Ca_v2.3 channel function and Zn²⁺-induced modulation: potential mechanisms and (patho)physiological relevance

ABSTRACT

Voltage-gated calcium channels (VGCCs) are critical for Ca²⁺ influx into all types of excitable cells, but their exact function is still poorly understood. Recent reconstruction of homology models for all human VGCCs at atomic resolution provides the opportunity for a structure-based discussion of VGCC function and novel insights into the mechanisms underlying Ca²⁺ selective flux through these channels. In the present review, we use these data as a basis to examine the structure, function, and Zn²⁺-induced modulation of Ca_v2.3 VGCCs, which mediate native R-type currents and belong to the most enigmatic members of the family. Their unique sensitivity to Zn²⁺ and the existence of multiple mechanisms of Zn²⁺ action strongly argue for a role of these channels in the modulatory action of endogenous loosely bound Zn²⁺, pools of which have been detected in a number of neuronal, endocrine, and reproductive tissues. Following a description of the different mechanisms by which Zn²⁺ has been shown or is thought to alter the function of these channels, we discuss their potential (patho)physiological relevance, taking into account what is known about the magnitude and function of extracellular Zn²⁺ signals in different tissues. While still far from complete, the picture that emerges is one where Ca_v2.3 channel expression parallels the occurrence of loosely bound Zn²⁺ pools in different tissues and where these channels may serve to translate physiological Zn²⁺ signals into changes of electrical activity and/or intracellular Ca²⁺ levels.

Introduction

Zn²⁺, the second most abundant trace metal ion in mammals, is a well-known component of many proteins and co-factor in various enzymes [1,2]. Apart from its structural and catalytic functions, Zn²⁺ has been identified as a modulator of neuronal transmission, neuroendocrine function, and other processes [2–5]. These modulatory actions depend on a small pool of endogenous Zn²⁺ that is almost exclusively located in synaptic or secretory vesicles and not tightly bound by zinc metalloproteins, so that it remains thermodynamically and kinetically accessible. Spontaneous and activity-dependent release of vesicular Zn²⁺ have been convincingly demonstrated in a number of tissues [3,4,6], although the exact magnitude of the resulting Zn²⁺ signals and their targets remain a matter of debate. Ca_v2.3 channels belong to the group of high voltage-activated (HVA) voltage-gated calcium channels (VGCCs) and are responsible for Ca²⁺ influx into neuronal, neuroendocrine and other cells. They mediate the major portion of native R-type currents, which are characterized by their resistance to most organic Ca²⁺ channel antagonists and sensitivity to the tarantula toxin SNX-482 [7–9]. In addition, Ca_v2.3 channels are among the most sensitive molecular targets of Zn²⁺ currently known and expressed in many Zn²⁺-enriched tissues, suggesting that they are involved in the modulatory actions of endogenous Zn²⁺. The aim of the present article is to provide a concise overview of Ca_v2.3 channel structure and function, with a special focus on Zn²⁺-induced modulation and its potential (patho)physiological relevance. To this end, section two will resort to a recently published homology model of human Ca_v2.3 channels at atomic resolution [10] to illustrate their structure and discuss different mechanisms by which Zn²⁺ has been shown or is thought to alter Ca²⁺ influx through these channels. The third section will briefly summarize current knowledge about their expression and role in neuronal, endocrine, and reproductive tissues, while the fourth section will consider the effects of Zn²⁺ in terms of their potential (patho)physiological relevance, taking into account what is known about extracellular Zn²⁺ signals in these tissues.

Ca_v2.3 channel structure, function and Zn²⁺ induced modulation

Membrane topology

Like other HVA VGCCs, Ca_v2.3 channels in native membranes form multi-subunit complexes made up of a pore-forming Ca_vα₁-subunit, one of several cytoplasmic Ca_vβ-subunits and one of several extracellular Ca_vα₂δ-subunits. The Ca_vα₁-subunit is a pseudotetrameric protein with four homologous repeats (I–IV), which are composed of six membrane-spanning helical segments (S1-S6) [11] (Figure 1(a, b). Four of the segments (S1-S4) in each repeat form a voltage-sensor module (VSM), while the remaining two segments (S5-S6) from all repeats make up most of the pore domain (PD) and the intracellular activation gate (Figure 1c). The extracellular pore mouth and the selectivity filter are formed by reentrant pore loops (p-loops) between segments S5 and S6 (Figure 1c), which partly fold back into the single-file region of the pore [12,13] (Figure 1d). The three inter-domain linkers and other cytoplasmic parts of the channel (not shown in Figure 1) are involved in inactivation, the association of auxiliary Ca_vβ-subunits, and intracellular modulation [14].

Figure 1.

Structure of human Ca_v2.3 voltage-gated calcium channels

Cartoon representation of a homology model of human Ca_v2.3 channels at atomic resolution. Depicted are (a) side view showing two of the four repeats, (b) top view showing all four repeats, (c) side view of repeat I with the different functional domains indicated below, and (d) side view of the pore-forming domains from all four repeats [10].

