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Flecainide blocks RyR2 channel activity at +40 mV. (A)  Records of open cha...
Published: 17 June 2022
Figure 1. Flecainide blocks RyR2 channel activity at +40 mV. (A) Records of open channel block by flecainide ( Mehra et al., 2014 ). Single channels from sheep RyR2 incorporated into bilayers with SR vesicles also containing triadin, junctin, and CSQ2. The bilayer potential was +40 mV (cytoplasm relative to lumen). Recordings were obtained before and after flecainide addition to the cytoplasmic bath at indicated concentrations. The cytoplasmic bath (cyto) also contained 2 mM ATP; free Ca2+ = 0.1 mM and free Mg2+ = 1 mM. The luminal bath contained 0.1 mM Ca2+. Channel openings appear as upward current transitions from the baseline (channel closed level). The dashed red line indicates a possible substate level corresponding to that described by Bannister et al. (2015) and shown in C. (B) RyR2 open probability (Po) expressed relative to Po in the absence of flecainide (relative Po) for individual channels, then average data plotted as a function of flecainide concentration. The cytoplasmic [Ca2+] and the side of flecainide addition are listed with appropriate symbols beside the graph. In addition, 1 mM Mg2+ was added (*open squares) or 1 mM Mg2+ with reduced cytoplasmic [Cs+] (**open triangles). In all conditions, the cytoplasmic solution also contained 1 mM ATP, the luminal [Ca2+] was 0.1 µM, the luminal [Cs+] was 250 mM, and the cytoplasmic [Cs+] was 250 mM except for the open triangles where it was reduced to 150 mM. A and B were modified from Fig. 1 of Mehra et al. (2014) under copyright license # 1174586-1. (C) Recordings from single purified recombinant human RyR2 channels in the presence of 20 µM EMD ( Bannister et al., 2015 ). The solutions were symmetrical, both containing 20 mM HEPES (pH = 7.4) and 610 mM KCl. In the tradition of this laboratory, channel openings at +40 mV are shown as downward transitions from the baseline, indicated by the short continuous line at the right-hand side of each recording. The flecainide-induced substate level is indicated by the short dashed line on the right side of each record and also by the superimposed dashed red line across each record in the presence of flecainide. (D) The effect of flecainide block on channel Po is plotted against flecainide concentration. The graph has been modified from the original, which showed the effect of flecainide on 1-Po, to facilitate a direct comparison with data obtained under very different conditions in B. There is a remarkable similarity to the concentration dependence of the open circle data in B. C and D were modified from Fig. 1 of Bannister et al. (2015) under copyright license #1174592-1. More
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A cartoon illustrating the hypothetical action of flecainide in occupying/b...
Published: 17 June 2022
Figure 2. A cartoon illustrating the hypothetical action of flecainide in occupying/binding in the RyR2 pore. (A) In the absence of flecainide Ca2+ flow from the SR to the cytoplasm is facilitated by a largely K+-mediated counter current from the cytoplasm into the lumen. (B) The positively charged flecainide when added to the cytoplasmic solution moves into the pore where it is too large to pass through the selectivity filter and remains trapped ( Bannister et al., 2021 ). In this situation, flecainide partially blocks the K+ counter current flowing into the SR during Ca2+ release and thus reduces Ca2+ flow through the channel. Bannister et al. (2021) suggest that retarding the charge compensation via partial/substate block of a cytoplasmic-to-luminal counter-current through RyR2 may result in inhibition of Ca2+ release. More
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Flecainide can exert a voltage-independent inhibitory action on high activi...
Published: 17 June 2022
Figure 3. Flecainide can exert a voltage-independent inhibitory action on high activity RyR2 channels. Data is shown for mouse RyR2 channels isolated from WT littermates of homozygous RyR2-P2328S+/+ mice ( Salvage et al., 2021a ). (A) Representative channel currents before addition of flecainide (upper) and with 5 µM flecainide (middle) and 50 µM flecainide (lower) at −40 mV, with channel opening downward (A, I) and +40 mV, with channel opening upward (A, II). Arrows indicate maximum open current level. (B) . Individual relative Po data at +40 mV (circles) and −40 mV (triangles). Data for each channel is represented by symbols of the same color. The data in this graph illustrates the voltage-independence of changes with flecainide and suggests a decline in activity as flecainide concentration increases in channels with high control Po values, in contrast to an increase in activity in channels with a lower control Po. The boxes indicate the average Po for each flecainide concentration. (C) Individual maximum relative Po values with flecainide, plotted against control Po. Black circles, +40 mV; purple triangles, −40 mV. Maximum relative Po is the highest value recorded over 60–90 s segments of activity with flecainide in each channel across all concentrations of the drug. Values below 1 indicate that there was no increase in activity at any concentration, but do not reflect the full decline in activity. The full decline in activity for individual channels is shown in B. The graph in C shows that all channels with an initial activity <0.02 demonstrated substantial periods of activation during exposure to flecainide, while channels with initial activity >0.08 did not show similar periods of increased activity. (C–E) The upper opening bracket indicates channels included in the low control activity group (E). The remaining channels are included in the high activity group in D. D and E show average relative Po values for high (D) and low (E) activity channels at individual flecainide concentrations. The high activity channels show a significant decline in activity with 5–50 µM flecainide. The low activity channels show significant increases in activity with 0.5–5 µM flecainide. Single-channel traces and graphs are from Salvage et al. (2021a) . More
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Substate activity is stabilized by flecainide in RyR2 channels from WT and ...
