The Cardiac Ryanodine Receptor Provides a Suitable Pathway for the Rapid Transport of Zinc (Zn²⁺)

Abstract

The sarcoplasmic reticulum (SR) in cardiac muscle is suggested to act as a dynamic storage for Zn²⁺ release and reuptake, albeit it is primarily implicated in the Ca²⁺ signaling required for the cardiac cycle. A large Ca²⁺ release from the SR is mediated by the cardiac ryanodine receptor (RYR2), and while this has a prominent conductance for Ca²⁺ in vivo, it also conducts other divalent cations in vitro. Since Zn²⁺ and permeant Mg²⁺ have similar physical properties, we tested if the RYR2 channel also conducts Zn²⁺. Using the method of planar lipid membranes, we evidenced that the RYR2 channel is permeable to Zn²⁺ with a considerable conductance of 81.1 ± 2.4 pS, which was significantly lower than the values for Ca²⁺ (127.5 ± 1.8 pS) and Mg²⁺ (95.3 ± 1.4 pS), obtained under the same asymmetric conditions. Despite similar physical properties, the intrinsic Zn²⁺ permeability (P_Ca/P_Zn = 2.65 ± 0.19) was found to be ~2.3-fold lower than that of Mg²⁺ (P_Ca/P_Mg = 1.146 ± 0.071). Further, we assessed whether the channel itself could be a direct target of the Zn²⁺ current, having the Zn²⁺ finger extended into the cytosolic vestibular portion of the permeation pathway. We attempted to displace Zn²⁺ from the RYR2 Zn²⁺ finger to induce its structural defects, which are associated with RYR2 dysfunction. Zn²⁺ chelators were added to the channel cytosolic side or strongly competing cadmium cations (Cd²⁺) were allowed to permeate the RYR2 channel. Only the Cd²⁺ current was able to cause the decay of channel activity, presumably as a result of Zn²⁺ to Cd²⁺ replacement. Our findings suggest that the RYR2 channel can provide a suitable pathway for rapid Zn²⁺ escape from the cardiac SR; thus, the channel may play a role in local and/or global Zn²⁺ signaling in cardiomyocytes.

1. Introduction

Zinc (Zn²⁺) is one of the most abundant metal cations in mammalian cells, with diverse functions in numerous physiological processes important for differentiation, growth and survival (reviewed in [1,2]). Although, Zn²⁺ has been generally considered to have moderate biological importance, significant advances in understanding Zn²⁺ biology in the past decade have changed this view. Reflecting the physiological relevance, both Zn²⁺ deficiency and excess have been documented in a wide range of pathological conditions, including cancer, diabetes and inflammatory, cardiovascular and neurodegenerative diseases, which have been the topic of several review articles [3,4,5,6,7,8,9]. While catalytic and structural functions of Zn²⁺ are well-established in a great number of metalloproteins [10,11], Zn²⁺ signaling capacities have only recently received extensive attention, mainly in a cellular and molecular context.

The Zn²⁺ affinity to protein metal binding sites is highly competitive towards Mg²⁺ and Ca²⁺, therefore, free intracellular Zn²⁺ concentration is tightly controlled and fluctuates in an extraordinarily narrow range (pM–nM), depending on the cell type [12,13,14,15]. In addition to cytosolic Zn²⁺-chelating proteins (reviewed in [16]), various Zn²⁺ transporters (carrier-type) are involved in controlling intracellular Zn²⁺, such as ZnT proteins exporting Zn²⁺ from the cytoplasm and ZIP proteins with the opposite function (reviewed in [17,18,19]).

ZnT and ZIP proteins have a distinct subcellular location, depending on their contribution to Zn²⁺ mobilization. In mammals, they are mostly localized to the plasma membrane [20,21,22,23,24,25,26] or in membranes of intracellular organelles, such as the ER and Golgi apparatus [27,28,29,30,31,32,33]. All these intracellular compartments obviously accumulate Zn²⁺ [34,35,36], but only some could be implicated in Zn²⁺ signaling, exemplified by Zn²⁺ transients and waves [35,37]. Although, there is no clear evidence, it can be proposed that the SR in cardiomyocytes acts as a dynamic storage for Zn²⁺ cations, readily available for release [8,38]. These results strongly imply the presence of Zn²⁺ transporters, and indeed ZIP and ZnT proteins have been evidenced in the heart tissue [27,39,40]. Moreover, their predominant subcellular localization in the SR membrane has been demonstrated [41].

It is well established that the release and accumulation of Ca²⁺ by the SR in cardiomyocytes is fundamental to the excitation-contraction coupling [42,43,44]. The cyclic release of Ca²⁺ required for contraction is mediated by the cardiac ryanodine receptor (RYR2) displaying a remarkable conductance for Ca²⁺ [45,46] compared to other Ca²⁺-permeable channels [47,48]. However, the RYR2 channel discriminates only slightly between divalent cations [46], and thus has been shown to be also permeable to Mg²⁺, Sr²⁺ and Ba²⁺ [46,49,50,51]. Since we noticed that both Mg²⁺ and Zn²⁺ possess similar physical properties relevant to permeation through ion channels [52,53,54], we examined whether the RYR2 channel provides a suitable permeation pathway for Zn²⁺. At the single-channel level, we established that Zn²⁺ greatly permeated the RYR2 channel in the lumen-to-cytosol direction, although with a lower conductance and intrinsic permeability than Ca²⁺ and even Mg²⁺. Our results indicate that the RYR2 channel may play a role in Zn²⁺ signaling in cardiomyocytes, and the channel itself could be a direct target for the localized Zn²⁺ increase, with the Zn²⁺ finger, a well-known Zn²⁺-binding site, extending into the cytosolic vestibular portion of the RYR2 permeation pathway [55].

2. Materials and Methods

2.1. Single-Channel Recordings

The sarcoplasmic reticulum (SR) microsomes enriched in RYR2 channels isolated from rat ventricular muscle were used for single-channel recordings as described previously [51] with some modifications. Planar lipid membranes (BLMs) were formed across a 50–70 μm aperture in the wall of a polystyrene cup separating two compartments, cytosolic and luminal.

