doses of LPS/IFNγ, nifedipine enhanced M1 activation, but with higher doses of LPS/IFNγ, nifedipine suppressed M1 activation (Wang et al., 2019). This alludes to a functional role of VGCCs in the M1/M2 transition, although the exact nature of the relationship has yet to be specified. VGCCs have also been examined through the lens of Alzheimer’s disease and Creutzfeldt-Jakob disease (Valerio Silei, 1999). PrP106–126 and β25–35 are synthetic fragments of prion protein (PrP) and Aβ, which are involved in pathogenesis for Creutzfeldt-Jakob and Alzheimer’s diseases, respectively. A cell culture of microglia treated with PrP106–126 and β25–35 exhibited enhanced phagocytic activity, a 65% increase in proliferation, and an increase in intracellular Ca²⁺ levels. Adding verapamil to the cell culture reduced the increase in Ca²⁺ levels by 50% and reduced the increase in proliferation by almost 100%, demonstrating the role of VGCCs in microglia activation and the progression of inflammatory disorders(Valerio Silei, 1999). These results may prove favorable for microglia associated with neurodegenerative diseases. VGCC blockers may reduce activation of the M1 state, reducing the neurotoxic effects of the disease. VGCC antagonists include either dihydropyridines, such as nifedipine and nimodipine, or non-dihydropyridines, such as verapamil and diltiazem. Using these to block VGCCs have been shown to enhance the anti-inflammatory effects of microglia M2 configuration but do not affect the M1 configuration (Espinosa-Parrilla et al., 2015; Huntula et al., 2019).

Parkinson’s Disease is a hypokinetic disorder caused by neurodegeneration in the substantia nigra pars compacta. Death of dopaminergic neurons leads to tremors and overall difficulty with movement (Dauer&Przedborski, 2003). Current treatments involve alleviating symptoms by manipulating the dopamine pathway, but there is now some evidence suggesting Ca²⁺ channel antagonists as a possible remedy (Athauda&Foltynie, 2015). One experiment used a genetic approach to analyze the effect of VGCCs on an MPTP model of Parkinson’s Disease. Experimental mice were treated with tamoxifen to inhibit the expression of Cacna1c, specifically in microglia. With a 40% reduction in Cav1.2 expression, it was found that there was much greater death of dopaminergic neurons in Cav1.2 knock down mice compared to the control (Wang et al., 2019) This contradicts previous results that present Ca²⁺ channel antagonists as a possible treatment for neurodegenerative disorders (Valerio Silei, 1999; Wang et al., 2019), and that Ca²⁺channel blockers reduce the risk of developing Parkinson’s Disease (Ritz et al., 2010), but this may be due to the timing of measurements in that particular experiment (Wang et al., 2019).

VGCCs on microglia also play a part in the pathophysiology of neuropathic pain. Neuropathic pain is a chronic pain caused by injury to nerve fibers or neuronal receptors (Saegusa&Tanabe, 2014). It includes hyperalgesia-an increased sensitivity to an already painful stimulus, allodynia-sudden pain response to normally non-painful stimuli, and spontaneous pain that occurs without a stimulus. Tamoxifen-induced suppression of the Cacna1b gene, which encodes Cav2.2, resulted in attenuated allodynia but no significant effect on thermal hyperalgesia in a spinal nerve ligation model of neuropathic pain (Saegusa&Tanabe, 2014). Spinal cords of Cav2.2 knockdown mice had significantly less microglia than those of wild type mice, suggesting that Cav2.2 plays a role in microglial activation and proliferation, and is involved in the pathogenesis of allodynia (Saegusa&Tanabe, 2014).

2.2.2. SOCE channels

A study on SOCE revealed a correlation between dysregulation of Ca²⁺ within microglia and a presentation of Alzheimer’s (McLarnon et al., 2005). Alzheimer’s Disease is characterized by the loss of cognitive function due to deterioration of brain regions. Alzheimer’s patients experience neuronal cell death and aggregation of Aβ, tau, and α-synuclein proteins (Braak&Braak, 1991). There is a connection between protein aggregates, such as Aβ, in microglia and the inflammatory effects of M1 microglia activation (McLarnon et al., 2005). SOCE is essential for Ca²⁺ signaling pathways within microglia (Hopp, 2020). SOCE inhibitors reduce microglial migration to damaged areas, and LPS-treated microglia exhibit lower levels of SOCE (Michaelis et al., 2015), indicating that SOC channels are implicated in the neuroprotective functions of microglia. Ca²⁺ influx due to both depletion of ER stores and SOC-mediated entry was lower for Alzheimer microglia and human fetal microglia treated with Aβ than for healthy microglia (McLarnon et al., 2005). Alzheimer microglia and Aβ-treated microglia displayed an amoeboid morphology, indicating an activated state, as opposed to the ramified morphology of healthy microglia (McLarnon et al., 2005). Ca²⁺ mobilized from ER stores acts on SOCs and causes an even greater influx of Ca²⁺; there is a possibility of an unknown pathway or molecule that connects the depletion of ER stores to SOC calcium influx, and this unknown might be defective in Alzheimer’s microglia (McLarnon et al., 2005). Although the specifics are unknown, these results show that Alzheimer pathology is likely related to Ca²⁺ signaling within microglia, perhaps due to SOCs themselves or due to an indirect mechanism.

