Zinc-Permeable Ion Channels: Effects on Intracellular Zinc Dynamics and Potential Physiological/Pathophysiological Significance

Abstract

Zinc (Zn²⁺) is one of the most important trace metals in the body. It is necessary for the normal function of a large number of proteins including enzymes and transcription factors. While extracellular fluid may contain up to micromolar Zn²⁺, intracellular Zn²⁺ concentration is generally maintained at a subnanomolar level; this steep gradient across the cell membrane is primarily attributable to Zn²⁺ extrusion by Zn²⁺ transporting systems. Interestingly, systematic investigation has revealed that activities, previously believed to be dependent on calcium (Ca²⁺), may be partially mediated by Zn²⁺. This is also supported by new findings that some Ca²⁺-permeable channels such as voltage-dependent calcium channels (VDCCs), N-methyl-D-aspartate receptors (NMDA), and amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPA-Rs) are also permeable to Zn²⁺. Thus, the importance of Zn²⁺ in physiological and pathophysiological processes is now more widely appreciated. In this review, we describe Zn²⁺-permeable membrane molecules, especially Zn²⁺-permeable ion channels, in intracellular Zn²⁺dynamics and Zn²⁺ mediated physiology/pathophysiology.

Introduction

Following iron (Fe²⁺), Zn²⁺ is the second most abundant trace metal in human and animals. Like other metals, Zn²⁺ is involved in various cellular processes contributing to normal physiology. Enzymes that mediate important cellular processes, for which Zn²⁺ is required, number more than 200 [1-4]. It is speculated that approximately 10% of proteins utilize Zn²⁺ to maintain their structure and/or function [5, 6]. Therefore, either a deficiency or excess of Zn²⁺ is expected to be detrimental. In fact, 23% of the world's population has been estimated to be Zn²⁺ deficient; this deficiency may cause illness such as pneumonia and diarrhea [7, 8]. Indispensable in maintaining membrane potential, especially in excitable cells, energy-dependent Na⁺/K⁺-pumps generate a 10 to 30-fold gradient for Na⁺ and K⁺ ions [9, 10]. In comparison, the intracellular concentration of Zn²⁺ is only about a thousandth of the extracellular concentration [11-13]. Therefore, effective homeostatic mechanisms must exist for zinc dynamics. Though unique among other ions Zn²⁺ is similar to Ca²⁺. Ca²⁺ is one of the most investigated metal ions because of its diverse functions in physiological/pathophysiological processes, its widespread abundance, and its high concentration gradient across cellular membranes [14-16]. While intracellular Ca²⁺ dynamics are well established, it is less so for Zn²⁺. Recent advances in the studies of Zn²⁺ permeable channels and Zn²⁺ transporters have increased our understanding of cellular mechanism in regulating intracellular zinc homeostasis. This review will focus on the role of Zn²⁺-permeable ion channels in intracellular Zn²⁺ dynamics and cellular physiology/pathophysiology.

Physical and chemical properties of Zn²⁺

Zn²⁺ is a very common element distributed in Earth's outer crust and is handily smelted and manufactured due to its low melting point [17, 18]. The International Union of Pure and Applied Chemistry definition states that Zn²⁺, like cadmium, is regarded as a typical element in which the d-block of electron shell is filled with electrons [19]. Therefore, like cadmium, there is no unpaired electron in the reactive d-block, resulting in less reactivity to redox reaction compared to other divalent metal ions such as iron and cobalt, which are listed in the same period of the periodic table [20]. Considering that the redox reaction is constantly occurring in cells, less reactivity is beneficial so that molecules, such as enzymes, can perform their functions more efficiently. Zn²⁺ is also utilized for acid-base reactions and as a transition metal it is classified as “borderline” for its polarity [21]. Its polarity limits coordination with nitrogen, oxygen and sulfur, which are regarded as soft bases.