The cytosolic compartment was filled with 1 mL of 50 mM KCl, 10 mM Tris and 20 mM HEPES (pH = 7.35). The free cytosolic Ca²⁺ concentration of 90 nM was obtained by including 1 mM ethylene glycol-bis(β-aminoethylether)–N,N,N′,N′-tetraacetic acid (EGTA) and 0.587 mM CaCl₂. In some cases, N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethylene diamine (TPEN) or nitrilotriacetic acid (NTA) was used to chelate Zn²⁺ impurities in the cytosolic solutions. The free Zn²⁺ ([Zn²⁺]_C) and Ca²⁺([Ca²⁺]_C) concentrations were determined by WinMaxc32 version 2.50 (http://www.stanford.edu/~cpatton/maxc.html, accessed on 24 April 2021). In control experiments, the luminal compartment was filled with 1 mL of 1–8 mM Ca(OH)₂, 50 mM KCl, 10 mM Tris and 22–43 mM HEPES (pH = 7.35). In mole-fraction experiments for luminal Mg²⁺ or Ca²⁺, prior to formation of the BLM, 4–7 mM of MgCl₂ or CaCl₂ was added to the luminal solution to obtain varied mixtures with 1–4 mM Ca(OH)₂ while maintaining the total divalent concentration at 8 mM. For pure Mg²⁺ or Ca²⁺, the luminal compartment was filled with 1 mL of 8 mM MgCl₂ or CaCl₂, 50 mM KCl, 10 mM Tris and 20 mM HEPES (pH = 7.35). The fusion of SR microsomes with the BLM was not successful enough when Zn²⁺ was present on the BLM luminal side, therefore, the RYR2 channel was exposed to 8 mM ZnCl₂, 50 mM KCl, 10 mM Tris and 20 mM HEPES (pH = 6.83) by perfusing the luminal compartment after channel reconstitution under control conditions. In mole-fraction experiments, 4–7 mM ZnCl₂ was also added to 1–4 mM Ca(OH)₂ after RYR2 reconstitution. In addition, mixtures of 8 mM Ca(OH)₂ with 8 mM ZnCl₂ or 8 mM CdCl₂ were also tested. In the control and for the MCl₂ additions (4–7 mM), 1–2 mM cytosolic caffeine was used to moderately activate RYR2 channels (M: Mg, Zn or Ca). Similarly, these caffeine concentrations were sufficient for the activation of channels exposed to 8 mM Ca(OH)₂ mixed with 8 mM ZnCl₂ or 8 mM CdCl₂. When ZnCl₂ or MgCl₂ was present in the luminal solution alone, caffeine concentration was increased to 6–7 mM to achieve a moderate RYR2 activity. The channel was incorporated into the BLM in a fixed orientation with the cytosolic side always exposed to the cytosolic solution because all RYR2 channels were sensitive to cytosolic caffeine [55,56,57,58]. At the end of experiments, the addition of ryanodine causing the characteristic transition to a half-conducting state was used to validate the channel identity [59].

2.2. Acquisition and Analysis of Single-Channel Recordings

Data acquisition and analysis of the open probability (P_O), frequency of opening (F), average open (T_O) and closed times (T_C) were performed as detailed in [51,60]. The conductance (G) and the reversal potential (E_rev) were determined from a linear regression of the current-voltage relationship, acquired by applying membrane voltage varying from −20 mV to +20 mV. The slope of the fitted line was equal to G, and E_rev was taken as the intersection of the voltage axis with the linear fit. To avoid misinterpretation of E_rev measurements, the E_rev value was further corrected for the measured offset voltage at the end of the experiment and liquid junction potentials (LJPs). The LJP values were determined for all tested luminal solutions according to Barry et al. [61] and were in the range from −0.18 mV to +0.76 mV. The current amplitude at 0 mV (I₀) was consequently corrected. For tested M²⁺ (M²⁺: Mg²⁺, Zn²⁺ or Ca²⁺), the dependence of E_rev on a composition of M²⁺/Ca²⁺ mixture on the RYR2 luminal face was fitted by the following equation to determine the relative Ca²⁺/M²⁺ permeability coefficient (P_Ca/P_M), referred to as intrinsic permeability [62]:

$${\mathrm{E}}_{\mathrm{rev}}=\frac{\mathrm{RT}}{2\mathrm{F}}\ln \frac{4{\mathrm{P}}_{\mathrm{Ca}}[{\mathrm{Ca}}^{2+}{]}_{\mathrm{C}}+{\mathrm{P}}_{\mathrm{K}}[{\mathrm{K}}^{+}{]}_{\mathrm{C}}+{\mathrm{P}}_{\mathrm{Tris}}[{\mathrm{Tris}}^{+}{]}_{\mathrm{C}}}{4{\mathrm{P}}_{\mathrm{Ca}}[{\mathrm{Ca}}^{2+}{]}_{\mathrm{L}}+{\mathrm{P}}_{\mathrm{K}}[{\mathrm{K}}^{+}{]}_{\mathrm{L}}+{\mathrm{P}}_{\mathrm{Tris}}[{\mathrm{Tris}}^{+}{]}_{\mathrm{L}}+4{\mathrm{P}}_{\mathrm{M}}[{\mathrm{M}}^{2+}{]}_{\mathrm{L}}},$$

(1)

Equation (1) is derived from the well-known Goldman—Hodgkin—Katz (GHK) equation [63,64]; where F is Faraday’s constant, R is the universal gas constant and T is the absolute temperature. At room temperature (25 °C), RT/F is ~25.693 mV. The values of P_Ca/P_Tris and P_Ca/P_K were, respectively, 29.545 [65] and 6.5 [46,66]. The subscripts C and L denote a cytosolic or luminal quantity, respectively. Similarly, the E_rev plotted against pure luminal Ca²⁺ ([Ca²⁺]_L) was fitted. For visualization purposes, the dependences of G and I₀ on [Ca²⁺]_L were fitted by the Michaelis—Menten equation.

Ion diffusion theory [67] was used to relate the local [Zn²⁺], Zn²⁺ current amplitude at 0 mV (I_0,Zn), and distance from an open channel pore (r) (Equation (2)):

$$\left[{\mathrm{Zn}}^{2+}\right]\left(\mathrm{r}\right)={\mathrm{e}}^{\frac{-\mathrm{r}}{\sqrt{\frac{{\mathrm{D}}_{\mathrm{Zn}}}{\mathrm{k}\left[\mathrm{B}\right]}}}}\frac{{\mathrm{I}}_{0,\mathrm{Zn}}}{4{\mathsf{\pi }\mathrm{FD}}_{\mathrm{Zn}}\mathrm{r}}.$$

(2)

Here, D_Zn = 7.03 × 10⁻¹⁰ m² s⁻¹ [68] is the diffusion coefficient for Zn²⁺, k is the rate for Zn²⁺ binding to the buffer B, [B] = 1 mM is the buffer concentration, F = 96,500 C mol⁻¹ is the Faraday constant. The only Zn²⁺ buffer present in most of our experiments was EGTA and its rate of Zn²⁺ binding, k, was 2.6 × 10⁶ M⁻¹ s⁻¹ [69].

The results are reported as the average ± SEM or the fitted value ± SEM. Statistical comparison of differences in mole-fraction experiments (I₀ and G) were made by two-way ANOVA with Tukey’s post hoc test. We also used one-way ANOVA with Tukey’s post hoc test to statistically compare changes in the pH of solutions when luminal M²⁺ was added and changes in the values of T_O, T_C, and F caused by luminal Zn²⁺. Unpaired Student’s t tests were performed to detect significant changes in the latency of RYR2 activity decay when [Cd²⁺]_L was added and differences in I₀, E_rev, or G values when pH was changed. Paired Student’s t tests were utilized for statistical comparison of differences in I₀, E_rev, or G values when 8 mM [Zn²⁺]_L or 8 mM [Cd²⁺]_L was mixed with 8 mM [Ca²⁺]_L, and changes in P_O when NTA or TPEN was added to chelate Zn²⁺ in the cytosolic solution. Differences were regarded to be statistically significant at p < 0.05.