2.2.3. TRP channels

TRP channels have also been shown to play a part in microglial activation and possibly some disease pathology. As we have seen previously, LPS/IFN-γ treatment causes an increase in the expression of the pro-inflammatory cytokines, but this increase is greatly attenuated in TRPM2 KO microglia (Haraguchi et al., 2012). One experiment showed that genetic deletion of TRPV1 caused a decrease in microglial activation caused by various pain models (Chen et al., 2009). Furthermore, a partial spinal nerve ligation induces microglial activation, demonstrated by a shift to an amoeboid morphology and the presence of phosphorylated p-38 signal (Haraguchi et al., 2012). These activated microglia exhibited increased expression of TRPM2 mRNA, and TRPM2 KO mice showed impaired microglia activation (Haraguchi et al., 2012). TRPM2 may also be linked to the inflammatory effects of Alzheimer’s Disease. WT microglia exposed to Aβ saw an increase in TNF-α expression and release (Alawieyah Syed Mortadza et al., 2018), which was eliminated in TRPM2 KO microglia and microglia treated with 2-APB, a TRPM2 antagonist (Alawieyah Syed Mortadza et al., 2018). Moreover, microglial activation caused by Aβ exposure was not detected in TRPM2 KO microglia nor 2-APB-treated microglia (Alawieyah Syed Mortadza et al., 2018). From this evidence, it can be concluded that Aβ-dependent microglial activation and TNF-α release are associated with TRPM2 activity.

Consistent with the finding that Alzheimer pathology is connected to calcium mobilization in microglia (McLarnon et al., 2005), it was found that WT microglia bathed in extracellular calcium saw an increase in intracellular Ca²⁺ after Aβ₄₂ exposure, and this elevated calcium was absent in TRPM2 KO microglia, microglia treated with 2-APB, and microglia exposed to Aβ_42-1 (Alawieyah Syed Mortadza et al., 2018), suggesting that Aβ has a causative role in opening TRPM2 channels, and TRPM2 antagonists could be a possible therapeutic strategy for Alzheimer’s Disease. Another TRP channel, TRPC6, also has a part in the Aβ inflammatory response. TRPC KD and treatment with TRPC antagonist, SKF, reduced the expression of TNF-α, IL-1β, IL-6, and COX-2 caused by Aβ accumulation in microglia (Liu et al., 2017), and Aβ-treated microglia with TRPC6 KD caused less neuronal death than WT microglia (Liu et al., 2017). Moreover, microglia treated with Aβ caused upregulation of microglial TRPC6 by increasing both mRNA and cell surface expression (Liu et al., 2017).

Vibroacoustic Disease is a CNS disease caused by long-term or extreme exposure to infrasound or low frequency noise (Branco&Alves-Pereira, 2004). One experiment sought a connection between the TRPV4 channel in microglia/astrocytes and vibroacoustic disease. It was discovered that infrasound exposure caused an increase in expression of TRPV4 mRNA, and it prompted the release of IL-β and TNF-α, which was greatly attenuated both in glia treated with RN1734-a TRPV4 antagonist-and TRPV4 KD microglia (Shi et al., 2013a). Infrasound exposure also caused an increase in intracellular Ca²⁺ that was reduced by transfection with trpv4 siRNA and treatment with RN1734 (Shi et al., 2013a).

2.2.4. NCX

NCX, specifically the NCX1 isoform, has been shown to play a role in microglial function pertaining to ischemic stroke (Boscia et al., 2009). In vivo and in vitro experiments demonstrated that there is up-regulation of NCX1 in microglia plasma membrane after a permanent middle cerebral artery occlusion model of a stroke (Boscia et al., 2009). This upregulation was specific to microglia displaying the phagocytic M2 phenotype. Brains exposed to hypoxic conditions also saw an increase in NCX1 protein levels, as well as microglial activation, observed as an increase in Iba1 expression and NO production (Boscia et al., 2009). NCX1 also exhibited increased activity in the forward direction, and even higher activity in the reverse direction, as evidenced by altered intracellular calcium levels, which was prevented with siRNA knockdown of the Ncx1 gene (Boscia et al., 2009).

3. Potassium (K⁺) channels in microglial function

3.1. Microglial K⁺ channels in physiology

K⁺ channels are important in microglia since they participate in microglial membrane hyper-polarizations and volume regulation (working together with Cl⁻ channels in setting up local osmotic gradients) and thereby in important cellular functions, such as shape changes, phagocytosis, and migration toward chemotaxic stimuli (Nguyen et al., 2017). Patch-clamp studies of microglial cells showed that a wide variety of potassium channels including inward rectifier K⁺ channels (K_ir) (described in rat, murine, bovine, and human microglia), voltage-gated K⁺ channels (K_V) (described in rat, mouse, and human microglia) and calcium-activated K⁺ channels (K_Ca) (described in murine, bovine, and human microglia) were expressed in microglial cells (Kettenmann et al., 2011; Dolga&Culmsee, 2012) (Table 1 and Fig. 1), some of which facilitate refilling of intracellular Ca²⁺ stores through inward rectifying Ca²⁺-release-activated-Ca²⁺-channels, purinergic receptors, and other Ca²⁺-permeable cation channels, thus maintaining high Ca²⁺ and timing of intracellular signaling events important for microglia activation and proliferation (Nguyen, 2017).