Voltage-dependent gating and gating modulation

The exact processes involved in Ca²⁺ channel gating are still incompletely understood, but its coupling to the membrane potential is thought to depend on the same mechanism as in all voltage-gated ion channels. At resting membrane potentials, the local electric field drags the positively charged S4 segments toward the cell’s interior and parts of the S5 and S6 segments occlude the conduction pathway. Depolarization results in outward movement of the S4 segments through the electric field, relocation of the S5 and S6 segments and formation of a crevice, which ions can traverse [15,16]. In electrophysiological recordings, these charge-moving conformational changes are reflected in a steep change of channel open probability over a narrow range of membrane potentials and, after block of ion conduction, as measurable gating currents. Co-expression of auxiliary subunits affects the voltage-dependence and kinetics of ionic but not gating currents, suggesting that they alter the coupling between voltage-sensor movement and channel opening [17–20]. In voltage-gated sodium channels, activation of the VSMs in repeats I–III is coupled to channel opening [21], while activation of the VSM in repeat IV gives rise to a short-lived second open state that precedes inactivation [22–24]. Interestingly, these findings are already implicit in the classical Hodgkin and Huxley model with its three activation and one inactivation particles for the sodium conductance [25,26]. Less is known about the situation in VGCCs, which have most commonly been described by a combination of two or three activation and one or more inactivation particles [27–30]. However, optical tracking of Ca_v1.2 channel voltage-sensor movement in combination with kinetic modeling revealed that, depending on the exact subunit composition, activation of two or three VSMs may indeed be sufficient for Ca²⁺ channel opening [31–33], which is reinforced by mutational studies [34,35], evidence for the existence of multiple open states [36–38] and a slow component of charge movement in gating current recordings [17,39,40]. Also, when a large set of Ca_v2.3+β₃ channel ionic and gating currents recorded in our lab was fitted with obligatory models in which activation of 2–4 VSMs was required for channel opening, a good agreement between experimental and modeled currents was only obtained when just two VSMs are coupled to channel opening [40]. In this context, it is interesting to note that structural determinants in two of the four repeats (III and IV) have been shown to be a prerequisite for SNX-482-induced shifts in Ca_v2.3 channel gating [9], suggesting that activation of the VSMs in these repeats could either be directly coupled to channel opening or associated with a stabilization of the open state.

Moderate acidification or low micromolar concentrations of Zn²⁺ and certain other trace metal ions potently suppress native and cloned Ca_v2.3 channels (Figure 2a) by shifting voltage-dependent gating to more depolarized test potentials (Figure 2b), and produce a dramatic slowing of macroscopic activation (Figure 2c) that cannot be accounted for by the shift in channel voltage-dependence alone [40–49]. This unique trace metal sensitivity has been convincingly linked to three non-conserved histidine residues (His₁₁₁, His₁₇₉ & His₁₈₃) on the repeat I VSM (Figure 2d), which could form a high-affinity trace metal-binding site. Replacement of these residues by site-directed mutagenesis significantly reduces the gating effects of Zn²⁺, Cu²⁺, Ni²⁺ and protons but does not prevent them completely [42,43,47]. In addition, a detailed analysis of the Zn²⁺ concentration-dependence for shift and slowing performed in our lab revealed two separable components with vastly different affinities (Figure 2e), which could also be distinguished based on their sensitivity to histidine modification with DEPC [40]. High-affinity shift and slowing were sensitive to DEPC pre-treatment (Figures 2c & e), could be well described by a common IC₅₀ value (2.3–4.4 µM with 2–4 mM free Ca²⁺) and reproduced with the simplified model mentioned above by assuming that Zn²⁺ binding to a single site leads to electrostatic modification and mechanical slowing of one of the VSMs. Low-affinity shift (IC₅₀ > 500 µM) and slowing (IC₅₀ ~ 100–200 µM), on the other hand, were insensitive to histidine modification (Figure 2e), suggesting that they are not related to the putative trace metal-binding site in repeat I. However, the low-affinity slowing appeared to be correlated with Zn²⁺ effects on permeation, which will be discussed below in the context of ionic block (see section 2.3). More importantly, we also found that, depending on the holding potential (HP), high-affinity binding could either inhibit or stimulate Ca²⁺ influx through Ca_v2.3 channels. Thus, at the very negative HPs typically employed to minimize HP-dependent inactivation in electrophysiological recordings, Zn²⁺ rapidly and exclusively suppressed Ca_v2.3 channel currents by shifting voltage-dependent activation to more depolarized test potentials and slowing activation (figure 2F). At HPs more positive than −80 mV, where Ca_v2.3 channels are subject to processes like ultra-slow closed-state inactivation [50,51], rapid suppression was accompanied by a time-dependent current increase that was also slow to reverse after removal of Zn²⁺, as reflected in a transient over-recovery of current amplitudes during washout (figure 2f). In the absence of Zn²⁺, the time-course and degree of current stimulation by a given concentration of free Zn²⁺ could be reproduced by a hyperpolarizing shift in HP equal to the depolarizing shift in channel voltage-dependence produced by that free Zn²⁺ concentration [40], suggesting that it reflects recovery from slow HP-dependent inactivation. That Zn²⁺ reduces slow inactivation would also be in line with the parallel Zn²⁺-induced changes in activation and fast inactivation (Figures 2(b, c) and could reflect the proposed coupling between activation and inactivation gating [52,53].