3. Results

RYR2 channels isolated from rat hearts were incorporated into the BLMs and recorded under voltage clamp conditions. The goal here was to measure the RYR2 current carried by Zn²⁺ in the lumen-to-cytosol direction. Data are presented that define permeation and gating properties including the intrinsic Zn²⁺ permeability of the RYR2 channel. Additionally, a potential role of the RYR2 Zn²⁺ current in local Zn²⁺ signaling was assessed by computing Zn²⁺ accumulation near potential molecular targets, such as neighboring RYR2 channels and the Zn²⁺ finger domain extending into the cytosolic vestibular portion of the RYR2 permeation pathway [55]. Finally, the ability of Cd²⁺ current in the lumen-to-cytosol direction to displace Zn²⁺ from the RYR2 Zn²⁺ finger was investigated.

Working with Zn²⁺ compounds is usually difficult, because they show a low solubility in water at the physiologically-relevant pH range [70,71]. We therefore tested more Zn²⁺ compounds and found that the chloride salt of Zn²⁺ was most appropriate for our purpose. In addition, 8 mM ZnCl₂ was the maximal concentration available for testing to avoid formation of precipitates and thus prevent overestimation of the Zn²⁺ content. Of note, K⁺ and Tris⁺ cations were also added to the solutions, although both permeate the RYR2 channel [65] and could potentially interfere with RYR2 permeation properties for Zn²⁺ cations. However, they were added symmetrically to both RYR2 sides to diminish their contributions. Since only the chloride salt of Zn²⁺ could have been used, a small Cl⁻ gradient (50 mM vs. 58–66 mM) was inevitably generated at 0 mV by Zn²⁺ additions to the luminal solution, thus providing a driving force for Cl⁻ transport through the BLM. Despite the presence of Cl⁻ channels in cardiac SR microsomes, we did not observe their activity under the conditions utilized in our study. The reason for this probably lies in the fact that Cl⁻ gradient was not sufficient to produce recordable Cl⁻ currents. Another possibility is that Cl⁻ channels were not incorporated into the BLMs together with RYR2 channels.

3.1. The RYR2 Conductance for Zn²⁺

Our initial aim was to directly record the Zn²⁺ current by building the Zn²⁺ gradient across the BLM. However, after several unsuccessful attempts to reconstitute RYR2 channels with Zn²⁺ in the luminal solution, we modified our experimental approach. The luminal compartment was perfused with the Zn²⁺ solution after RYR2 channels were incorporated into the BLMs in the presence of 8 mM [Ca²⁺]_L. Representative RYR2 recordings measured in the presence of 8 mM [Zn²⁺]_L are shown in Figure 1A. The channels were solely activated by 6–7 mM cytosolic caffeine in the presence of non-activating free [Ca²⁺]_C (90 nM) to record the longer and fully resolved open and closed channel events required for the unbiased determination of the Zn²⁺ current values (I_Zn). When the membrane voltage changed in 5 mV incremental steps from −10 mV to +10 mV, the RYR2 I_Zn increased from 0.31 ± 0.15 pA to 1.86 ± 0.16 pA. At 0 mV, the typical value of I_0,Zn was 0.927 ± 0.093 pA. To estimate Zn²⁺ conductance (G_Zn), linear regression was performed on the current-voltage relationship (Figure 1B). Under asymmetrical conditions when the pure Zn²⁺ current was driven by the 8 mM gradient, the value of G_Zn was 81.1 ± 2.4 pS. Moreover, the reversal potential (E_rev) was −11.8 ± 1.1 mV.

3.2. Mole-Fraction Experiments under Non-Saturating Conditions

To evaluate the Zn²⁺ permeability coefficient relative to Ca²⁺, we investigated the mole-fraction behavior of I₀, E_rev, and G in the following Zn²⁺/Ca²⁺ mixtures on RYR2 luminal face. Particularly, 4, 5, 6 and 7 mM [Zn²⁺]_L was mixed with 4, 3, 2 and 1 mM [Ca²⁺]_L, respectively. We focused on the luminal solutions with the highest mole fraction of Zn²⁺ because if there were differences from the control (1–8 mM [Ca²⁺]_L), they should be the most pronounced. Figure 2 depicts the representative RYR2 recordings at 0 mV in the control and when luminal Zn²⁺ was present. RYR2 channels were moderately activated by 1–2 mM caffeine and only for 8 mM [Zn²⁺]_L, caffeine concentration was increased to 6–7 mM because luminal Ca²⁺, in contrast with other divalent cations, has been shown to be a strong sensitizer of the RYR2 channel to caffeine [50,51]. Since RYR2 gating is also strongly affected by luminal Ca²⁺ [51], the RYR2 channel exhibited a much faster gating, manifested by the increased number of open and closed events at the similar channel activity when only 8 mM [Zn²⁺]_L was present (Figure 2). At the quantitative level, this finding was supported by gating parameter calculations. Results collected for the frequency of opening (F), average open (T_O) and closed (T_C) times are summarized in

Table 1. Effects of luminal M²⁺ (M²⁺: Mg²⁺, Zn²⁺ or Ca²⁺) on gating parameters of the RYR2 channel at P_O ~ 0.5.

Luminal M2+	PO	F (Hz)	TO (ms)	TC (ms)
8 mM [Ca2+]L	0.4947 ± 0.0059	10.29 ± 1.04 #	49.03 ± 4.73 #	49.81 ± 7.04 #
8 mM [Mg2+]L	0.5137 ± 0.0040	38.4 ± 2.4 *	13.54 ± 0.77 *	12.94 ± 0.99 *
8 mM [Zn2+]L	0.526 ± 0.010	36.99 ± 1.03 *	14.74 ± 0.64 *	12.17 ± 0.51 *
1 mM [Ca2+]L + 7 mM [Zn2+]L	0.495 ± 0.015	11.43 ± 0.66 #	41.8 ± 1.7 #	45.22 ± 4.37 #

Data are presented as average ± SEM of 5 different experiments. * Significantly different from 8 mM [Ca²⁺]_L; ^# significantly different from 8 mM [Zn²⁺]_L (one-way ANOVA with Tukey′s post hoc test).