The resting membrane potential of microglia cells in situ is typically around 45 mV, with a range from 20 mV to 60 mV (Izquierdo et al., 2019). This depolarized membrane potential in resting microglia is maintained primarily by two-pore domain halothane-inhibited K⁺ channel type 1 (THIK-1) K⁺ channels, which are tonically active, but can also be potentiated by extracellular ATP and ADP via P2Y12 receptors to hyperpolarize microglia. Voltage-gated, Ca²⁺-gated, and inwardly rectifying K⁺ channels may contribute to a negative potential in activated microglia (Izquierdo et al., 2019).

The K_ir channels have only two membrane-spanning domains and are responsible for stabilization of the resting membrane potential, and blockade of K_ir channels depolarizes the cell and decreases the driving force for inwardly transported Ca²⁺ in macrophages or microglial cells (Tsai et al., 2013). There are 7 subfamilies of K_ir channels, denoted as K_ir1 - K_ir7, in which K_ir3 belongs to G protein-coupled inwardly-rectifying K⁺ channels and K_ir6 belongs to ATP-sensitive K⁺ channels (K_ATP). Muessel et al. (2013) reported that the K_ir2.1 channel is mediated by the small GTPases, Rac and Rho, that regulate actin cytoskeleton. Stromal cell-derived factor (SDF)-1α chemokine rapidly increased the K_ir2.1 current amplitude and murine microglia cell spreading which was blocked by inhibiting the Rac1by transiently transfecting DNA encoding dominant-negative Rac1 (17N) into primary microglia. In contrast, bioactive lysophospholipids such as lysophosphatidic acid (LPA) activated the RhoA that led to decreased K_ir2.1 currents and stimulated microglia contraction. The inhibition of K_ir2.1 with chloroethylclonidine produced cell contraction independently of chemokine action. This suggests that K_ir2.1 channel is essential for the morphological phenotype and functioning of microglia (Muessel et al., 2013). The K_ATP channels are a type of K⁺ channel that are gated by intracellular nucleotides, ATP and ADP. ATP-sensitive K⁺ channels are composed of K_ir6.x (6.1 or 6.2) subunits and sulfonylurea receptor (SUR1 or SUR2) subunits (Du et al., 2018). As a metabolic sensor, K_ATP channels are widely expressed in most metabolically active tissues, including brain, heart and pancreatic β-cells. Within the brain, K_ir6.2 is predominantly expressed in neurons while K_ir6.1 is mainly expressed in microglia that is essential for M2 microglia polarization (Du et al., 2018). However, little is known about the role of G protein-coupled inwardly-rectifying K⁺ channels in microglia activation, further study should be investigated. The K_V channels belong to one of the largest and highly evolutionarily conserved ion channel families. Each K_V channel contains 4 similar or identical pore-forming α-subunits, and it may also contain auxiliary β-subunits that could affect the channel function and/or localization (Gonzalez et al., 2012). The K_V channels are also important regulators for its role of regulating resting membrane potential and repolarization (Zhang et al., 2020a). The K_Ca channels share their regulation by the intracellular Ca²⁺,and are divided into three principle varieties that differ with regards to their biophysical and pharmacological features (Weaver et al., 2006). Large conductance calcium-activated potassium (BK) channels, such as K_Ca1.1, exhibit a very high single-channel conductance (100–300 pS) which are activated by the concerted influences of membrane depolarization and increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (Vergara et al., 1998). Intermediate conductance calcium-activated potassium (IK) channels derive from a closely related gene (K_Ca3.1) that gives rise to channels of ~20–60 pS conductance, such as a value intermediate to small (SK) and big conductance (BK) channels. Small conductance calcium-activated potassium (SK) channels belong to the KCNN gene family and have a relatively small conductance of ~5–10 pS (Weaver et al., 2006). SK channels are predominantly expressed in the nervous system and are gated only by rises in [Ca²⁺]_i (Weaver et al., 2006). Following differentiation with LPS or a combination of LPS and IFN-γ, classically activated M1-like microglia exhibited high Kv1.3 current densities (Nguyen et al., 2017; Di Lucente J, 2018)and virtually no K_Ca3.1 (Blomster et al., 2016) and K_ir currents, while alternatively activated M2-like microglia differentiated with IL-4 exhibited large K_ir2.1 currents (Nguyen, 2017). K_Ca3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (Nguyen, 2017). Genetic knockout of Kv1.3 abolished LPS-induced microglial activation exemplified by Iba-1 immunoreactivity and expression of pro-inflammatory mediators IL-1β, TNF-α, IL-6 and iNOS (Di Lucente J et al., 2018). Moreover, Kv1.3 knockout mitigated the LPS-induced impairment of hippocampal long-term potentiation (hLTP), suggesting that Kv1.3 activity regulates pro-inflammatory microglial neurotoxicity (Di Lucente J et al., 2018). Blockade of Kv1.3 or K_Ca3.1 also inhibited pro-inflammatory cytokine production and iNOS expression (Nguyen et al., 2017). The effect of the above microglial K⁺ channels on brain diseases will be discussed in the next section.

