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

Figure 1. The RYR2 channel is permeable to Zn2+. (A) Representative RYR2 currents (digitally filtered at 250–400 Hz), shown as upward deflections from the marked zero-current level, in the presence of 8 mM [Zn2+]L when Zn2+ was only the charge carrier. Current recordings were conducted at −10, −5, 0, +5, +10 mV and are from the same channel. RYR2 channels were activated by 6–7 mM caffeine in the presence of 90 nM free [Ca2+]C. (B) Current-voltage relationship of the RYR2 channel was well-fitted by a straight line with the GZn value of 81.1 ± 2.4 pS. Data shown are average ± SEM of 5–7 experiments and error bars are shown only when SEM is larger than symbol size.

Figure 2. Luminal Zn2+ mixed with Ca2+ increases RYR2 currents under non-saturating conditions. Representative RYR2 currents (digitally filtered at 250–400 Hz), shown as upward deflections from the marked zero-current level, in the absence (control, left five traces) and after luminal Zn2+ exposure (right five traces). In total, 4, 5, 6, 7 mM [Zn2+]L was added to 4, 3, 2, 1 mM [Ca2+]L (control), respectively, to keep the total divalent concentration constant at 8 mM. Representative RYR2 recordings for pure 8 mM [Ca2+]L (control) and 8 mM [Zn2+]L are also shown. RYR2 channels were activated by 1–2 mM caffeine in the presence of 90 nM free [Ca2+]C. In the presence of 8 mM [Zn2+]L, caffeine concentration was increased to 6–7 mM. Current recordings were conducted at 0 mV and are from different channels.

Figure 3. M2+/Ca2+ mole-fraction experiments under non-saturating conditions. The I0 (A), Erev (B), and G (C) plotted against a composition of M2+/Ca2+ mixture on the RYR2 luminal face (M2+: Mg2+–green square, Zn2+–red triangle, or Ca2+–blue circle). In total, 4, 5, 6, 7 mM [M2+]L was added to 4, 3, 2, 1 mM [Ca2+]L, respectively, to keep the total divalent concentration constant at 8 mM. Data obtained for pure 8 mM [M2+]L are also included. As the control, the [Ca2+]L-dependence of Erev, G and I0 is shown (black circle). * Significantly different from the respective control values; # significantly different from the control value obtained for 8 mM [Ca2+]L (two-way ANOVA with Tukey’s post hoc test). The solid lines in (B) are the best fits using Equation (1) and in (A) and (C) using the Michaelis—Menten equation. The dashed lines in (A) and (C) are drawn point to point. Data shown are average ± SEM of 5–15 experiments and error bars are shown only when SEM is larger than symbol size.

Figure 4. Changes in pH caused by the Zn2+ additions have no effects on RYR2 permeation properties. (A) Effects of the M2+ additions on pH of M2+/Ca2+ mixture (M2+: Mg2+−green square, Zn2+–red triangle, or Ca2+–blue circle). In total, 4–7 mM [M2+]L was added to 1–4 mM [Ca2+]L, keeping the total divalent concentration constant at 8 mM. As the control, the [Ca2+]L-dependence of pH is shown (black circle). Data obtained for the pure 8 mM [M2+]L (chloride salt) or 8 mM [Ca2+]L (hydroxide) are also included. * Significantly different from the respective control values (one-way ANOVA with Tukey’s post hoc test). (B) The I0, Erev and G values obtained for pH of 6.83 and 7.35 when 8 mM [Ca2+]L (hydroxide) was present (unpaired Student’s t test). Data shown are average ± SEM of 5–7 experiments, and error bars in (A) are shown only when SEM is larger than symbol size. Data points shown in (B) are individual measurements.