. The F value was increased more than three-fold when 8 mM [Ca²⁺]_L was completely replaced by 8 mM [Zn²⁺]_L. Because F is the reciprocal of a summation of T_O and T_C, a significant shortening in both T_O and T_C was observed. As we expected, the presence of even 1 mM [Ca²⁺]_L precluded any change in RYR2 gating behavior when luminal Zn²⁺ was added. This is fully consistent with findings gained for various mixtures of luminal Mg²⁺ or Ba²⁺ with Ca²⁺ [51], thus further highlighting a specific ability of luminal Ca²⁺ to slow down RYR2 gating. Moreover, no appreciable differences were noted in all three gating parameters calculated for 8 mM [Zn²⁺]_L and 8 mM [Mg²⁺]_L, similar to that found previously for luminal Mg²⁺ and Ba²⁺ [51].

Further, it is evident from the raw current traces that for all tested Zn²⁺ additions the I₀ substantially increased (Figure 2). The averaged results are presented in Figure 3A (middle panel). In the control, the I₀ grew with increasing [Ca²⁺]_L. The dependence of I₀ on [Ca²⁺]_L was well fitted by the Michaelis—Menten equation. The significant increase in I₀ by ~45% and ~100% was caused by the addition of 6 mM and 7 mM [Zn²⁺]_L, respectively. For lower [Zn²⁺]_L, an increasing trend was also detected; however, it did not achieve significance. To demonstrate the validity and reliability of our Zn²⁺ measurements, we generated the same dataset for Mg²⁺ and Ca²⁺ (chloride salts). Mg²⁺ and Zn²⁺ share the intrinsic physical properties such as ionic size, charge density and hydration enthalpy (

Table 2. Physical characteristics of divalent cations implicated in permeation through ion channels.

Divalent Cation	Ionic Radius a (pm)	Charge Density b (C mm−3)	Enthalpy of Hydration c (kJ mol−1)
Ca2+	99	79	−1577
Mg2+	65	278	−1921
Zn2+	71	214	−2046
Cd2+	91	102	−1807

^a Ionic radius taken from [72]. ^b Charge density was calculated according to the formula 2e/(4/3)πr³, where r is the ionic radius and e is 1.602 × 10⁻¹⁹ C. ^c Enthalpy of hydration taken from [73].

) relevant to permeation through ion channels [52,53,54]; therefore, we would expect similar passage of these cations through the RYR2 pore. As the positive control, varied CaCl₂ concentrations were added to the luminal RYR2 side to reach the total 8 mM [Ca²⁺]_L. Here, M²⁺ stands for Mg²⁺, Zn²⁺ or Ca²⁺ for simplicity. Luminal Zn²⁺ had less ability to affect I₀ carried by Ca²⁺ than luminal Mg²⁺. This was particularly noted when 7 mM [Mg²⁺]_L caused more than three-fold increase in the RYR2 current driven by the 1 mM Ca²⁺ gradient (Figure 3A, left panel). Lower [Mg²⁺]_L had a smaller but still significant impact. Because the RYR2 I₀ was considerably lower when 8 mM [Ca²⁺]_L was replaced by 8 mM [Mg²⁺]_L, the dominance of Mg²⁺ (6–7 mM) in the mixture with Ca²⁺ was not sufficient to reach the value of I₀ with pure 8 mM [Ca²⁺]_L. Therefore, only the Ca²⁺ additions (chloride salt) caused such a large increase that the I₀ values became similar to those obtained for both 8 mM Ca(OH)₂ (2.835 ± 0.094 pA) and 8 mM CaCl₂ (2.69 ± 0.11 pA) (Figure 3A, right panel). I₀ data indicate that the RYR2 channel apparently has a lower conductance and intrinsic permeability for Zn²⁺ than for Ca²⁺ and Mg²⁺.

We tested this possibility by determining E_rev and G from current-voltage relationships when the membrane voltage changed in 5 mV incremental steps from −20 mV to +20 mV. All collected current-voltage plots were Ohmic (data not shown) and well fitted by a straight line. In the control, the E_rev values substantially decreased with raising [Ca²⁺]_L from 1 mM to 8 mM (Figure 3B). Equation (1) well reproduces the luminal Ca²⁺-dependence of E_rev, thus validating the accuracy of our E_rev determination. When luminal Zn²⁺ was added, the E_rev decreased, as expected for the addition of permeant cation, while E_rev for pure 8 mM [Zn²⁺]_L was −11.8 ± 1.1 mV (Figure 3B, middle panel). To estimate the intrinsic Zn²⁺ permeability of the RYR2 channel, the dependence of E_rev on a composition of Zn²⁺/Ca²⁺ mixture was then fitted by Equation (1) yielding the relative Ca²⁺/Zn²⁺ permeability coefficient of 2.65 ± 0.19 (P_Ca/P_Zn). In comparison, the E_rev data collected for Mg²⁺/Ca²⁺ mixtures were well fitted with Equation (1) when P_Ca/P_Mg was 1.146 ± 0.071 (Figure 3B, left panel). This is in good agreement with the intrinsic Mg²⁺ permeability reported for the RYR2 channel [46]. As expected, the E_rev measured in Ca²⁺/Ca²⁺ mixtures did not depend on a mixture composition because 8 mM [Ca²⁺]_L was always present on the RYR2 luminal face. The fitted value for P_Ca/P_Ca was equal to 1.035 ± 0.061 (Figure 3B, right panel).

In general, ion translocation through the channel pore is characterized by a specific conductance, therefore, we examined G as a function of the Zn²⁺/Ca²⁺ mixture composition (Figure 3C, middle panel). For the pure luminal Ca²⁺ solutions, the G values plotted against [Ca²⁺]_L were fitted by the Michaelis—Menten equation with almost full saturation at 8 mM [Ca²⁺]_L (Figure 3C). When luminal Zn²⁺ was added, we revealed a significant increase in G only for the addition of 7 mM [Zn²⁺]_L to 1 mM [Ca²⁺]_L (Figure 3C, middle panel), evidently because of the weaker intrinsic Zn²⁺ permeability (P_Ca/P_Zn = 2.65 ± 0.19) and a significantly lower RYR2 conductance for Zn²⁺ (81.1 ± 2.4 pS for 8 mM [Zn²⁺]_L vs. 127.5 ± 1.8 pS for 8 mM [Ca²⁺]_L). The situation for luminal Mg²⁺ was slightly different because the Mg²⁺ additions resulted in a greater change in the G values (Figure 3C, left panel). This was despite that the RYR2 channel exhibited a significantly lower G for Mg²⁺ than for Ca²⁺ when testing 8 mM gradients (95.3 ± 1.4 pS vs. 127.5 ± 1.8 pS, respectively). When luminal Ca²⁺ was added, two identical cations were mixed, and therefore the G values were not changed (Figure 3C, right panel). In summary, the I₀, G and E_rev results collectively indicate that the passage of Zn²⁺ through the RYR2 channel is less effective than even for Mg²⁺, despite having similar physical properties (Table 2).