Figure 2.

Modulation of Ca_v2.3 channel function by low micromolar Zn²⁺ concentrations

(a) Concentration-dependent suppression by low micromolar Zn²⁺ of Ca²⁺ currents carried by 4 mM free Ca²⁺ and recorded during a 30 ms voltage-step to 10 mV in HEK293 cells stably transfected with cloned human Ca_v2.3+ β₃ channel complexes. (b) Activation (right) and pre-pulse inactivation (left) curves determined in the absence (black squares) and presence (orange circles) of 2.3 µM free Zn²⁺ under the same conditions as in a. (c) Macroscopic Ca_v2.3+ β₃ channel currents recorded during a 30 ms voltage-step to 10 mV in the absence (black) or presence (orange) of 2.3 µM free Zn²⁺ and scaled to the same amplitude to illustrate kinetic changes. Also shown are the effects of the same concentration of Zn²⁺ after pre-treatment of cells with the histidine modifying reagent DEPC (red). Note the Zn²⁺-induced slowing of activation but not deactivation kinetics, which was completely prevented by histidine modification with DEPC. (d) Homology model of Ca_v2.3 repeat I with the three histidine side chains implicated in high-affinity Zn²⁺-induced modulation shown as ball and stick representation. (e) Concentration-dependence of Zn²⁺-induced slowing determined under the same conditions as in a (open squares) or after pre-treatment of cells with the histidine modifying reagent DEPC (red squares). Solid lines are the combination of two binding isotherms for simple biomolecular reactions, while dotted lines indicate the corresponding high- and low-affinity components. (f) Holding potential (HP)-dependence of Zn²⁺ effects on the amplitude of Ca²⁺ currents evoked by repetitive depolarization to 10 mV.