Zn²⁺ has been known to be involved in a large number of cellular processes as described below and most commonly it plays a role through its interaction with proteins. Among amino acids, the imidazole functional group of histidine, the carbonyl oxygen of aspartate and glutamate, and the thiol functional group of cysteine are prevalent molecules facilitating Zn²⁺ coordination [5, 22]. With such large variations of coordination, it is of no surprise that Zn²⁺ has a broad spectrum of functions. The functional role of Zn²⁺ in proteins can be classified into two main categories: structural and catalytic. Structurally, Zn²⁺ is normally surrounded by four amino acids preventing solvent interaction. Motifs, such as Zn²⁺ fingers, facilitate not only the stabilization of peptides, but also structural maintenance of the whole protein by mutual interaction with multiple local contextures [1, 4, 5]. In catalytic sites, Zn²⁺ is positioned in active center of the catalytic reaction. Solvents such as water occupy one ligation site of the catalytic Zn²⁺ [5]. The Zn²⁺ metalloenzymes include the family of nucleases, superoxide dismutases, and carbonic anhydrase II (see below) [4].

Physiologic and pathophysiolologic functions of Zn²⁺

Zn²⁺ is an essential factor for growth of higher organisms [23]. Its importance for growth, first established in plants in 1869 [24], has been revealed across many species [23, 25]. In humans, gonadal dysfunction and growth retardation, seen in the Middle East during the 1960's, was found to be mediated by Zn²⁺deficiency [26]. Meanwhile, various enzymes were found to act in a Zn²⁺-dependent manner and the pathophysiological symptoms related to the dysfunction of those enzymes have been described [13, 27-30]. A well recognizable pathophysiology associated with Zn²⁺ dependent enzymes, acrodermatitis enteropathica, is especially egregious [31]. It is characterized by initial skin lesions (e.g. eruption, erosion), subsequent intestinal disorders (e.g. diarrhea) among weaning infants, and growth and mental retardation. Recent reports suggest that high doses of Zn²⁺ can improve clinical outcomes in both inherited and acquired cases of acrodermatitis enteropathica, as some cases implicated ZIP4, one of the Zn²⁺ transporters, as an aberrant gene [32, 33]. Abnormal zinc homeostasis has also been shown to be involved in other pathophysiological conditions. Insults that result from traumatic brain injury, brain ischemia and epilepsy, for example, contribute to excessive Zn²⁺ levels [34-36]. Dysfunctional Zn²⁺ regulation in the mitochondria, leads to a disruption of cell metabolism [37-39]. The energy-generating mitochondrial oxidation-reduction reaction, on the other hand, requires the presence of intracellular Zn²⁺; mitochondrial complexes are not functional when Zn²⁺ is chelated, essentially abating ATP generation [40].

Zn²⁺ is relatively less toxic compared to other metals and Zn²⁺ poisoning rarely occur through normal dietary intake. However, absorption of Zn²⁺ through fumes, in gaseous form, and contamination from Zn²⁺-coated containers may lead to abnormally high concentrations of Zn²⁺ within different organ systems, which may result in disorders of nervous system, intestinal and renal systems [41]. Presently, over-the-counter oral supplementation of vitamins and minerals are common. However, excessive supplementation or iatrogenic errors may cause Zn²⁺ poisoning [42, 43].

Intense research of Zn²⁺ has been conducted in the nervous system although Zn²⁺ is important in other organ systems as well. One reason for systematic study of the nervous system is that neurologic symptoms are often produced by Zn²⁺-related disorders [26, 41, 44]. Different from other organ systems, the nervous system is surrounded by cerebrospinal fluid in which the Zn²⁺ concentration is much lower than the surrounding interstitial fluid [45-47]. Interestingly, neurons can release Zn²⁺ from their synaptic terminals [48, 49]. Low physiologic concentrations and steep gradients of Zn²⁺ surrounding cells may indicate a tight control mechanism of which Zn²⁺ plays important regulatory role in neuronal functions.