3.3. Effects of pH When Luminal Zn²⁺ Was Added

The chloride salt of Zn²⁺ has been reported to change the pH of solutions because it is a weak acid [74]. Such changes could substantially affect measurements of the RYR2 G and intrinsic permeability [75,76]. To evaluate this potential source of inaccuracy, we first investigated the effects of the Zn²⁺ additions on the pH of the luminal solutions, while retaining the same volumes used for the single-channel recordings. Mg²⁺ and Ca²⁺ were examined in a similar manner. The summary data are plotted as a function of M²⁺/Ca²⁺ mixture content in Figure 4A. While Mg²⁺ and Ca²⁺ additions (4–7 mM) induced no significant changes in the pH from the control, luminal Zn²⁺ had a substantial effect with increasing its mole fraction. The pH of the luminal solution with no Ca²⁺ present (10 mM Tris, 20 mM HEPES, 50 mM KCl) dropped from 7.34 ± 0.007 to 6.83 ± 0.017 when 8 mM [Zn²⁺]_L was added. In contrast, there was no pH change with 8 mM [Mg²⁺]_L or [Ca²⁺]_l. Importantly, experimental conditions with 8 mM [Zn²⁺]_L were limited to a lower pH of 6.83 because, otherwise, Zn²⁺ precipitates occurred. To test if the large decrease in pH observed for the addition of 8 mM [Zn²⁺]_L might produce a shift in I₀, E_rev and G values, we tested 8 mM [Ca²⁺]_L (hydroxide) buffered to pH values of 6.83 and 7.35. Summary results presented in Figure 4B show that pH decrease was essentially without any significant effect. These data indicate that all results of mole-fraction experiments collected for luminal Zn²⁺ can be indeed interpreted as a manifestation of the intrinsic Zn²⁺ permeability displayed by the RYR2 channel.

3.4. Effects of Luminal Zn²⁺ on RYR2 Permeation Properties under Near-Saturating Conditions

As seen in Figure 3C, the [Ca²⁺]_L-dependence of the RYR2 G almost saturated at 8 mM. This indicates that all mole-fraction experiments shown in Figure 3 were performed at [Ca²⁺]_L, which is not at the saturating portion of the G-[Ca²⁺]_L curve. Under such conditions, luminal Zn²⁺ would not strongly compete with Ca²⁺ for occupancy of the RYR2 pore, if at all [77,78]. To further examine the mechanisms of Zn²⁺ permeation, we extended the I₀, E_rev and G measurements to include near-saturating conditions. Because 8 mM concentration was the highest possible [Zn²⁺]_L available for testing, we could use only a mixture with 8 mM [Ca²⁺]_L to allow a reasonable competition between these cations [79]. Figure 5A shows representative current traces of the same caffeine-activated RYR2 channel recorded at 0 mV in the absence and presence of luminal Zn²⁺. One can see that 8 mM [Zn²⁺]_L slightly reduced the current amplitude carried by 8 mM [Ca²⁺]_L. Average results are presented in Figure 5B (left panel). While the reduction in the I₀ was small but significant, E_rev changed only negligibly by ~−1.35 mV (Figure 5B, middle panel). This small shift toward more negative values was also predicted by Equation (1) for the estimated P_Ca/P_Zn of 2.65 ± 0.19 (−23.97 mV theoretical vs. −23.59 ± 1.03 mV experimental). Inevitably, the G significantly decreased (Figure 5B, right panel). Of note, the aforementioned changes were not related to pH, although it dropped from 7.35 ± 0.04 to 7.058 ± 0.029 by the luminal addition of 8 mM [Zn²⁺]_L. As shown in Figure 4B, an even larger decrease in pH from 7.35 to 6.83 did not produce any significant effect. Taken together, the decrease in I₀ and G values by Zn²⁺ addition to almost saturating [Ca²⁺]_L could indeed be interpreted as a consequence of competition between two permeant cations inside the RYR2 pore, which exhibits a different intrinsic permeability and/or conductance for each cation.

3.5. The Zn²⁺ Finger Located within the C-Terminus of the RYR2 Channel as a Potential Target of the Zn²⁺ Current

In an attempt to identify a potential target of the RYR2 Zn²⁺ current, we focused on small protein domains termed the Zn²⁺ fingers whose structure is highly organized upon Zn²⁺ complexation [80]. C2H2 type with two Cysteines (Cys or C) followed by a pair of Histidines (His or H) is one of the most ubiquitous coordination sites for Zn²⁺ (Figure 6A). This configuration has been recognized within the C-terminal tail of the RYR2 channel [55] and the inositol-1,4,5-trisphosphate receptor isoform-1 (IP₃R1) [81,82]. The IP₃R1 channel participates in Ca²⁺ transport across the ER membrane in all cell types (reviewed in [83]). The classical C2H2 topology has the consensus sequence C-X_2–5-C-X₁₂-H-X_3–4-H (X is for any amino acid) (reviewed in [84]). To determine and compare spacing in the C2H2 Zn²⁺ fingers of RYR2 and IP₃R1 channels, we computed the alignment of their C-terminal sequences using the ClustalX method (Figure 6B; [85]). Instead of focusing on rat channels, we extended our analysis to other mammalians such as mouse, dog and human, which are commonly used as a source of RYR2 and IP₃R1 channels for BLM experiments. Within this set, the first Cys and the second Cys are separated by two residues and this spacing is well conserved across mammalian RYR2 and IP₃R1 channels. Further, the interhistidine spacing of four residues is conserved as well. Up to here, the topology of RYR2 and IP₃R1 Zn²⁺ fingers matches the classical C2H2 pattern. However, we revealed one deviation in the distance between Cys-Cys and His-His pairs. The spacing of 16 residues is uncommon (Figure 6B). According to the consensus sequence, 12 residues are allowed, albeit, it has been shown that 10–14 residues could also be tolerated (atypical C2H2, Figure 6A). In the RYR2 sequence, specifically, we located one additional conserved His that is 11 residues away from the Cys-Cys pair and four residues from the following His. It is thus conceivable that this newly-identified His residue could act as the third Zn²⁺ ligand matching the overall spacing of the atypical C2H2 topology.