Selective permeation and ionic block

When electrophysiological recordings are performed in the absence of Ca²⁺ and other polyvalent cations, VGCCs can mediate large currents carried by monovalent ions like sodium. Addition of micromolar Ca²⁺ potently suppresses these currents in a voltage-dependent manner, which can be well described by assuming that Ca²⁺ blocks the pore through interaction with a high-affinity site located in the membrane electric field [54–58]. Cd²⁺ and lanthanoid block of Ca²⁺ currents have almost the same voltage-dependence and are thought to result from blocker occupation of the same principle intra-pore site [59–65]. In single-channel recordings, these cations produce discrete, long-lasting blocking events [61,66–69], which are consistent with an occlusion of the pore that prevents ion transfer through open channels. Mutational studies have convincingly linked high-affinity Ca²⁺ binding in the pore to a conserved ring of glutamate (and in LVA channels aspartate) residues (EEEE/EEDD-locus) located at equivalent positions in each of the four p-loops [13,70–72]. How high-affinity binding can be reconciled with the high rates of flux observed in the presence of millimolar Ca²⁺ concentrations is less well understood, but thought to involve ion–ion interactions due to simultaneous occupation of the pore by multiple ions [73]. A potential mechanism has been elucidated by construction and crystallographic analysis of the homotetrameric bacterial model channel Ca_vAb and its derivatives [74], in which the selectivity filter is formed by three successive ion-binding sites in the single-file region of the pore (Figure 3a). A central high-affinity Ca²⁺ binding site (site 2) is formed by carboxylate-bearing side chains of aspartate (D₁₇₇) residues equivalent to the EEEE/EEDD-locus in vertebrate channels and backbone carbonyl groups of neighboring leucine (L₁₇₆) residues. Consistent with the data on vertebrate channels, site 2 can coordinate a single, hydrated Ca²⁺ (Figure 3a left) or Cd²⁺ (Figure 3b) ion with sufficient affinity for block. It is flanked by two lower-affinity sites formed by carboxylate-bearing side chains of aspartate (D₁₇₈) residues at the outer edge of the selectivity filter (site 1) and backbone carbonyl groups of threonine (T₁₇₅) residues on the internal side (site 3) near the central cavity (Figure 3a). High-affinity binding to the central site is thought to be destabilized by repulsive ion–ion interactions when Ca²⁺ ions occupy the two flanking, lower-affinity sites, so that the channel mainly oscillates between two low-energy states in which either one Ca²⁺ ion occupies the central site (Figure 3a left) or two Ca²⁺ ions occupy the two flanking sites (Figure 3a right). Comparison with the modeled human Ca_v2.3 channel (Figures 3c-e) shows that sites 2 and 3 are conserved in sequence and 3D space, whereas the architecture of site 1 is distinct from the fourfold symmetric planar ring in Ca_vAb. Thus, in hCa_v2.3 (and other HVA channels), only one of the four aspartate residues corresponding to site 1 in Ca_vAb is preserved (D₆₁₅ in repeat II, Figures 3d&e) and has been shown to directly participate in Ca²⁺ interaction with the pore [75]. The remaining three side chains involved in the formation of site 1 in hCa_v2.3 appear to be provided by two glutamate (E₆₁₈ in repeat II & E₁₃₂₇ in repeat VI) and one glutamine residue (Q₁₀₁₈ in repeat III) from the so-called DCS-locus (Figure 3d), a ring of channel-specific charged residues that participates in selective permeation and ionic block [76,77] and corresponds to residue 181 in Ca_vAb and its derivatives (Figures 3(a, b). Importantly, these structural features could explain a number of previous findings on Ca²⁺ channel block by group IIB metal ions like Zn²⁺ or Cu²⁺, which has a much shallower voltage-dependence than Cd²⁺ or lanthanoid block and is markedly subunit-dependent, suggesting that it does not take place at the conserved EEEE-locus but at a more superficial, channel-specific site [73]. In addition, these metal ions produce a characteristic slowing of macroscopic activation and deactivation of all native HVA VGCCs, which is closely correlated with their blocking effects but not directly related to the intrinsic properties (i.e. time- and voltage-dependence) of block itself [44,45]. Instead, the slowing has been proposed to involve an allosteric effect on opening and closing that is linked to occupation of the blocking site in the pore. Consistent with this assumption, we have found that both, Zn²⁺-induced voltage-dependent block (Figure 4a) and the Zn²⁺-induced low-affinity slowing of cloned Ca_v2.3 channels (Figure 4b) described in the preceding section could be reasonably reproduced by assuming that Zn²⁺-binding to a single site (K_Zn~100 µM) in the pore blocks the channels and slows the opening and closing transitions [40]. Given the many simplifying assumptions of this model, it remains to be determined if both effects are truly mediated by a single site and, if so, whether this site corresponds to site 1 as hypothesized above. Interestingly, our experiments also revealed that Zn²⁺-induced block is accompanied by a voltage-independent and incomplete suppression of Ca²⁺ influx through open channels with much higher affinity (IC₅₀ = 12–16 µM with 4 mM Ca²⁺) [40]. Histidine-modification with DEPC reduced the degree of high-affinity suppression but increased the degree of block (Figure 4c), suggesting that occupation of the underlying sites may be mutually exclusive. In this context, it is interesting that protons have been shown to reduce Ca_v2.3 channel unitary conductance by interaction with a non-conserved histidine residue (His₂₄₁) in the p-loop of repeat I [47], which is located directly adjacent to site 1 (Figures 4(d, e). This makes it tempting to speculate that interaction of Zn²⁺ with the same residue might reduce the net charge density and/or cross-sectional area available for passage of other ions without complete occlusion of the pore, which could explain the DEPC-sensitivity and incomplete nature of the high-affinity component. Moreover, considering its close spatial proximity to site 1, Zn²⁺ occupation of His₂₄₁ would be expected to reduce the probability that another Zn²⁺ ion occupies site 1. That said, the multiple effects of Zn²⁺ on Ca_v2.3 channel gating and permeation are evidently complex, and further studies will be required for a clear separation and throughout understanding of the underlying mechanisms. Thus, a putative EF-hand motif in the p-loop of domain III of all HVA channels (Figure 4d) has previously been implicated in their differential sensitivity to Zn²⁺-block as well [78,79] and its absence in LVA channels could explain why a prominent Zn²⁺-induced slowing is only observed in HVA channels. On the other hand, the superficial location of this site (Figure 4d) is difficult to reconcile with an occlusion of the pore, so that it may not be directly involved in Zn²⁺-induced block. Another relevant question that will be discussed in section 4 is if voltage-dependent block and low-affinity slowing could ever become relevant under (patho)physiological conditions, or if they are simply phenomena that have to be taken into account when studying the effects of trace metals on Ca_v2.3 channel function.

Figure 3.