Intracellular Zn²⁺ balance and biochemistry

Over 90% of Zn²⁺ is thought to be bound tightly to intracellular molecules; the remaining intracellular Zn²⁺ is labile [1]. Homeostasis of intracellular Zn²⁺ dynamics generally include the following processes: (1) Zn²⁺ incorporation into intracellular organelles and molecules such as mitochondria and metallothioneins (MTs) [50]; (2) Zn²⁺ extrusion into extracellular space or secretion by vesicles [2, 36]; and (3) Zn²⁺ is transported by Zn²⁺ transporters and Zn²⁺ -permeable ion channels (Figure 1). The existence of Na⁺/Zn²⁺ and Ca²⁺/Zn²⁺ exchangers are suggested, but have not been identified at the molecular level [51, 52]. Although recent works have uncovered the importance of extracellular Zn²⁺, for example, in regulating Zn²⁺ -potentiated ectoenzymes [53-55], in Zn²⁺ -sensing receptors, and in Zn²⁺ -activated channels [56-58], intracellular Zn²⁺ is still considered to play a leading part in cellular functions.

Figure 1.

Schematic illustration of the molecular mechanisms involved in intracellular Zn²⁺ homeostasis. Over 90% of intracellular Zn²⁺ binds to molecules. The remaining loosely bound Zn²⁺ affects the equilibrium state and zinc signaling. A fraction of the variable free Zn²⁺ is brought through Zn²⁺-permeable ion channels. Excessive accumulation of labile Zn²⁺ disrupts cellular Zn²⁺ homeostasis, resulting in cellular dysfunction. Hypothetical exchangers are depicted with a dashed arrow.

MTs are small molecules, which have about 70 amino acids; many of them contain about twenty cysteine residues that can trap up to seven Zn²⁺ ions through metal-thiolate bonds [59, 60]. The binding or release of Zn²⁺ ions is regulated by redox status and intracellular pH. Besides MTs, Zn²⁺ is sequestered within the cytoplasm by enzymes and transcription factors. Another main system to maintain intracellular Zn²⁺ levels is the elimination of cytoplasmic Zn²⁺ constituents. In this regard, a number of molecules for Zn²⁺ elimination have been recently cloned [61-63]. Zrt- and Irt- like proteins (ZIPs), which mainly transport Zn²⁺ into cells, and the family of Zn²⁺ transporters (ZnTs), which transport Zn²⁺ out of cytoplasm, seem to work independently within different tissues, and have varied intracellular distribution and transport activity. Some reports have shown that these molecules are involved in human diseases. Although important in Zn²⁺ homeostasis, these transporters are beyond the scope of this review. Readers may obtain valuable information on the subject from other reviews [63-66].

Zn²⁺ is essential for many enzymes which are fundamental to normal cell function. One noteworthy enzyme, carbonic anhydrase II, which catalyzes the reversible hydration of carbonic dioxide, was first to be found as dependent on Zn²⁺ for its activity [4, 67, 68]. These reports were the first to demonstrate important physiological functions of Zn²⁺. Interestingly, one newly uncovered pathophysiological aspect of Zn²⁺ is that it may act on amyloid precursor protein (APP) and play a role in Alzheimer's disease [69]. APP excretes irons from cytoplasm as iron transporter and also works as ferroxidase, which oxidizes Fe²⁺ to Fe³⁺. It interacts with ferroportin, which binds to oxidized iron, to facilitate iron transport. Extracellular Zn²⁺, which is involved in aggregated β-amiloid, is suggested to inhibit the ferroxidase activity, resulting in intracellular iron retention [69].

Zn²⁺ -permeable channels

Zn²⁺ influx across the cellular membrane, through ion channels, is a key factor for the increase of intracellular Zn²⁺ concentration and activation of zinc signaling pathways. The activities of various channels are, in turn, modified by Zn²⁺. For example, NMDA and γ-aminobutric acid – type A (GABA_A) receptor-gated channels are sensitive to Zn²⁺ inhibition, which results in reduced current flux [70-72]. While the activities of many ion channels are modified by Zn²⁺, some cation-permeable channels are reported to be Zn²⁺ permeable. For example, recent studies have demonstrated that voltage-gated Ca²⁺ channels and Ca²⁺-permeable ionotropic glutamate receptors conduct Zn²⁺ (see section Voltage-dependent Ca²⁺ channels). It has also been established that Zn²⁺ flux plays an important role in physiological and pathophysiological processes such as long term potentiation and ischemic stress [62, 73, 74]. Here, we provide an overview of Zn²⁺ -conducting ion channels and their potential functions in physiological/pathophysiological processes. The Zn²⁺ -permeable channels are summarized in Table 1.