3.2. Microglial K⁺ channels in brain diseases

Upon CNS insult, microglia respond by increasing regulation of Ca²⁺-dependent functions, including activation of small- and intermediate-conductance Ca²⁺-activated K⁺ channels such as K_Ca3.1 (also known as KCNN4, SK4, IK1), a calcium-activated potassium channel highly prevalent in the microglia of rat brains (Skaper, 2011a). K_Ca3.1 plays a role in general cellular processes such as intracellular Ca²⁺ regulation, cell volume regulation, and cell migration (Schilling et al., 2004; Sforna et al., 2018) and has also been implicated in the regulation of glial immune response in numerous studies (Khanna et al., 2001; Kaushal et al., 2007). One such study demonstrated that inhibition of K_Ca3.1 channel by selective blocker TRAM-34 in LPS-M1-state-induced rat microglia results in a statistically significant neuroprotective effect in neural cell cultures, with TRAM-34 treated cultures displaying levels of apoptotic marker TUNEL at levels similar to control group cells (Kaushal et al., 2007). Similarly, in a rat model of traumatic brain injury, it was found that rats treated with a K_Ca3.1 inhibitory triazole (1-[(2-chlorophenyl) diphenylmethyl]-1,2,3-triazole) and cyclohexadiene (methyl4-[4-chloro-3-(trifluoromethyl)phenyl]-6-methyl-3-oxo-1,4,7-tetrahydroisobenzofuran-5-carboxylate) during induced subdural hematoma surgery experienced significantly less severe edema, intracranial pressure, and infarct volume than control group subjects (Mauler et al., 2004).

Additionally, research has shown that respiratory burst, the process by which reactive oxygen species (ROS) are released following microglial activation in the CNS, is significantly reduced in rat microglial cell cultures treated with channel inhibitors (names in parentheses) for SK2 (apamin), SK4 (clotrimazole and charybdotoxin), and Kv1.3 (agitoxin-2) channels. Respiratory burst was simulated through addition of phorbol 12-myristate 13-acetate to cultures and was measured through fluorescent imaging using respiratory marker dihydrorhodamine-123 (Khanna et al., 2001). In contrast to these findings, microglia treated with IL-4 to induce the M2 modulation state display KCNN4 transcript up-regulation, leading to an increase in K_Ca3.1 current that plays a neuroprotective role in the CNS by increasing migratory capacity of the alternatively activated glial cells (Ferreira et al., 2014). This dichotomy in which the K_Ca3.1 channel can act in either a neurodegenerative or neuroprotective role depending on the activated phenotype of the glial environment illustrates the relevancy and potential of microglial phenotype modulation therapy as a means of addressing neurophysiological disorders in the future.

As one of the most common neurodegenerative disorders of our time, Parkinson’s disease (PD) is an example of a neuropathology that has garnered interest amongst the scientific community within the context of microglial activation-state modulation therapy. PD is characterized by the death of dopaminergic neurons (DA) within the substantia nigra compacta (SNc) as well as a marked increase in pro-inflammatory activity of microglia in patients suffering from the disorder (Du et al., 2018). In an experimental PD model induced by MPTP, it was reported that the Kir6.1-containing ATP-sensitive potassium (Kir6.1/K-ATP) channel is involved in the polarization state of mouse microglia. When compared to Kir6.1+/+ mice, Kir6.1 ± specimens treated with MPTP showed a significantly greater decrease in Tyrosine hydroxylase-stained SNc DA neurons as well as a markedly greater increase in M1 activation state marker ionized calcium-binding adaptor molecule 1 (IBA-1). Kir6.1 ± mice exhibited increased M1 activation and reduced M2 polarization, which is accompanied with worsened DA neurodegeneration within the SNc (Du et al., 2018). These findings suggest that increased Kir6.1/K-ATP channel expression and activation increase activation of the M2 microglial phenotype. Conversely, decreased Kir6.1/K-ATP channel expression was concluded to lead to exaggerated M1 microgliosis via disinhibition of the p38 MAPK-NF-κB microglial activation pathway (Du et al., 2018). Western blotting analysis of post-mortem PD brain samples has shown that PD brains contain a high Kv1.3 channel concentration in their substantia nigra (SN) relative to controls. Immunostaining showed increased immunoreactivity of Kv1.3 and IBA1 colocalization mainly in the prefrontal cortex and SN of PD brains (Sarkar et al., 2020).

Researchers have also managed to draw connections between Alzheimer’s disease (AD) and microglial K⁺ channels. Specifically, in an immunohistochemical semi-quantitative analysis of cryopreserved human AD patient brains, it was observed that-relative to controls-AD patient brains contain a significantly higher Kv1.3 staining density and positive cell density (p = 0.03 for both) in microglia of the frontal cortex (Rangaraju et al., 2015). A more recent study showed that PAP-1 inhibition of Kv1.3 channels in AD model APP/PS1 transgenic mice from 9 to 14 months of age resulted in decreased neuroinflammation and cerebral amyloid load, increased hippocampal neuronal plasticity, and improved behavioral deficits of treatment group mice with respect to the control group (Maezawa et al., 2018). In both investigations, Kv1.3 was identified as playing a central role in the activated M1 phenotype response in microglia, mediating activities such as amyloid-mediated microglial priming and ROS production while contributing to chronic neuroinflammation (Rangaraju et al., 2015; Maezawa et al., 2018).