Despite a little doubt about the composition of the RYR2 Zn²⁺ finger, its location at the joint between the CTD (C-terminal domain) and the last transmembrane segment (S6) remains indisputable [55]. Since the Zn²⁺ finger domain is oriented toward the channel permeation pathway it is feasible to test the possibility that this domain is directly targeted by the Zn²⁺ current (Figure 6B). We began by calculating the free [Zn²⁺] profile around an open RYR2 pore. Considering the extremely high binding affinities of numerous Zn²⁺ fingers in the femtomolar range [86,87,88], Figure 6C shows that an extremely small Zn²⁺ current (from 1.5 × 10⁻¹² pA to 1.5 × 10⁻⁹ pA) would be sufficient to achieve femtomolar [Zn²⁺] around the Zn²⁺ finger located ~1.8 nm from the channel pore (Figure 6D, top panel; [55]). Since the RYR2 channel has been shown to be sensitive to cytosolic Zn²⁺ [89,90] and the channel itself seems to be resistant to its own current [91], we extended our calculations also to the neighboring RYR2 channels that might be targeted via Zn²⁺ diffusion. This scenario should also receive attention because in the heart, RYR2 channels operate in small packed clusters [92,93] and at the single channel level, two or more RYR2 channels can be occasionally reconstituted, perhaps at their normal cellular spacing. Recent BLM studies have presented evidence that Ca²⁺-activated RYR2 channels are substantially modulated by cytosolic Zn²⁺ (100 pM–100 nM) [89,90]. To reach this magnitude change over the entire cytosolic domain of the neighboring channel, where Zn²⁺ binding sites must be located, the I_0,Zn from 4.1 × 10⁻⁶ pA to 4.1 × 10⁻³ pA is required (Figure 6C). Center-to-corner distances of ~18 nm and ~44 nm were taken from Liu et al. [91] and are indicated in Figure 6D (bottom panel). Such I_0,Zn values are 122- to 122,000-fold lower in comparison to the physiologically relevant amplitude of the Ca²⁺ current (~0.5 pA) flowing through the RYR2 channel [77,78]. Thus, our calculations suggest that RYR2 function might be significantly affected by almost negligible Zn²⁺ current of more than or equal to 4.1 × 10⁻⁶ pA, which could be reachable in cardiomyocytes.

3.6. Displacement of Zn²⁺ from the RYR2 Zn²⁺ Finger

To further test the idea that the Zn²⁺ current supplies Zn²⁺ cations essential for the stabilization of the RYR2 Zn²⁺ finger, we assessed the accessibility of this Zn²⁺ binding site from either channel side (cytosolic and luminal) by displaying Zn²⁺ from its structure. As a nondestructive readout for this event, we monitored RYR2 P_O because it has been evidenced that the Zn²⁺ finger is fundamental to the channel activation by caffeine [55]. First, we tested whether Zn²⁺ chelating agents such as NTA or TPEN could compete for Zn²⁺ when applied to the RYR2 cytosolic side. Based on the purity assays of ultrapure chemicals (Sigma-Aldrich) and the strong binding affinity of EGTA to Zn²⁺, we estimated that ~2.13 pM [Zn²⁺]_C should have always been present in the cytosolic solution. When 5 mM NTA was added, [Zn²⁺]_C dropped down to 0.5 pM. During 20-min incubation, the RYR2 P_O did not change significantly (Figure 7A, left panel). Although, 5 µM TPEN decreased free [Zn²⁺]_C to the attomolar range (7.2 aM = 0.0072 fM), no significant change in the channel activity was observed during 20-min exposure (Figure 7A, right panel). Insufficiently high Zn²⁺ affinities of NTA and TPEN appear to be unlikely because binding affinities of numerous Zn²⁺ fingers are in the femtomolar range [86,87,88]. Presumably, a substantially longer incubation (it could reach hours) was needed to remove obviously tightly bound Zn²⁺ in the RYR2 Zn²⁺ finger, which could also be less approachable sitting deep in the RYR2 cytosolic vestibule. A short BLM lifetime, however, limited such demanding conditions. In a further attempt to displace Zn²⁺, we assessed whether a well-known competition between Zn²⁺ and Cd²⁺ in different eukaryotic Zn²⁺ fingers [94,95,96,97] could be a more direct approach. Given the comparable physical properties of Cd²⁺ with those of permeant Ca²⁺ and Mg²⁺ (Table 2), Cd²⁺ passage through the RYR2 channel might be expected. Indeed, we found that the luminal addition of 8 mM [Cd²⁺]_L to 8 mM [Ca²⁺]_L significantly decreased the RYR2 G, thus reflecting Cd²⁺ permeation (Figure 7B), as we evidenced for Zn²⁺ under the same conditions (Figure 5). This finding was crucial because it allowed us to approach the Zn²⁺ finger from the RYR2 luminal side by the Cd²⁺ current in the lumen-to-cytosol direction. Importantly, a drop in pH from 7.350 ± 0.010 to 7.234 ± 0.016 when 8 mM [Cd²⁺]_L was added did not contribute to a decrease in G because a much larger pH change from 7.35 to 6.83 was essentially without effect (Figure 4B). In Figure 7C (top trace), the Ca²⁺ current was only passing the caffeine-activated RYR2 channel in the presence of 8 mM [Ca²⁺]_L. After the addition of 8 mM [Cd²⁺]_L, a sudden decay of channel activity occurred after 8-min incubation and persisted within 20 min of exposure (Figure 7C, bottom two traces). Figure 7D shows that the latency of RYR2 activity decay varied considerably with [Cd²⁺]_L. As [Cd²⁺]_L increased from 8 mM to 16 mM, the channel activity was suppressed within a shorter time due to a greater ability of Cd²⁺ to substitute for Zn²⁺. Importantly, RYR2 activity was not recovered when caffeine concentration was substantially increased to 6–7 mM. This indicates that RYR2 sensitivity to caffeine was not attenuated as a result of Cd²⁺/Ca²⁺ competition directly at the RYR2 luminal side, as previously shown for Sr²⁺, Mg²⁺, or Ba²⁺ (when competing with 1 mM luminal Ca²⁺) [51]. Rather, the Cd²⁺ current destabilized the Zn²⁺ finger by replacing Zn²⁺ and thus promoting the decay of channel activity.

4. Discussion

There has been a rapidly growing interest in the field of Zn²⁺ biology in the last ten years because of significant advances in the knowledge of Zn²⁺ chemistry and biochemistry. Moreover, defective Zn²⁺ cellular homeostasis has been implicated in the pathophysiology of cancer and diabetes, common diseases which greatly affect global health. This has further stimulated an intensive research effort to understand the importance of Zn²⁺ in a diverse spectrum of biological functions. In mammalian cells, large amounts of Zn²⁺ accumulate in various cellular compartments and organelles [34,35,36], mainly as a structural or catalytic factor directly regulating a great number of protein functions. To be candidates for Zn²⁺ signaling, intracellular Zn²⁺ storages must have proteins for both Zn²⁺ release and reuptake. Although, Zn²⁺ transporters such as ZIP and ZnT with subcellular distribution have been identified in various mammalian cells (reviewed in [17,18,19]), a limited number of studies have been conducted in cardiomyocytes. However, at least one member of the ZIP family responsible for Zn²⁺ release to the cytoplasm and one member of the ZnT family mediating Zn²⁺ transport in the opposite direction have been found [27,40] and localized to the cardiac SR membrane [41]. Although, these transporters assumingly participated in Zn²⁺ signaling originating from the SR [8,38], we put forward the possibility that the cardiac ryanodine receptor (RYR2) located in the SR membrane also contributes. It has been known for many years that the RYR2 channel is responsible for a massive release of Ca²⁺ from the SR required for muscle contraction (reviewed in [98]). Its role in Zn²⁺ signaling has however been overlooked. Tuncay et al. [8] revealed a significant suppression of Zn²⁺ transients by ryanodine in electrically stimulated cardiomyocytes. Ryanodine is a plant alkaloid that binds to all three isoforms of the RYR channel with high affinity and either holds the channel open in a state of reduced conductance or causes a complete inhibition, depending on its concentration [59,99,100,101]. According to Tuncay et al. [8], Zn²⁺ transients mostly resulted from an increase in [Ca²⁺]_C and subsequent Zn²⁺ displacement from intracellular binding sites. In such a scenario, Zn²⁺ transients would be suppressed in the presence of ryanodine, because Ca²⁺ release from the SR mediated by the RYR2 channel would be compromised. However, considering similar physical properties of Zn²⁺ with those of permeant Mg²⁺ (Table 2), the Zn²⁺ release through the RYR2 channel which would inevitably accompany Ca²⁺ release, could be an alternative explanation. Our study was therefore designed to investigate whether Zn²⁺ permeates the RYR2 channel reconstituted into the BLM.