Architecture of the selectivity filter in bacterial Ca_vAb and human Ca_v2.3 channels

(a-b) Ball and stick representation of residues forming the three ion-binding sites in the crystal structure of the bacterial model channel Ca_vAb and its derivatives with (a) one hydrated Ca²⁺ ion bound to the central high-affinity site (left) or two hydrated Ca²⁺ ions bound to the two flanking low-affinity (right) sites (pdb: 4MS2) or (b) one hydrated Cd²⁺ ion bound to the central high-affinity site (pdb: 4MVS). (c) Cartoon representation of the p-loops from three repeats in the homology model of hCa_v2.3 with residues corresponding to the three ion-binding sites shown as ball and stick representation. (d) Close-up view of hCa_v2.3 channel residues corresponding to the three ion-binding sites. (e) Sequence comparison of residues forming the selectivity filter in Ca_vAb and hCa_v2.3.

Figure 4.

Modulation of Ca_v2.3 channel function by high micromolar Zn²⁺ concentrations

(a) Concentration-dependent suppression by Zn²⁺ of instantaneous currents carried by 4 mM free Ca²⁺ and recorded after channel opening by a depolarizing prepulse in HEK-293 cells stably transfected with cloned human Ca_v2.3+ β₃ channel complexes. (b) Macroscopic Ca_v2.3+ β₃ channel currents recorded during a 30 ms voltage-step to 10 mV in the absence (black) or presence (green) of 101.6 µM free Zn²⁺ and scaled to the same amplitude to illustrate kinetic changes. Also shown are the effects of the same concentration of free Zn²⁺ after pre-treatment of cells with the histidine modifying reagent DEPC (red). Note the DEPC-resistant Zn²⁺-induced slowing of both activation and deactivation kinetics. (c) Concentration-dependence of Zn²⁺-induced suppression of the instantaneous slope conductance (G_IIV) between −40 mV and +20 mV, determined under the same conditions as in A (open squares) or after pre-treatment of cells with the histidine modifying reagent DEPC (red squares). Solid lines are the combination of two binding isotherms for simple biomolecular reactions, while dotted lines indicate the corresponding high- and low-affinity components. (d) Cartoon representation of the p-loops from three repeats in the homology model of Ca_v2.3 with putative residues that could be involved in the blocking and low-affinity gating effects of high micromolar Zn²⁺ shown as ball and stick representation. (e) Close-up view of residues forming the putative blocking site for group IIB metal ions (orange = site 1 in Fig. 3d) and the nearby histidine residue (green), which has previously been linked to proton-induced changes in unitary conductance.

Ca_v2.3 channel expression and relevance in different tissues

Ca_v2.3 channels are widely expressed throughout the central and peripheral nervous system [37,64,80,81] and in endocrine [80,82,83], cardiovascular [84], gastrointestinal [80] and reproductive tissues [85,86]. Despite this widespread expression, Ca_v2.3-deficient mice display a surprisingly subtle phenotype with no major pathologies but altered nociception [87], glucose homeostasis [49,88,89] and spatial memory [90], impaired autonomic control [84,91], increased anxiety [92] and reduced susceptibility to chemically induced seizures [93,94].

Ca_v2.3 channels in the central nervous system

In the brain, highest densities of Ca_v2.3 channels have been detected in the hippocampus and other limbic regions, in the neocortex and in the retina [95,96]. They are expressed in pre- and post-synaptic neurons [97,98] and have been shown to play a role for neurotransmitter release, synaptic plasticity, action potential burst firing and somatodendritic integration [99–106]. In the retina, Ca²⁺-influx through Ca_v2.3 channels has been linked to GABA-ergic reciprocal inhibition of rod ON-bipolar cells as part of the scotopic visual pathway [107]. The exact physiological relevance of Ca_v2.3 channels in most other brain regions remains elusive, but they have been implicated in a number of pathophysiological conditions. Most notably, Ca_v2.3 channels are thought to play a pro-ictogenic role in convulsive-generalized tonic-clonic and hippocampal seizures, since their genetic ablation reduces the susceptibility to chemically induced seizures [93,94,108] and abrogates the effects of several broad-spectrum antiepileptic drugs known to suppress cloned Ca_v2.3 channels [94,109,110]. More recently, de novo gain-of-function mutations in Ca_v2.3 channels have also been linked to developmental and epileptic encephalopathies in human patients, which are associated with epileptiform EEG activity, intractable seizures, and developmental impairments [111,112].