With respect to neuroinflammatory responses, it was found that in response to LPS stimulation, the presence of Kv1.3 blockers (PAP-1/ShK-186) and K_Ca3.1 blockers (TRAM-34) in cultured mouse microglial cells decreased microglial expression and secretion of the pro-inflammatory IL-1β and TNF-α relative to controls (per qPCR and ELISA quantification, respectively) (Nguyen et al., 2017). Moreover, these treated microglial cells displayed decreased iNOS expression and NO production at 24 and 48 h after LPS stimulation. Especially, Western blotting at the 48-h timepoint demonstrated a significant reduction in iNOS as well as COX-2 protein expressions in microglial cultures that were administered with PAP-1 (H.M. Nguyen et al., 2017). Recent research using flow cytometry has shown that activation of the M1 phenotype via LPS or αSyn_Agg treatment results in markedly increased surface expression of Kv1.3 channels concurrent with whole-cell patch clamp current density changes typical of Kv1.3 channel activity in cultured mouse microglia. qRT-PCR and Western blotting similarly showed increases in Kv1.3 mRNA and protein expression in αSyn_Agg-treated cells (Sarkar et al., 2020). Kv1.3 has thus emerged as one of the foremost membrane channels to garner interest from scientists in the realm of microglial neuroinflammatory research.

4. Chloride (Cl⁻) channels and transporters in microglial function

4.1. Microglial Cl⁻ channels and transporters in physiology

Chloride channels play a vital role in cellular physiology including stabilization of cell membrane potential, transepithelial transport, maintenance of intracellular pH, cell proliferation, fluid secretion and regulation of cell volume (Gururaja Rao et al., 2020). Chloride channels expressed in microglia can be classified as members of the volume (or swelling)-regulated Cl⁻ channels (VRCCs) and chloride intracellular channels (CLICs) (Table 1 and Fig. 1). VRCCs are of particular importance in regulating the cell volume and give rise to the swelling-activated Cl⁻ current (I_Cl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling (Kittl et al., 2019). Harl et al. (2007) found that phagocytose of apoptotic and necrotic cells by microglia cells during the physiology and pathology process increased cellular volume and activated VRCCs in volume regulation (Furtner et al., 2007). Blockade of VRCCs by flufenamic acid (200 μM) or 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 200 μM), eliminated uptake of microspheres almost completely. SITS (1 mM), which blocks VRCCs and to some extend K–Cl cotransporters (KCC), had only a moderate inhibiting impact on particle uptake. DIOA, a compound that inhibits KCC as well as VRCCs, did not inhibit particle uptake in BV-2 cells (Furtner et al., 2007). However, osmotic challenge by hypoosmotic saline (60% saline) elicited a swelling-activated chloride channel sensitive to SITS (1 mM) and flufenamic acid (200 μM) (Furtner et al., 2007). Harl et al. (2013) further tested whether VRCCs, required for global cell volume (CV) regulation, also contributes to local expansion and retraction of engulfment pseudopodia in microglial BV-2 cells (Harl et al., 2013). Exposure of BV-2 cells to microbeads (MBs) in Cl⁻-free extracellular solution attenuated MB uptake and the Cl⁻-channel blockers DIOA, flufenamic acid, NPPB and DCPIB suppressed the uptake of MBs which is indicating that I_Cl,swell contributes to formation of engulfment pseudopodia and participates in engulfment and particle uptake in microglial cells (Harl et al., 2013).

The CLIC family consists of six evolutionarily conserved proteins in humans (Al Khamici et al., 2015). CLIC1 is the most widely expressed and studied channel of this family, in both physiological and pathological conditions, including brain functioning and cancer cell proliferation (Barbieri et al., 2019). The functional expression of CLIC-1 proteins was identified in primary rat neonatal microglial cultures (Kettenmann et al., 2011) and presents with the unique feature of being able to convert between a soluble cytoplasmic conformation and a transmembrane isoform (tmCLIC1), a property termed “metamorphic” (Skaper et al., 2013). The activation of microglia with LPS orbasic fibroblast growth factor affected neither the expression nor the functional properties of these Cl⁻ channels. However, inhibition of CLIC-1 channels with the specific blockerIAA-94(R(+)-[(6,7-dichlor-o-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy]acetic acid) suppressed microglial proliferation and reduced neurotoxicity of Aβ-treated microglial cells (Kettenmann et al., 2011; Barbieri et al., 2019). The detailed information about microglial CLIC-1 that involved in the pathophysiology of AD will be discussed in the following section.

The modulation of intracellular Cl⁻ was also regulated by potassium-chloride cotransporter (KCC2) and sodium-potassium-chloride cotransporter (NKCC1). KCC2 is neuron-specific and will not be further discussed here. NKCC1 is widely distributed throughout the body including many regions of the brain. However, little is known about the role of NKCC1 in microglia cells. A recent study showed that microglia secreted cytokine IL-18 induced depression-like behaviors may be mediated by the IL-18R/NKCC1 signaling in the late phase of mice with post-stroke depression (Wu et al., 2020) which will be further discussed in the following section.