4.1. Permeation Properties of the RYR2 Channel for Zn²⁺

In this study, the permeation properties of the rat RYR2 channel were examined under various ionic conditions. As shown in Figure 1, the Zn²⁺ currents through the RYR2 channel in the luminal-to-cytosol direction were recorded for the first time. Our results are compatible with several studies reporting a weak Zn²⁺ conductance for voltage-gated Ca²⁺ channels [102,103,104,105]. When 8 mM [Zn²⁺]_L was present and used as a sole charge carrier, the RYR2 I_0,Zn was 0.927 ± 0.093 pA. It is comparable to, but significantly smaller than, those obtained for luminal Ca²⁺ (2.835 ± 0.094 pA) or even Mg²⁺ (1.822 ± 0.079 pA) under similar conditions. Accordingly, the G values fall in the sequence Ca²⁺ (127.5 ± 1.8 pS) > Mg²⁺ (95.3 ± 1.4 pS) > Zn²⁺ (81.1 ± 2.4 pS). Such differences between Zn²⁺ and Mg²⁺ were unexpected as these cations have similar physical properties relevant to ion channel permeation (Table 2). To gain a better understanding, we evaluated the Zn²⁺ permeability coefficient relative to Ca²⁺. We performed mole-fraction experiments with a mixture of Zn²⁺ and Ca²⁺ on the luminal side (8 mM total concentration) and monitored the changes in RYR2 permeation properties. Zn²⁺ additions to the luminal solution resulted in specific changes when we used non-saturating concentrations of luminal Ca²⁺ in respect to the RYR2 conductance (<8 mM). The I₀ and G were increased while the E_rev was shifted towards more negative values (Figure 3). The changes were more pronounced in mixtures with the highest mole fractions of Zn²⁺. Under near-saturating conditions, where a competition between Zn²⁺ and Ca²⁺ could occur within the RYR2 permeation pathway [79], the I₀ and G were significantly decreased, but no change was revealed for E_rev when 8 mM [Zn²⁺]_L was added to 8 mM [Ca²⁺]_L (Figure 5). Since the I₀ and G did not drop below the values obtained for pure luminal Zn²⁺, this phenomenon cannot be interpreted as the anomalous mole fraction effect occurring when the values of I₀ or G are lower in a mixture of two ions than in the pure solutions of individual ions at the same total concentration. To make such conclusion, we assumed that the permeation properties of the RYR2 channel at 8 mM and 16 mM [Zn²⁺]_L (the latter is not reachable at pH ~7.00) are similar. This was not an unreasonable proposal, as 8 mM concentration of divalent cations was found to be near-saturating.

In comparison to permeant Mg²⁺, Zn²⁺ displayed much less ability to contribute to permeation properties. The permeability coefficient for Zn²⁺ (P_Ca/P_Zn = 2.65 ± 0.19), estimated by fitting dependence of E_rev on a composition of Zn²⁺/Ca²⁺ mixture by Equation (1), was found to be ~2.3-fold lower than that of Mg²⁺ (P_Ca/P_Mg = 1.146 ± 0.071). Because the permeability coefficient has been shown to be a diffusion component of channel selectivity and reflects how well particular ions pass through the channel pore [106], our results clearly indicate that the RYR2 channel can differentiate, albeit only moderately, between Zn²⁺ and Mg²⁺ or Ca²⁺, providing a slower path to Zn²⁺. In respect to G, it has been proven that this parameter contains information not only about ion movement throughout the channel pore, but it also measures how well particular ions enter and exit the channel [62,106]. Since Zn²⁺ and Mg²⁺ exhibit a similar hydration enthalpy (Table 2), critically implicated in ion entry into the narrowest region of the channel pore [52,62], and as Zn²⁺ moves through the RYR2 channel more slowly than Mg²⁺, a lower G_Zn is an inevitable consequence.

4.2. Physiological Implications

Ion channels are predominantly involved in rapid signal transduction because ion movement through ion channels is usually much faster than passive transport down the electrochemical gradient mediated by carrier-type transporters [107]. During each heartbeat, RYR2 channels are responsible for the release of a huge amount of Ca²⁺ from the SR lumen. Our study shows that the same route is also suitable for Zn²⁺ mobilization from the SR, and thus Zn²⁺ might contribute to the RYR2 current of ~0.5 pA [77,78] during cardiac excitation-contraction coupling, albeit primarily driven by a 10,000-fold Ca²⁺ gradient across the SR membrane (100 nM in the cytosol, 1 mM in the SR lumen [108,109]). This seems reasonable, if we consider the extremely low free Zn²⁺ level of ~100 pM in the cytosol of cardiomyocytes [3,110] and more than 60-fold higher free [Zn²⁺] in the cardiac SR lumen (>6 nM, [110]). The existence of a Zn²⁺ gradient, albeit ~166-fold smaller than for Ca²⁺, together with a considerable RYR2 permeability for Zn²⁺, is compatible with the biological significance of the Zn²⁺ current through the RYR2 channel. At the cellular level, Tuncay et al. [8] support this idea by visualizing ryanodine-sensitive Zn²⁺ transients in cardiomyocytes having similar kinetics to those of Ca²⁺. Furthermore, both Ca²⁺ and Zn²⁺ transients required a small Ca²⁺ influx from the extracellular space through the L-type Ca²⁺ channel in the plasma membrane. Thus, it seems reasonable to propose that the RYR2 channel has a real Zn²⁺ transport function in cardiomyocytes (Figure 8), but the cellular target for this Zn²⁺ release has not been established. Certainly, the release does not trigger contraction because Zn²⁺ only negligibly interacts with proteins of the contractile machinery [111]. However, 100 pM–100 nM Zn²⁺ has been shown to shape Ca²⁺ release by amplifying the Ca²⁺-induced RYR2 activity [89,90]. According to our simple calculations, the Zn²⁺ current of more than or equal to 4.1 × 10⁻⁶ pA (~0.0008% of the RYR2 current in cardiomyocytes) should be sufficient for the building up of activating [Zn²⁺] near neighboring RYR2 channels (Figure 6C). This estimation suggests that almost negligible Zn²⁺ current mediated by one RYR2 channel could amplify Ca²⁺ release through neighboring RYR2 channels. This regulatory process might be impaired in cardiovascular diseases, which are often associated with Zn²⁺ deficiency [39,112,113,114]. It may arise from low intake, malabsorptive syndromes, or the administration of various medications (reviewed in [115]). In Zn²⁺-deficient cardiomyocytes, intracellular Zn²⁺ stores including the SR have been indeed found to be substantially depleted of Zn²⁺ [116]. One would then expect that the Zn²⁺ gradient across the SR membrane will be reduced, resulting in a compromised Zn²⁺ release. This implies that appropriate Zn²⁺ supplementation might be beneficial in the management of cardiovascular diseases, directly affecting Ca²⁺ signaling fundamental to the cardiac contraction. So far, a few studies have reported an improvement in symptoms of heart failure with Zn²⁺ supplementation [116,117,118]. Some clues on the physiological relevance of the RYR2 Zn²⁺ current, can also be found in work of Atar et al. [104], where changes in free [Zn²⁺]_C have been linked to the activation of transcription in electrically stimulated cardiomyocytes, given the catalytic and structural roles of Zn²⁺ in DNA- and RNA-binding proteins.