Ca_v2.3 channels in the endocrine pancreas

A number of studies have linked Ca_v2.3 channels to α-cell glucagon [49,113], β-cell insulin [88,89,114], and δ-cell somatostatin (SST) secretion [115] in pancreatic islets, suggesting that they are involved in the regulation of blood glucose homeostasis. During hyperglycemia, glucose homeostasis is mainly maintained by glucose-stimulated insulin secretion (GSIS), which can be separated into a first phase mediated by fast secretion of insulin from a readily releasable pool of vesicles and a second phase of sustained release that is thought to involve mobilization of a reserve pool of vesicles [114]. Genetic or pharmacological ablation of Ca_v2.3 channels in mice has been shown to selectively reduce the second-phase insulin response [113,114], suggesting that they are involved in the mobilization of insulin vesicles from the reserve pool. In addition, SST has been shown to inhibit cloned Ca_v2.3 channels [116] and SNX-482 prevents SST inhibition of insulin secretion from insulinoma cells [117], indicating that β-cell Ca_v2.3 channels could also play a role for the regulation of insulin secretion by SST. According to the intra-islet insulin hypothesis, insulin released from β-cells during hyperglycemia could tonically suppress glucagon release from downstream α-cells, so that cessation of insulin release during hypoglycemia serves as a switch-off signal that initiates α-cell glucagon secretion [118,119]. Interestingly, glucose-induced suppression of glucagon release is severely impaired in Ca_v2.3-deficient mice [114], in isolated islets from Ca_v2.3-deficient mice [113,114] and in SNX-482 treated islets from wild-type mice [114,120], but SNX-482 fails to suppress glucagon secretion from wild-type islets in low glucose and fasting glucagon levels are significantly higher in Ca_v2.3-deficient compared to wild-type mice [49]. Taken together, these findings suggest that Ca_v2.3 channels are important for suppression of glucagon release during hyperglycemia but not strictly required for the release of glucagon during hypoglycemia.

Ca_v2.3 channels in reproductive tissues

During passage of sperm through the female reproductive tract, spermatozoa undergo a series of chemical and physiological changes collectively called capacitation, which are required for acrosome exocytosis (AE) and the subsequent steps of oocyte fertilization. Capacitation is associated with changes in membrane lipid composition, increased intracellular Ca²⁺ levels, membrane hyperpolarization, activation of second messenger systems and a unique sperm motility pattern [85,121–124]. The underlying mechanisms are still incompletely understood, but Ca²⁺ influx through Ca_v2.3 channels secondary to focal enrichment of the ganglioside G_M1 has been shown to promote capacitation [85] and sperm from Ca_v2.3-deficient mice exhibits altered Ca²⁺ responses, aberrant motility, reduced acrosome exocytosis, and a strong sub-fertility phenotype [85,125].

4. (Patho)physiological implications of Zn²⁺ induced Ca_v2.3 channel modulation

Although the role of vesicular Zn²⁺ has been most extensively studied in the central nervous system, loosely bound Zn²⁺ is also important in the peripheral nervous system and in neuroendocrine and reproductive tissues. Due to a large concentration-gradient favoring passive entry, released Zn²⁺ can usually gain access to the intracellular compartment as well, which is under study as a potential neuronal signaling pathway [126] and has been implicated in cell death under certain pathophysiological conditions [4,127,128]. Since Ca_v2.3 channels are unlikely to play a role for cellular Zn²⁺ entry [73,129], the rest of the present article will focus on the role and effects of extracellular loosely bound Zn²⁺ in different tissues.