4.2. Microglial Cl⁻ channels and transporters in brain diseases

Chloride channels are involved innumerous steps in the microglial activation process, such as chemotaxis, ramification, proliferation and production of pro-inflammatory cytokines and neurotoxic factors as well as phagocytosis that related to neurodegenerative diseases (Kjaer et al., 2009). Proliferation of activated microglia is dependent on chloride channel activation and specifically in response to β-amyloid (Aβ) exposure (Kjaer et al., 2009). Activation of microglia by Aβ peptide resulted in an overall increase in CLIC1 protein with resultant increase in CLIC1 activity (Skaper, 2011b). Furthermore, both IAA94, a CLIC1 chloride channel blocker and knockdown of CLIC1 by RNA interference acted broadly to inhibit the production of pro-inflammatory and neurotoxic products elaborated by Aβ-stimulated microglial cells and function as neuroprotective tools in neuron-microglia co-cultures (Skaper, 2011b). CLIC1 exposed to Aβ underwent acute translocation from the cytosol to the plasma membrane of microglia, resulting in the appearance of an anion conductance which was essential for reactive oxygen species generation by NADPH oxidase, and was itself regulated by it, thus defining a fundamental role for CLIC1 in Aβ-induced oxidative stress (Skaper, 2011b). Indeed, Aβ fragment (25–35)(Aβ_25–35) stimulation of either primary rat microglia or the microglial cell line BV2 not only up-regulated CLIC1 protein but also led to a change in membrane conductance and blockade of CLIC1 channel limited injury to hippocampal neurons in co-culture with microglia and Aβ_25–35 (Skaper et al., 2013). Kjaer et al. (2009) also found that CLIC1channel blockers such as NPPB, IAA-94, (Z)-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine (tamoxifen), 2-[3-(3-trifluoromethylphenyl)ureido] benzoic acid (NS1652) and N-[3,5-bis(trifluoromethyl)phenyl]-N^’-[4-bromo-2-(1H-tetrazol-5-yl)phenyl]urea (NS3728) inhibited iNOS expression and NO production in IFNγ-stimulated microglial BV2 cells (Kjaer et al., 2009). However, little is known about the effect of microglial VRCCs on brain diseases. Microglial NKCC1 was recently reported to be involved in IL-18/IL18 receptor-induced depressive behaviors (Wu et al., 2020). IL-18R was detected in immunoprecipitated protein by anti-NKCC1 antibody, suggesting that IL-18R and NKCC1 may form a complex or interact in the cellular membrane (Wu et al., 2020). Blockade of NKCC1 with its inhibitor bumetanide significantly decreased immobility time in tail suspension test and in forced swimming test in IL-18-induced depression mice (Wu et al., 2020).

5. Sodium (Na⁺) channels and transporters in microglial function

5.1. Microglial Na⁺ channels and transporters in physiology

Sodium channels are essential for cell membrane depolarization to initiate and propagate action potentials in the excitable cells (Mercier et al., 2018). As the non-excitable glia cells, microglia also express voltage-gated sodium channels (Nav) as the main sodium channels to participate in fast-activating/inactivating Na⁺ currents for regulation of their functions, such as migration, phagocytosis, and secretion of cytokines (Pappalardo et al., 2016) (Table 1 and Fig. 1). Microglia express a variety isoforms of Nav, including Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.5, Nav 1.6, Nav 1.7, Nav 1.8, and Nav 1.9, and Nav 2.1 in either microglial cell line (the BV2 cells) or primary microglial culture (Hossain et al., 2017). These Nav channels can be categorized into two different types based on their sensitivity to the Nav-specific antagonists tetrodotoxin (TTX): Nav 1.1, Nav 1.2, Nav 1.3, Nav 1.4, Nav 1.6, and Nav 1.7 are sensitive to nanomolar levels of TTX (TTX-sensitive), while Nav 1.5, Nav 1.8, and Nav 1.9 are only responsive to micromolar levels of TTX (TTX-resistant) (Pappalardo et al., 2016; Mercier et al., 2018). Using advanced techniques such as the whole-cell voltage clamp, depolarization-induced Na⁺ currents were detected in microglia, which was completely blocked by 0.3 μM TTX, indicating the presence and functionality of TTX-sensitive Nav in mediating Na⁺ load in microglia (Pappalardo et al., 2016). Nav activity is especially important for the short-term/immediate Na⁺ load in LPS-treated microglia, while long-term Na⁺ load (6–24 h) is dependent on Na⁺/H⁺ exchanger (NHE) activity (Hossain et al., 2013). The TTX-sensitive but not TTX-resistant Nav channels are involved in microglial phagocytosis function as blockade of Nav by TTX significantly reduced their phagocytic activity of fluorophore-conjugated latex beads at 0.3 μM, while 10 μM TTX did not further suppress the microglial phagocytosis (Black et al., 2009). In addition, the TTX-sensitive Nav channels, especially Nav 1.6, played an important role in the lamellipodial protrusions for microglial migration following ATP stimulation (Black et al., 2009; Persson et al., 2014), mainly by modulating Rac1 and ERK1/2 but not p38 or JNK dependent pathways (Persson et al., 2014).