Published: 17 June 2022
Figure 4. Substate activity is stabilized by flecainide in RyR2 channels from WT and homozygous RyR2-P2328S +/+ mouse hearts. Examples of flecainide-stabilized substate activity seen in channels analyzed for datasets shown in Figs. 3 and 6 . (A and B) Data for WT channels (A) and data for the P2328S channels (B). In A and B, examples of single channel recordings at −40 mV are shown with channel opening downward. The all-points frequency histograms for each channel are shown on the left. The upper record in each panel shows control activity and the lower recording shows activity from the same channel after exposure to 10 µM flecainide. The red lines in the records and red arrows in the histograms indicate the main substate level. The blue lines and blue arrows indicate the maximum channel conductance levels. The black lines in the records show the zero current closed level. More
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A model showing multiple binding sites for flecainide in the RyR2 channel p...
Published: 17 June 2022
Figure 5. A model showing multiple binding sites for flecainide in the RyR2 channel protein. The RyR2 channels, consisting of four subunits, are shown embedded in the SR membrane. The channel in the upper half of the figure is shown with flecainide bound to a peripheral activation site on one of... More
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Significant voltage-independent activation of low activity P2328S channels ...
Published: 17 June 2022
Figure 6. Significant voltage-independent activation of low activity P2328S channels observed with flecainide concentrations of 0.5 to 50 µM, while inhibition of high-activity channels was less consistent than in WT channels. Related to Fig. 3 . Data is shown for single RyR2 channels ( Salvage et al., 2021a ). (A) Representative channel currents before addition of flecainide (upper) and with 5 µM flecainide (middle) and 50 µM flecainide (lower) at −40 mV, with channel opening downward (A, I) and +40 mV, with channel opening upward (A, II). Arrows indicate maximum open current level. (B) Individual relative Po data at +40 mV (circles) and −40 mV (triangles). Data for each channel is represented by symbols of the same color. The data in this graph illustrates the voltage independence of changes with flecainide and suggests a decline in activity as flecainide concentration increases in channels with high control Po values, in contrast to an increase in activity in channels with a lower control Po. The boxes indicate the average Po for each flecainide concentration. (C) Individual maximum relative Po values with flecainide, plotted against control Po. Black circles, +40 mV; purple triangles, −40 mV. Maximum relative Po is the highest value recorded over 60–90 s segments of activity with flecainide in each channel across all concentrations of the drug. Values below 1 indicate that there was no increase in activity at any concentration, but do not reflect the full decline in activity. The full decline in activity in individual channels is shown in B. (C–E) The upper opening bracket and arrow indicates channels included in the low control activity group (D), while the lower closing bracket and arrow indicate channels included in the high control activity group (E). The control Po dependence of relative Po seen in WT channels ( Fig. 3 ) was not apparent in the P2328S channels. Nevertheless, the P2328S data was separated into low activity (Po < 0.08) and high activity (Po > 0.08) groups for comparison with WT channel data. D and E show average relative Po values for low- (D) and high-activity (E) channels at individual flecainide concentrations. The low-activity channels show significant increases in activity with 0.5–50 µM flecainide and a small but significant decline with 100 µM flecainide. The high-activity P2328S channels show a less consistent decline in Po when compared with that in WT channels ( Fig. 3 ). Single-channel traces and graphs are from Salvage et al. (2021a) . More
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Cooperative gating between as few as eight coupled RyR2s in a dyadic juncti...
Published: 17 June 2022
Figure 7. Cooperative gating between as few as eight coupled RyR2s in a dyadic junction (couplon) dramatically increases the steepness of the predicted relationship between P o and [Ca 2+ ] i . The solid black line shows the predicted Po versus [Ca2+]i relationship for a single or rogue nonjunctional RyR2. The filled brown circles show the relationship predicted for eight cooperatively gated coupled junctional RyR2s. As a consequence of cooperativity, Po of nonjunctional rogue RyR2s increase as [Ca2+]i increases through much of the diastolic range, concentrations which have little effect on Po of the clustered RyR2s. The graph shows predicted effects of changing [Ca2+]i on Po. The Po and [Ca2+]i values are within the physiological range but have not been experimentally verified in vivo because the technology to do so does not yet exist. The figure is modified from Fig. 2 of Sobie et al. (2006) under copyright license 1210751-1. More
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Isolation of Cm28 peptide from the venom of <em>C. margaritatus</em>...