Table 1.

Zinc-permeable channels in vertebrates

Channels/receptors	Cells/organs/tissues	Endogenous function & significance	References
VDCCs	muscle, neurons, mast cells, pancreatic b cells	Zn2+ toxicity in neurons is enhanced by high K+. Zinc wave mediated by L-type calcium channel affects expression of IL-6 and TNFα genes.	[78, 80-82, 88]
NMDA-Rs	neurons	Zn2+ toxicity is enhanced by NMDA. Field excitatory postsynaptic potentials might be enhanced by Zn2+ entry through NMDA-Rs.	[94, 97]
AMPA-Rs	neurons	Zn2+ toxicity is enhanced byAMPA.	[34, 82, 84]
nAch-Rs	muscle endplate, chromaffin cells	N/A	[108, 110]
TrpM6/7	Ubiquitous (TrpM7) Kidney (TrpM6)	Zn2+ toxicity in neurons is inhibited by silencing TrpM7 in neurons.	[116, 118, 120, 121, 123, 126]
TrpM3	Pancreatic b cells	Zn2+ is possibly taken up for subsequent exocytosis.	[134]
TrpA1	DRG neurons	Zn2+ causes nociception through TRPA1	[142]
TrpV6	Intestine, kidney, placenta	N/A	[110]
TrpC6	Ubiquitous	N/A	[157]
TRPML1	Ubiquitous	TRPML1 knockout results in abnormal lysosomal Zn2+ homeostasis.	[166, 167]

Voltage-dependent Ca²⁺ channels (VDCCs)

VDCCs are expressed mainly in excitable cells such as muscle and neuronal cells and contribute to cellular Ca²⁺ homeostasis [75-77]. They are inactive at resting membrane potentials but are activated when membrane potentials are depolarized, resulting in Ca²⁺ entry. Intracellular Ca²⁺ increase elicits contraction in muscles and activation of signaling pathways in neurons [77]. VDCCs were initially considered to be the main entry gate for Zn²⁺ from the extracellular space into cytoplasm [78, 79].

Zn²⁺ influx into cells was first described in invertebrates 35 years ago [80]. Shortly after that, VDCCs were found to be a route for Zn²⁺ entry as demonstrated by the inhibition of Zn²⁺ influx with VDCC blockers, such as verapamil [81]. Like invertebrates, VDCCs are also permeable to Zn²⁺ in mammalian neurons, cardiac myocytes, pancreatic β cells, and chromaffin cells [78, 81-85]. Variations in VDCC configuration and subunit composition likely affect the Zn²⁺ permeablity of these channels. Inhibition of Zn²⁺ influx by ω-conotoxin and nimodipine [82, 83, 86, 87], which block different types of VDCCs, suggests that the extent of Zn²⁺ permeability may be determined by the presence of specific subunits. Although VDCCs conduct Zn²⁺, interestingly, they are also known to be blocked by Zn²⁺ [75-79]. For example, it has been demonstrated that Zn²⁺ blocks Ca²⁺ entry through the same channels [87]. In addition, extracellular acidic pH enhances Zn²⁺ entry through VDCCs and modifies the ratio of Zn²⁺/Ca²⁺ flux [87], which may suggest that an increased Zn²⁺ influx contributes to neurotoxicity in conditions such as brain ischemia and lactic acidosis. Interestingly, recent reports have indicated that VDCCs located on intracellular endplasmic reticulum also contribute to zinc signaling and the downstream gene expression in mast cells [88].