In addition, acid-sensing ion channels (ASICs) has been recently identified to express in microglia (Yu et al., 2015; Lin et al., 2018) (Fig. 1). ASICs belong to the degenerin/epithelial Na⁺ channel (DEG/ENaC) superfamily, which display high Na⁺ permeability and sensitivity to amiloride blockade (Liu et al., 2015b). ASICs are positively associated with inflammatory conditions, where microglia are the key players of inflammatory responses in the CNS (Yu et al., 2015). While there are seven subunits of ASICs identified (ASIC1a, ASIC1b1, ASIC1b2, ASIC2a, ASIC2b, ASIC3, and ASIC4), encoded by four different genes respectively (Accn1–4), only ASIC1, ASIC2a, and ASIC3 have been reported to be expressed in cultured or in situ rat microglia so far (Yu et al., 2015; Lin et al., 2018), where ASIC1 consists of 94% of the total ASICs expression in microglia. Both ASIC1 and ASIC2a expressions were upregulated upon LPS or scrape stimulation, while ASIC3 expression remained unchanged in both models (Yu et al., 2015). ASIC1 regulates microglia Na⁺ load in response to extracellular acidosis, as administration of an unspecific ASICs blocker amiloride, or a specific ASIC1a antagonist PcTx1, both attenuated the pH drop in LPS-treated microglia by 56–63%, revealed by whole-cell patch-clamp recording in cultured rat microglia (Yu et al., 2015). ASIC1 and ASIC2a, but not ASIC3, also contributed to microglial migration via the activation and phosphorylation of ERK signaling pathway in a scrape stimulation model, as both amiloride and PcTx1 reduced migrating microglia to the stimulation site by 54–57%, while there was no change with the ASIC3 inhibitor APETx2 (Yu et al., 2015). Moreover, silencing of ASIC1 using siRNA almost completely abolished iNOS and COX-2 expressions in the LPS-treated microglia, confirming the involvement of ACIS1 in microglial inflammatory responses (Yu et al., 2015).

5.2. Microglial Na⁺ channels and transporters in brain diseases

Microglial activation is dependent on Nav activity, as pharmacological blockade of Nav by various inhibitors (i.e. anti-epileptic drugs oxcarbazepine and rufinamide, anti-convulsant drug safinamide, and analgesic drug HYP-17) attenuated microglial activation in various disease models of ischemic stroke (Park et al., 2017, 2020; Ahn et al., 2019), Parkinson’s disease (Hossain et al., 2018), and neuropathic pain (Lee et al., 2017). Especially, intrathecal injection of Nav 1.7 shRNA reduced Nav 1.7 expression as well as microglial activation in a cancer pain model (Pan et al., 2015), indicating Nav 1.7 could play an important role in microglial activation.

Inhibition of Nav also reduced the release of IL-1alpha, IL-1beta, TNF-alpha, as well as ROS, H2O2, and gp91 (phox) of NADPH oxidase (NOX) from activated microglia (Black et al., 2009; Hossain et al., 2013, 2017), indicating an involvement of Nav in microglial inflammatory responses and oxidative damages. Indeed, pharmacological blockade of Nav by an anti-epileptic drug significantly reduced microglial release of IL-1beta and TNF-alpha in ischemic stroke models (Park et al., 2020). Moreover, as Nav can be the additional off-targets of monoamine oxidase B (MAOB) inhibitor used to treat Parkinson’s disease (PD), anti-convulsant drugs (i.e. zonisamide, safinamide, etc.) effectively reduced microglial Nav 1.6 protein expression with concurrently reduced level of pro-inflammatory cytokines TNF-alpha and gp91 (phox) in both mouse models and post-mortem human brains of PD (Hossain et al., 2018), as well as increased arginase-1 protein expression for a transitioned anti-inflammatory microglial phenotype (Sadeghian et al., 2016). Moreover, the Nav-mediated microglial inflammation also contributed to motor fiber injury for neuropathic pain (Liu et al., 2016), while pharmacological blockade of Nav significantly reduced levels of pp38MAPK and p-JNK in microglia in a rat model of neuropathic pain (Lee et al., 2017).

6. Proton (H⁺)-sensitive channels and exchangers in microglial function

6.1. Microglial H⁺ channels and exchangers in physiology

Regulation of proton current through H⁺ channels or exchangers mediates local pH change and the removal of positive charge hyperpolarizes the membrane potential. The dominant mechanisms for H⁺ regulation in microglia include the voltage-gated proton channel (Hv1) and Na⁺/H⁺ exchanger (NHE) (Table 1 and Fig. 1), where the Hv1 gene expression is higher relative to NHE1 in microglia (Lam et al., 2013). Hv1 is selectively expressed in microglia but not neurons or astrocytes in the mouse brains (Wu, 2014), and elicits H⁺ efflux activity upon sensing voltage changes in response to membrane potential fluctuations (Morera et al., 2015). Hv1 can also act as a pH sensor and regulator, to sense the pH gradient across membrane and results in an outward H⁺ current to maintain the pH homeostasis (DeCoursey, 2018), but only when accompanied with a strong depolarization, such as increased extracellular K⁺ concentration, due to the high voltage threshold for its activation (Wu, 2014). On the other hand, NHE1 is ubiquitously expressed in all cell types in the CNS, and can be activated upon intracellular acidosis or cell shrinkage and mediates electroneutral transport of H⁺ extrusion in exchange of Na⁺ at an 1:1 ratio (Song et al., 2020). NHE1 is important for maintaining the basal intracellular pH (pH_i) in microglia, as pharmacological inhibition of NHE1 using its potent inhibitor HOE642 acidified the pH_i in resting microglia from 7.19 to 6.82 (Liu et al., 2010), as we recently reviewed here (Song et al., 2020). On the contrary, Hv1-deficient microglia did not show baseline pH_i change with or without intracellular acidification by NH₄Cl washout (Wu et al., 2012), but exhibited pH_i recovery in the presence of high Na⁺, which was abolished upon Na⁺ removal, further corroborating that the microglial pH_i regulation is mainly through NHE1 but not Hv1 activity (Wu et al., 2012). The NHE1-mediated pH_i regulation is closely linked to microglial migration, as assembly of new actin filaments requires an alkaline pH_i (Shi et al., 2013b). NHE1 also directly interacts with the ezrin/radixin/moesin (ERM) protein through its c-terminal binding site and anchors the actin cytoskeleton protein to the plasma membrane to stimulate microglial migration upon bradykinin stimulation (Shi et al., 2013b). In contrast, Hv1 does not participate in ATP-induced chemotaxic microglial migration because ATP-activated P2Y receptor couples K⁺ channels which induces hyperpolarization that suppress Hv1 activity (Wu, 2014). In addition, because the action of NHE1 couples H⁺ extrusion with an inward Na⁺ current, NHE1 activation also plays a vital role in cell swelling (Nishioka et al., 2016) and triggers the Ca²⁺-dependent signaling via reversed action of the Na⁺/Ca²⁺ exchanger (NCX), as we reviewed recently (Song et al., 2020).