Figure 5. Luminal Zn2+ decreases the RYR2 current under near-saturating conditions. (A) Representative RYR2 currents, shown as upward deflections from the marked zero-current level, for 8 mM [Ca2+]L (control, top trace) and when 8 mM [Zn2+]L was added (bottom trace). RYR2 channels were activated by 1–2 mM caffeine in the presence of 90 nM free [Ca2+]C. Current recordings were conducted at 0 mV and are from the same channel. (B) The I0, Erev and G values obtained in the absence and after luminal Zn2+ exposure. * Significantly different from the control values when only luminal Ca2+ was present (paired Student’s t test). Data shown are average ± SEM of 6 experiments. Data points shown in (B) are individual measurements (Ca2+–black circle and Zn2+/Ca2+–red triangle).

Figure 6. The RYR2 Zn2+ finger might be locally controlled by the Zn2+ current. (A) A schematic representation of the C2H2 Zn2+ finger consisting of two Cysteines (Cys or C) and two Histidines (His or H), which are coordinated by Zn2+ cation. Usually, 12 residues separate the last Cys and the first His residues (classical C2H2 pattern). In atypical C2H2 Zn2+ fingers which still retain the ability to bind Zn2+, 10–14 residues are tolerated. (B) Sequence alignment of the Zn2+ finger motifs identified within the C-terminal tail of the rat and mouse IP3R1 channels [81,82] and the RYR2 channel [55] from indicated mammals. The sequences were taken from the UniProtKB database. The Cys and His residues of the Zn2+ finger domains with uncommon spacing (16 residues separating the Cys-Cys pair from the His-His pair) are boxed in red. The conserved His in the RYR2 sequences located 11 residues from the Cys-Cys pair, but not in the IP3R1 sequence, is highlighted in black. The membrane topology model of two opposing RYR2 subunits with six transmembrane domains was generated from recent work on the near-atomic resolution structure of the RYR2 isoform [55]. The putative Zn2+-finger domains (ZF) within the…

Figure 7. Zn2+ displacement from the RYR2 Zn2+ finger. (A) Effects of NTA (left panel) and TPEN (right panel) added to the cytosolic solution on RYR2 PO as a consequence of a decrease in [Zn2+]C (paired Student’s t test). (B) The effect of Cd2+ addition (8 mM) on the RYR2 G under near-saturating conditions when 8 mM [Ca2+]L was present. * Significantly different from the control values when only luminal Ca2+ was present (paired Student’s t test). (C) Representative RYR2 currents, shown as upward deflections from the marked zero-current level, for 8 mM [Ca2+]L (control, top trace) and when 8 mM [Cd2+]L was added (two bottom traces). RYR2 channels were activated by 1–2 mM caffeine in the presence of 90 nM free [Ca2+]C. Current recordings were conducted at 0 mV and are from the same channel. (D) Latency of RYR2 activity decay as a function of [Cd2+]L. # Significantly different from 8 mM [Cd2+]L (unpaired Student’s t test). Data shown are average ± SEM of 5–7 experiments. Data points shown in (B) and (D) are individual measurements (Ca2+–black circle and Cd2+/Ca2+–white circle).

Figure 8. The RYR2 channel provides a novel pathway for rapid Zn2+ transport in cardiomyocytes. Schematic illustrating three pathways (one novel) for Zn2+ transport across the cardiac SR membrane. Two Zn2+ transporter protein families, ZIP and ZnT, play an essential role in cardiac Zn2+ homeostasis and mediate a slow-rate transport. In the heart, Zn2+ release through the RYR2 channel, accompanying a robust Ca2+ release triggered by small Ca2+ entry through the L-type Ca2+ channel, could also contribute to global and/or local Zn2+ signaling which requires a fast-rate transport. At the local scale, the Zn2+ current flowing through the RYR2 channel might target its intrinsic Zn2+ finger situated within the C-terminal tail of all four RYR2 subunits. In addition, the RYR2 channel (colored gray) might be stimulated by Zn2+ cations emanating from the neighboring RYR2 channel (colored white). Arrows indicate the direction of Zn2+ (red) and Ca2+ (black) mobilization.