Glutamate receptors

Glutamate is the major excitatory neurotransmitter in the central nervous system and is released into the synaptic cleft when stimulated by an action potential at the synaptic terminus. The binding of glutamate to its receptor on the postsynaptic membrane activates the receptor-gated cation channels. Glutamate receptors are divided into two major groups: ionotropic receptors, coupled to ion channels, and metabotropic receptors, coupled to G-protein mediated signaling pathways (mGluRs) [89-93]. The ionotropic receptors include N-methyl-D-aspartate receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs), and kainate receptors. When these receptors are activated by glutamate, influx of cations, such as Na⁺ and Ca²⁺, is facilitated. Activation of glutamate receptor-gated channels is important for physiological functions such as neurotransmission. Zn²⁺ entry through NMDA-Rs has been shown to enhance field excitatory postsynaptic potentials [94]. Over activation of these channels are also known to contribute to neurotoxicity under a varitey of pathophysiological contions such as stroke and epilepsy [95].

NMDA-Rs were first reported to be blocked by Zn²⁺ [71, 72, 96]. Later, they were found to be Zn²⁺ -permeable although the permeablilty to Zn²⁺ is much lower than Ca²⁺ [97]. Physiological and pathophysiological events mediated by Zn²⁺ via NMDA-Rs seem to be dependent more on Zn²⁺ modulation of the channel rather than Zn²⁺ conductance [98, 99].

Early studies reported that Zn²⁺ can modulate the activity of AMPA-Rs by an unknown mechanism [71]. Weiss and colleagues later found that Zn²⁺ permeates AMPA-Rs, producing toxic effects [82]. They also demonstrated a clear relationship between Zn²⁺ toxicity and the activation of AMPA-Rs, which plays a role in ischemic neuronal injury [34]. Additional studies suggests that Zn²⁺ entry mediated by AMPA-Rs contributes to neurotoxicity more than VDCCs and NMDA-Rs [84, 100]. Interestingly, in neocortex, interneurons are more sensitive to AMPA-Rs mediated Zn²⁺ toxicity than other neurons [101]. Furthermore, Zn²⁺ inhibits gene expression of GluR2, a subunit of AMPA-Rs that regulates Ca²⁺ permeability; this inhibibition results in Ca²⁺ and Zn²⁺ toxicity [102, 103]. Unlike VDCCs, Ca²⁺ entry through AMPA-Rs are not influenced by Zn²⁺ [104].

Nicotinic acethylcoline receptors (nAch-Rs)

There are two types of acetylcholine receptors (Ach-R): ionotropic and metabotropic ones. Ionotropic Ach-Rs are also called nicotinic receptors (nAch-Rs). They are expressed mainly in muscle endoplate, at neuro-muscular junctions and throughout the nervous system in vertebrates [105, 106]. Neuronal nAch-Rs have a large variety of subunit components depending on their anatomical distribution. Their major role is to modulate neuronal function. In contrast, muscular nAch-Rs play an exlusive role in neuro-muscular transmission. Although neuronal nAch-Rs are in general highly Ca²⁺-permeable, muscular ones are less permeable to Ca²⁺ [107]. Therefore, secondary activation of VDCCs follwing nAch-Rs-mediated depolarization is required for Ca²⁺ accumulation and muscle contraction. Regardless of its relatively low Ca²⁺ permeability, Zn²⁺ conductance has been reported for nAch-Rs found on endplates in muscles [108] and in adrenal chromaffin cells [109]. Similar to native nAch-Rs, Zn²⁺ permeability was demonstrated in cloned nAch-Rs [110]. It has been shown that γ- and ε-subuits are Zn²⁺ -permeable whereas other subunits are not [110]. The physiological and/or pathophysiolological sigificance of Zn²⁺ permeability in nAch-Rs, however, remains to be elucidated.

Transient Receptor Potential (Trp) channels

First member of Trp channels was discovered in D. melanogaster, where a genetic mutation of the gene induced an abnormal photosensitivity [111, 112]. The Trp superfamily of ion channels now includes nearly thirty genes [113], which are divided into 6 subfamilies [113, 114]. Sequcence homology is conserved between flies and higher organisms and members of the Trp family are cation-permeable. Trp channels have widespread distribution and play important roles in various physiological and pathophysiological conditions. For example, local thermal sensation is largely attributable to certain Trp channels and genetic mutations of some Trp channels elicit a variety of diseases such as epilepsy, leukemia and hypertension [113, 115]. Recent studies of cation permeation using electrophysiology or fluorescent Zn²⁺ imaging have demonstrated that several members of the Trp family are Zn²⁺ permeable.