Ca_v2.3 channels as a target for synaptic Zn²⁺ signals in the brain

Loosely bound Zn²⁺ accounts for roughly 10% of total brain Zn²⁺, and is almost exclusively located in the presynaptic vesicles of so-called zinc-enriched neurons [3,4]. These neurons are not associated with a single neurotransmitter and mainly located in the limbic system, the neocortex and the retina [130–132], which coincides almost exactly with the expression pattern of Ca_v2.3 channels in the brain. Resting levels of loosely bound Zn²⁺ in the brain extracellular fluid are in the order of 5–25 nM [133], but these values are frequently exceeded at the synapses of zinc-enriched neurons, although the exact magnitude of synaptic Zn²⁺ signals remains a matter of debate. Based on experiments in acute brain slices, some have estimated that peak cleft concentrations at hippocampal mossy fiber synapses could be in the order of 10–20 µM during basal synaptic activity and in excess of 100 µM during more intense, LTP-inducing stimulation [134], while others have questioned that they could even exceed 1 µM [135,136]. However, based on a large number of studies [126,137–146] and taking into account that Zn²⁺ release is substantially reduced in acute brain slices [147], it seems reasonable to assume that physiological Zn²⁺ signals in the brain are in the low micromolar concentration range, so that Ca_v2.3 channel modulation occurs mainly via high-affinity binding. The exact functional relevance of synaptic Zn²⁺ remains controversial as well, but it has been implicated in several physiological and pathophysiological processes. For example, using the rapid Zn²⁺ chelator ZX1 and different strains of knockout mice, synaptic Zn²⁺ has been shown exert a dual control of non-NMDA receptor-dependent long-term potentiation (LTP) in hippocampal mossy fiber-CA3 synapses, where it promotes induction of presynaptic and inhibits induction of postsynaptic LTP [148]. Presynaptic LTP induction at these synapses involves Ca²⁺ influx through Ca_v2.3 channels [100,149], suggesting that they could play a role for the effects of Zn²⁺. Interestingly however, Zn²⁺ chelation by ZX1 [148] produced the same effects as genetic or pharmacological ablation of Ca_v2.3 channels [100,149], namely a decrease of LTP induction without changes in LTP expression, basal transmission, paired-pulse facilitation or frequency facilitation. These results are difficult to explain by a ZX1-induced reversal of Zn²⁺-induced suppression but could be reconciled by our recent finding that low micromolar Zn²⁺ can also stimulate Ca_v2.3 channel function [40]. Thus, assuming that the (local) resting membrane potential (RMP) is sufficiently positive (>-70 mV) that a significant fraction of the channels is inactivated, synaptic Zn²⁺ could increase Ca²⁺ influx by increasing the fraction of channels available for activation, in which case Zn²⁺ chelation should mimic the effects of Ca_v2.3 channel ablation. The dual effects of Zn²⁺ on mossy fiber LTP have been proposed to support physiological functions while preventing hyperexcitability due to excessive activation of CA3 pyramids [44,45]. A number of experimental and clinical findings support the view that synaptic Zn²⁺ could serve as an intrinsic anticonvulsant that dampens excitability not only in limbic brain regions. For example, synaptic release and extracellular accumulation of Zn²⁺ in mice have been demonstrated to limit the propagation of cortical spreading depression in vitro and in vivo [150] and Zn²⁺ treatment reduced dentate granule cell hyperexcitability in human epileptic patients [151]. Zn²⁺ deficiency or chelation have conversely been shown to increase the susceptibility to chemically induced seizures in mice or rats [152,153] and to induce seizures and excitotoxicity in healthy rats [154] and rats subjected to non-lesioning over-excitation [155]. Given their apparently pro-ictogenic role, Ca_v2.3 channels would represent an optimal target for the anticonvulsant action of vesicular Zn²⁺ under pathophysiological conditions. Zn²⁺ levels in the extracellular space during chemically induced epileptiform discharges have been estimated to be in the order of 300 µM [139], so that pore block and low-affinity slowing could significantly contribute to Zn²⁺-induced Ca_v2.3 channel modulation under pathophysiological conditions. The latter could be important to prevent excessive Ca²⁺ influx when over-excitation leads to a depolarization of the neuronal RMP, as it would counteract the reversal of RMP-dependent inactivation mediated by high-affinity Zn²⁺ binding. However, given its many molecular targets, the net effect of Zn²⁺ treatment or chelation on brain function can clearly not be attributed to changes in Ca_v2.3 channel gating and permeation alone. For example, Zn²⁺ has also been shown to slow the deactivation kinetics of LVA Ca_v3.3 channels [156] and even submicromolar concentrations potently inhibit Ca_v3.2 channels [42,156,157] as well as certain neurotransmitter receptors [158,159] and possibly other targets.

Ca_v2.3 channels as a target for paracrine Zn²⁺ signals in the pancreas

β-cell insulin secretory granules contain high (millimolar) concentrations of Zn²⁺, which forms 2Zn-hexameric complexes with insulin and is well known to be required for proper insulin biosynthesis, maturation, and secretion [160,161]. After release into the periportal circulation, the higher pH of blood leads to rapid dissociation into free Zn²⁺ and insulin monomers [161,162], which has been estimated to transiently raise the local Zn²⁺ concentration to several hundreds of micromolar [128]. Based on these and other findings, Zn²⁺ has been proposed as a candidate signal for non-insulin paracrine intra-islet crosstalk to nearby α-cells [5]. Thus, according to the Zn²⁺ switch-off hypothesis, β-cell Zn²⁺ release during hyperglycemia could tonically suppress glucagon secretion by opening α-cell K_ATP and inhibiting α-cell Ca²⁺ and Na⁺ channels, whereas cessation of Zn²⁺ supply during hypoglycemia could lead to closure of K_ATP channels and Na⁺ and L-type Ca²⁺ channel-dependent firing of action potentials (APs), during the peaks of which local Ca²⁺-influx through P/Q-type Ca²⁺ channels triggers glucagon secretion. Support for this hypothesis is provided by findings that Zn²⁺ suppresses pyruvate- or glucose-induced glucagon release in static secretion and electrophysiological experiments with isolated rodent islets and purified α-cells [163,164]. In addition, Zn²⁺ but not Zn²⁺-free insulin was reported to inhibit α-cell glucagon secretion in the perfused rat pancreas [162,165], although some studies failed to detect Zn²⁺-induced suppression of glucagon secretion [166,167]. Consistent with a role of Zn²⁺ and Ca_v2.3 channels for the regulation of glucagon release, we have previously shown that in vivo Zn²⁺ chelation produces a correlated increase of blood glucose and serum glucagon levels in wild-type mice, and that this response is severely blunted in Ca_v2.3-deficient mice [49]. In addition, fasting glucose and glucagon levels were significantly higher in Ca_v2.3-deficient compared to wild-type mice [49], suggesting that deficiency of these channels impairs the coupling between blood glucose levels and glucagon secretion without reducing glucagon release per se. One explanation for these findings could be that Zn²⁺ dis-inhibition of Ca_v2.3 channels serves to amplify the effects of K_ATP channel closure and alters P/Q-type channel-dependent exocytosis by modifying the threshold or peak height of α-cell APs, without being a critical requirement for α-cell glucagon secretion per se. Alternatively, considering the relatively depolarized α-cell RMP, Zn²⁺ release during hyperglycemia could simultaneously suppress Ca_v2.3 channels and increase their availability for activation at rest, so that rapid reversal of Zn²⁺-induced suppression upon cessation of Zn²⁺ supply allows for a brief burst of Ca²⁺ influx prior to time-dependent reentry into inactivated states. The latter could be an attractive mechanism by which (cessation of) Zn²⁺ release from β-cells synchronizes the pulsatile release of insulin and glucagon. Finally, it has also been proposed that the suppression of glucagon secretion during hyperglycemia results from depolarization-induced inactivation of Na⁺ and Ca²⁺ channels due to α-cell glucose metabolism and closure of K_ATP channels, which reduces the AP peak height and thus P/Q-type Ca²⁺ channel-mediated exocytosis, while electrical activity and global Ca²⁺ oscillations remain preserved [120,168,169]. In this view, the Zn²⁺-induced depolarizing shift in Ca_v2.3 channel voltage-dependence might allow them to remain active during hyperglycemia, so that they could be involved in maintaining α-cell electrical activity and global Ca²⁺ oscillations. In any case, we interestingly also found that glucose tolerance is preserved in vehicle-treated but severely impaired in Zn²⁺-chelated Ca_v2.3-deficient mice [49], suggesting that adequate Zn²⁺ levels are critical for maintaining glucose tolerance under conditions of Ca_v2.3 channel dysfunction. The latter finding could have important clinical implications, as the region of the human Ca_v2.3 gene has been linked to impaired glucose homeostasis in human patients with type 2 diabetes mellitus [170–172], which could possibly benefit from Zn²⁺ supplementation.