6.2. Microglial H⁺ channels and exchangers in brain diseases

Microglial H⁺ homeostasis is important for sustaining the optimal function of NADPH oxidase (NOX) for generation of reactive oxygen species (ROS) during the phagocytic respiratory burst, where microglia utilize the generated free radicals to kill and degrade the engulfed pathogens (Lam et al., 2013). Thus, the common regulation on H⁺ current by Hv1 and NHE1 deems them both as essential regulators for the NOX-mediated oxidative damage (Lam et al., 2013; Wu, 2014; Morera et al., 2015). Blockade of Hv1 by either pharmacological administration of Hv1 inhibitor Zn²⁺ or genetic knockout using Hv1^−/− mice abolished microglial ROS production upon ex vivo PMA stimulation or in vivo ischemic stroke using the middle cerebral artery occlusion (MCAO) model (Wu et al., 2012), where the Hv1^−/− mice concurrently exhibited significantly reduced infarct volume and improved neurological functions (Wu et al., 2012). Deletion of Hv1 also shifted the microglial polarization from pro-inflammatory to anti-inflammatory with reduced iNOS and CD16 expressions, along with increased CD206 and arginase expression in the Hv1^−/− mice using a photo-thrombosis model of ischemic stroke (Tian et al., 2016). However, the suppressive effect of Hv1^−/− on ROS production appeared to be dependent on the age of animals, as aged Hv1^−/− mice (6 months old) exhibited drastically increased ROS production compared to younger animals (1 day–3 weeks old) (Kawai et al., 2017). Similarly, compared to adult mice (2–3 months old), aged animals (18 months old) also exaggerated the microglia-mediated inflammatory responses with markedly increased pro-inflammatory CD16/32 expression co-labelling with elevated Hv1 in microglial cells after peripheral surgical intervention (Zhang et al., 2020b). The microglial Hv1-mediated ROS production and inflammatory responses contribute greatly to white matter damages. In both in vitro oxygen glucose deprivation (OGD) and in vivo bilateral common carotid artery stenosis (BCAS) models, the Hv1^−/− mice showed enhanced oligodendrocyte progenitor cells (OPCs) proliferation and differentiation into mature oligodendrocytes through the PI3K/Akt signaling pathway, along with preserved MAG and MBP expression as well as attenuated SMI32 elevation at 28 days post-hypoperfusion (Yu et al., 2018, 2020). The Hv1^−/− mice also exhibited reduced demyelination and improved motor functional recovery in a cuprizone-induced demyelination model even after 2 weeks of returning to normal chow (Liu et al., 2015a). These studies clearly demonstrated that Hv1 activation promotes microglial ROS production and inflammatory responses for brain tissue damages under both hypoperfusion or demyelination conditions.

The roles of microglial NHE1 activation in brain diseases have recently been reviewed (Song et al., 2020). Briefly, NHE1 activation is required for proinflammatory microglial activation with increased IL-1β, IL-6, TNF-α, iNOS, as well as oxidative injury with elevated NOX p40 and gp91 expressions, and ROS and H₂O₂ productions after ischemic stroke (Liu et al., 2010; Shi et al., 2011; Hossain et al., 2013; Song et al., 2018). Blocking NHE1 activity with either its potent inhibitor HOE642 (cariporide) or genetic knockout attenuated the proinflammatory responses and promoted the transformation of activated microglia to restorative phenotypes for ischemic brain tissue repair (Song et al., 2020). In addition, upregulation of NHE1 activity contributed to the activation of tumor-associated microglia (TAM) with increased iNOS,IL-1β, IL-6, TNF-α, arginase-1, IL-10, and TGF-β in glioma, and promoted glioma proliferation and migration (Zhu et al., 2016; Guan et al., 2018; Song et al., 2020). Our recent study also demonstrated that blocking NHE1 protein with either pharmacological inhibitor or genetic knockout in TAM drove a shift of metabolism profile from the aerobic glycolysis to mitochondrial oxidative phosphorylation (OXPHOS), which stimulated anti-tumor immunity and sensitivity to glioma treatment when combined with temozolomide and anti-PD-1 therapies (Hasan et al., 2020).