TrpM6/7

TrpM7 was the first among Trp channels found to be Zn²⁺ permeable [116]. TrpM6/7 are unique among channels because they also possess a kinase domain [117, 118]. TrpM7 plays a key role in intracellular Mg²⁺ homeostasis and in neuronal Ca²⁺ toxicity in ischemic conditions [118-122]. Monteilh-Zoller et al. demonstrated that the permeability of TrpM7 channels for Zn²⁺ ions is four-fold higher compared to Ca²⁺ [116]. TrpM7-overexpressing cells showed intracellular Zn²⁺ accumulation as indicated by increased fluorescence of FluoZin-3, a recently developed Zn²⁺ fluorescent indicator [123]. This Zn²⁺ accumulation contributes to, at least in part, functional gene expression in neuronal cells through the activation of metal-responsive element, which is important for monitoring intracellular metal levels [123-125]. Thus, not only because of abnormal cellular function (see above), but also because of changes in various gene expression, Zn²⁺ overload through TrpM7 will facilitate cytotoxicity [123, 126]. TrpM6, a homolog of TrpM7, also has a kinase domain. It has been implicated in Mg²⁺ homeostasis in the intestines and kidneys [120, 127]. Similar to TrpM7, TrpM6 has also been shown to be Zn²⁺ permeable [128].

TrpM3

TrpM3, a member of TRP channels highly expressed in brain and in pancreatic β cells [129, 130], is important for insulin release [131]. Zn²⁺ has been found in the vesicles of these cells, which is co-released with insulin and contributes to insulin function [131, 132]. While ZnT8 was speculated to facilitate Zn²⁺ uptake from cytoplasm into vesicles [133], Zn²⁺ permeability has recently been shown in one of the TrpM3 variants in native pancreatic β cells [134]. TrpM3 appears to contribute to Zn²⁺ permeation comparable to VDCCs [134]. Since activation of TrpM3 produces depolarization of β cells, which can lead to additional Zn²⁺ influx through VDCCs, further segregation of functional significance of Zn²⁺ entry between TrpM3 and VDCCs may be explored in the near future.

TrpA1

TrpA1 channels are highly expressed in dorsal root ganglia neurons [135, 136]. TrpA1 is stimulated by irritants such as mustard oil and noxious cold [135-139]. They are also expressed in the respiratory tract and play a role in hypersensitivity and inflammatory reactions associated with components of tobacco that activate the TrpA1 channels [140, 141]. Hu and colleagues demonstrated that TrpA1 channels act as a feedforward system which utilizes Zn²⁺ to further activate the channels [142]. TrpA1 deficient mice seem to feel less pain by Zn²⁺ injection whereas Zn²⁺ injection leads to apparent painful behavior in wild-type mice. This finding suggests that TrpA1 channels expressed in peripheral nerves are associated with pain sensation. Further studies showed that Asp 915 plays an important role in Zn²⁺ entry through TrpA1 while C641 and Cys 1021 are involved in the sensitivity of TrpA1 channels to intracellular Zn²⁺ activation [142].

TrpV6

TrpV6 is highly expressed in intestines, kidney and placenta [143-145]. Although TrpV6 is thought to contribute to Ca²⁺ absorption through its high Ca²⁺ conductance, the in vivo function of this channel still remains largely unknown. Some reports indicate that significantly higher TrpV6 expression is found on prostate cancer cells and its expression is associated with cancer progression [146-148]. Zn²⁺ also permeates TrpV6 channel [149], at least in TrpV6-overexpressing cells. Of note, TrpV5/6 in fishes are also known to transport Zn²⁺ [150, 151] and showed a transient decrease of gene expression during Zn²⁺ excess [152], suggesting that permeability of Zn²⁺ might play a role in all vertebrates.