Ca_v2.3 channels as a target for Zn²⁺ signals in reproductive tissues

When compared to blood plasma (~11-23 µM total and 0.1–1 nM loosely bound Zn²⁺) [173,174], human seminal plasma contains exceptionally high concentrations of Zn²⁺ (~2-3 mM total Zn²⁺) [175,176], which is mainly secreted by the prostate [177] and plays a vital role in sperm capacitation [176]. The high Zn²⁺ concentrations in seminal plasma are thought to be an important gatekeeper for maintaining sperm in a metabolically quiescent state, whereas the decrease in Zn²⁺ concentration due to filtering out of seminal fluid in the female reproductive tract could promote capacitation [176]. In addition, sperm-induced Zn²⁺ release from the oocyte, the so-called Zn²⁺ sparks, could be involved in preventing the entry of more than one spermatozoon at fertilization [176,178,179]. Unfortunately, the exact concentration of loosely bound Zn²⁺ in seminal plasma has not been determined, but estimated to be in the order of at least 3 µM [175], suggesting that high-affinity modulation of Ca_v2.3 channels could contribute to the effects of Zn²⁺. In this context, it is especially interesting that acrosome exocytosis has been shown to depend on spatiotemporal information encoded by a transient burst of Ca²⁺ influx through Ca_v2.3 channels and not the mere presence or amplitude of Ca²⁺ waves [85]. Based on the depolarized RMP of non-capacitated sperm, Zn²⁺ could not only suppress Ca_v2.3 channels but also increase their availability for activation. During the passage of the female reproductive tract and filtering out of seminal fluid, rapid reversal of Zn²⁺-induced suppression could allow for a transient influx of Ca²⁺ through these channels, which is subsequently terminated by time-dependent inactivation, as exemplified by the time-course of changes upon Zn²⁺ removal in (figure 2F). Presumably, however, a complex interplay exists between gradual changes in Zn²⁺ concentration, RMP, and membrane lipid composition during passage of the female reproductive tract, which could fine-tune the temporal profile of Ca²⁺ influx through Ca_v2.3 channels during different stages of the reproductive process.

Conclusion

Although the exact functional role of Zn²⁺-induced Ca_v2.3 channel modulation in most tissues remains to be firmly established, the picture that emerges is one where Ca_v2.3 channel expression parallels the occurrence of loosely bound Zn²⁺ pools, and where these channels may serve to translate physiological Zn²⁺ signals into changes of electrical activity and/or intracellular Ca²⁺ levels. Improved tools for investigation of endogenous Zn²⁺ signals and genetic approaches to identify and manipulate the different putative Zn²⁺ binding sites will be required to understand their function and contribution to Zn²⁺-induced Ca_v2.3 channel modulation in different tissues.

Disclosure statement

No potential conflict of interest was reported by the authors.