Numerous proteins with a variety of functions, including transcription, protein degradation, and DNA repair harbor small structured domains, the Zn²⁺ fingers, whose stability is dramatically improved by Zn²⁺ binding. The C2H2 Zn²⁺ finger with two Cys (C) followed by a pair of His residues (H) (Figure 6A) has also been identified within the C-terminal tail of structurally related IP₃R1 [81,82] and RYR2 channels [55] (Figure 6B). When individual or combined Cys and His residues were mutated, IP₃R1 function was completely abolished highlighting the critical role of the C-terminus in channel gating [81,82]. The C-terminal region is highly conserved in both the IP₃R and RYR families [119] and therefore it is not surprising that similar results were obtained for the RYR2 channel. The channel completely lost its ability to gate when both Cys residues and only the first of a His-His pair were individually substituted [55]. Surprisingly, mutation of the final His residue caused no change and the RYR2 channel retained its function. This is in sharp contrast to the IP₃R1 channel, where a critical importance of the final His residue in the Zn²⁺ finger sequence has been reported [82], albeit the spacing between the Cys-Cys and His-His pairs is uncommon (16 instead of classical 12 or atypical 10–14 residues, Figure 6B). The same deviation has also been demonstrated for the RYR2 channel [55], although we found one nearer conserved His, 11 residues away from the Cys-Cys pair (atypical C2H2 pattern), that could potentially serve as a third Zn²⁺ ligand when pairing with the following His (Figure 6B). To address such uncertainty in the composition of RYR2 Zn²⁺ finger, the mutation of the newly-identified His residue should be studied in the context of RYR2 function and Zn²⁺ binding properties.

The Zn²⁺ finger domain of each of the four RYR2 subunits impinges toward the central vertical axis of the channel permeation pathway, thus it may be well supplied with the Zn²⁺ current to ensure a proper channel function (Figure 8). In agreement with this prediction, the Cd²⁺ current passing the RYR2 channel in the lumen-to-cytosol direction caused a sudden decay of RYR2 activity. Since this action was specific for Cd²⁺ because the luminal additions of other divalent cations never had such detrimental effects [50,51], it was highly likely due to Zn²⁺ to Cd²⁺ replacement in the RYR2 Zn²⁺ finger. Obviously, Zn²⁺ displacement by Cd²⁺ was not structurally tolerated, and therefore RYR2 function was abolished. If the RYR2 Zn²⁺ current was fundamental to stabilization of the Zn²⁺ finger structure, one could ask why the channel function was not impaired in the absence of luminal Zn²⁺ when only Ca²⁺ or Mg²⁺ currents were carried by the channel. The answer could be found in Zn²⁺ impurities coming from otherwise ultrapure chemicals. We estimated that ~2.13 pM and ~0.8–1.0 nM free [Zn²⁺] was present in the cytosolic and luminal solutions, respectively. In generally, eukaryotic Zn²⁺ fingers possess the extremely high binding affinities in the femtomolar range [86,87,88], therefore, picomolar [Zn²⁺] on the RYR2 cytosolic side should have been sufficient to saturate the Zn²⁺ finger, albeit the deep position of this Zn²⁺-stabilized domain within the cytosolic portion of the RYR2 permeation pathway could considerably impair this process. We tested this idea by adding strong Zn²⁺ chelators such as NTA and TPEN to reach the atto-femtomolar free [Zn²⁺]_C, and thus facilitate Zn²⁺ displacement from the channel. This strategy was, however, not successful. One reason was probably the specific binding properties of the RYR2 Zn²⁺ finger. It is reasonable to assume that Zn²⁺ has to be tightly bound even under conditions of a transient depletion in Zn²⁺ because the RYR2 channel plays a dominant role in cardiac excitation-contraction coupling [42,43,44], and thus a proper RYR2 function is of particular importance. On the other hand, the ~470-fold Zn²⁺ gradient across the BLM, which was always present due to Zn²⁺ impurities, also likely contributed. Our calculations suggest that almost negligible Zn²⁺ current (≥1.5 × 10⁻¹² pA) is required to accumulate femtomolar [Zn²⁺] near the Zn²⁺ finger position (~1.8 nm from the pore). In cardiomyocytes, this extremely small current is seemingly also reachable, albeit Zn²⁺ movement will be driven by a smaller Zn²⁺ gradient compared to BLM experiments. Taken together, it appears that the RYR2 Zn²⁺ finger is more accessible from the luminal than the cytosolic side of the channel since Cd²⁺ current (from lumen to cytosol) replaced Zn²⁺ bound in the Zn²⁺ finger domain even in the presence of saturating [Zn²⁺]_C in the cytosolic solution. In contrast, a substantial decrease in [Zn²⁺]_C to the non-saturating range had no effect on Zn²⁺-RYR2 interactions when a subtle Zn²⁺ current in the lumen-to-cytosol direction occurred.

5. Conclusions

Here, we identify the RYR2 channel as a novel Zn²⁺ transporting protein in the SR membrane that might play a role in local and/or global Zn²⁺ signaling in cardiomyocytes, considering much faster ion movement through ion channels than the passive transport mediated by carrier-type transporters. The results demonstrate that the RYR2 channel itself could be regulated by its own Zn²⁺ current. Since Zn²⁺-binding domains, such as the Zn²⁺ fingers, often display extremely high binding affinities in the femtomolar range a subtle Zn²⁺ current in the lumen-to-cytosol direction was predicted to be sufficient for the saturation of the Zn²⁺ finger situated within the C-terminal tail of each of the four RYR2 subunits. It is not an unreasonable proposal, because the RYR2 Zn²⁺ finger is directed toward the permeation pathway, and thus it will inevitably be targeted by the Zn²⁺ current.