TrpC6

TrpC6 channels are expressed ubiquitously [153, 154]. It is known that Ca²⁺ is much more permeable than Na⁺ through these channels and that intra- and extracellular Ca²⁺ potentiates and inhibits the channel activity, respectively [154]. Because of its broad distribution, TrpC6 channels are suggested to play a significant role in various organ systems including kidney and brain [155, 156]. The Zn²⁺ permeability of these channels has recently been demonstrated in neurons and in transfected HEK293 cells [157]. Interestingly, TrpC6 has been suggested to be associated with Zn²⁺ accumulation in the nucleus. Zn²⁺ may also regulate TrpC6 channel activity by modifying the effect of Ca²⁺. Although the physiological/pathophysiological significance of Zn²⁺ permeability for TrpC6 is still unclear, a recent report indicates that its overexpression facilitates Zn²⁺ accumulation in endo/lysosomes and upregulation of MTs [158]. However, considering that inhibition of TrpC6 degradation ameliorates ischemic brain injury [156], Zn²⁺ permeation through this channel does not seem to exacerbate or produce neurotoxicity.

TRPML1

TRPML1, also known as mucolipin-1, is localized to endosomes and lysosomes [159, 160]. Mutation of TRPML1 gene is associated with mucolipidosis, which is a rare autosomal recessive neurodegenerative disorder [161-163]. The clinical symptoms are severe with growth and psychomotor impairment in early stage of development (mostly 0 – 3 years). Most patients can not walk and speak over their lifetime, while half of the patients also have aberrant iron metabolism and anemia [164, 165]. Dong et al. discovered that TRPML1 is an iron-permeable channel and that changes in cellular iron level may play an important role in hereditary mucolipidosis [166]. They also found that TRPML1 conducts Zn²⁺ [166]. Recent findings have shown that knockdown of TRPML1 leads to abnormal cellular zinc localization and accumulation, for example, TRPML1 knockout mice have a significantly increased level of Zn²⁺ in brain tissues [167, 168]. These findings suggest that abnormal zinc homeostasis derived from TRPML1 mutation may contribute to neurological dysfunction.

Prospective

The activities of a number of channels are modulated by Zn²⁺ [169-171]. Addition of Zn²⁺ may enhance or inhibit the channel activity, resulting in increased or decreased current amplitude. For some channels, changes in current amplitude maybe mediated by Zn²⁺ permeation. As mentioned above, amino acids such as histidine, cysteine, glutamate and aspartate are likely to be preferable for Zn²⁺ coordination of catalytic activities and protein architecture [5, 22]. Amino acid substitution may therefore change the activity and function of channels that are modulated by Zn²⁺ [172-178]. However, one may also consider the possibility that certain amino acids are required for Zn²⁺ permeation and an examination of existing channels may facilitate the discovery of other Zn²⁺ -permeable channels. For instance, Zn²⁺ permeability might be revealed in family members of Trp channels, most of which are found to permeate various divalent cations [113, 114].

Recent advancement in fluorescent Zn²⁺ indicators has made the investigation of intracellular Zn²⁺ dynamics much easier in comparison to previous probes. Historically, fluorescent Zn²⁺ indicators such as zinquin have been used for many decades [83, 179-184]. However, zinquin-like indicators are limited in specificity or have low affinity for Zn²⁺. Some of them have high affinity for Ca²⁺ [51, 83, 185], or are influenced by Ca²⁺ [186-188]. In contrast, FluoZin-3, which was utilized to study Zn²⁺ permeation in TrpA1 and TrpM3, has a higher affinity (Kd = 3 ~ 15 nM) for Zn²⁺, which is close to the intracellular Zn²⁺ concentration in resting conditions [11, 189, 190]. Of special note, FluoZin-3 is insensitive to Ca²⁺, which has a significant advantage over previous indicators that cannot differentiate between Zn²⁺ and Ca²⁺ signals. With the emergence of new indicators, examinations of Zn²⁺ and Ca²⁺, simultaneously, using different fluorescent indicators have been reported [191, 192]. These advancements are likely to provide a better understanding of Zn²⁺ regulation, conductance and functional importance.