Published: 14 June 2022
Figure 1. Isolation of Cm28 peptide from the venom of C. margaritatus and primary sequence determination. (A) Final step reverse-phase HPLC separation of venom components from fraction FII.6 (see Results for details). A linear gradient of 0–60% acetonitrile over 60 min was run to elute the peptides. Cm28 peptide was eluted at a retention time of 24.5 min, indicated with a red arrow. (B) Full-length primary structure of Cm28 was acquired by automatic Edman degradation using the conditions described in Materials and methods. Cysteine residues are highlighted in magenta. MW, molecular weight. More
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Multiple alignment of Cm28 with other representative KTx.  Len, mature chai...
Published: 14 June 2022
Figure 2. Multiple alignment of Cm28 with other representative KTx. Len, mature chain length; %ID, percent amino acid identity. Conserved cysteine residues are highlighted in yellow. Identical positions to those in Cm28 are indicated by dots, and * indicates that the C-terminal is amidated. More
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Phylogenetic analysis of Cm28.  Maximum likelihood tree topology obtained f...
Published: 14 June 2022
Figure 3. Phylogenetic analysis of Cm28. Maximum likelihood tree topology obtained from the analysis of Cm28 and other related KTxs (Log-likelihood = −4445.324618). The numbers below the nodes indicate bootstrap support values (UFBoot) >50. More
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Inhibition of K <sub> V </sub> 1.2 and K <sub> V </sub> 1.3 currents by Cm2...
Published: 14 June 2022
Figure 4. Inhibition of K V 1.2 and K V 1.3 currents by Cm28. (A and B). Whole-cell currents of KV1.2 (A) and KV1.3 (B) were recorded on transiently transfected CHO cells and activated human peripheral T lymphocytes, respectively, by applying 15-ms-long voltage pulses to +50 mV from a Vh of −120 mV every 15 s. Representative current traces show the K+ current in control solution (black), at equilibrium block in the presence of 14 nM ChTx for KV1.2, and 10 mM TEA+ for KV1.3, (blue traces in A and B, respectively) as a perfusion control, at equilibrium block upon application of 2 nM Cm28 (purple for KV1.2, red for KV1.3) and after recovery from the block upon application of toxin-free solution (green, wash-out). (C and D) Time course of development and recovery of the K+ current inhibition. Normalized peak currents were plotted as function of time. Data points in purple (C, KV1.2) and in red (D, KV1.3) represent the application of 2 nM of Cm28 to the bath solution. Upon reaching the block equilibrium, cells were perfused with toxin-free solution (arrow, wash out) to demonstrate reversibility of the block (data points in black). (E and F) Concentration-dependent block of KV1.2 (E) and KV1.3 (F) by Cm28. A Hill equation (see Materials and methods for details) was fitted to the RCF values calculated at different toxin concentrations (solid lines). The best fit resulted in Kd = 0.96 nM, H = 1.04 for KV1.2 (E) and Kd = 1.3 nM, H = 0.93 for KV1.3 (F). Error bar represents SEM and n = 3–5. More
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Mechanism of blocking K <sub> V </sub> 1.2 and K <sub> V </sub> 1.3 by Cm28...
Published: 14 June 2022
Figure 5. Mechanism of blocking K V 1.2 and K V 1.3 by Cm28. (A and B) Instantaneous I-V relationship for KV1.2 (A) and KV1.3 (B). Cells were held at −120 mV and depolarized to +50 mV in 200 ms using a voltage ramp protocol (at a rate of 0.85 mV/ms). Test pulses were applied every 15 s. Representative traces show the average of three traces either in the control solution (black) or at equilibrium block in the presence of 2 nM Cm28 (purple for KV1.2 in A and red for KV1.3 in B). Arrows indicate the activation threshold voltages. (C and D) G-V curve for KV1.2 (C) was constructed from isochronal tail current amplitudes recorded in CHO cells at −120 mV. The currents were activated by 300-ms-long depolarizing test potentials ranging from −70 to +80 mV in 10 mV increments from a Vh of −120 mV. Tail currents were normalized to maximum and plotted as a function of membrane protentional (Em). To obtain the G-V curve of KV1.3 (D), whole-cell currents were measured in activated human T lymphocytes by applying voltage pulses ranging from −70 to +50 mV in 10 mV steps from Vh of −120 mV. Then, normalized conductance was calculated using the chord-conductance equation (see Materials and methods) and plotted against membrane potential (Em). The Boltzmann sigmoidal equation was fitted to the averaged data points (solid lines). The voltage dependence of steady-state activation curve was determined in the absence (filled up triangles in black) or in the presence (empty circles in purple for KV1.2 and filled circles in red for KV1.3) of 2 nM Cm28. (E) V50 values from G-V curves of individual cells were averaged and plotted as bar graphs. Symbols indicate individual data points obtained in the absence (triangles) or in the presence (empty circles in purple for KV1.2 and filled circles in red for KV1.3) of 2 nM Cm28. Mann–Whitney test, P = 0.23 (KV1.2) and P = 0.57 (KV1.3). (F) Effect of Cm28 concentrations on the binding kinetics to KV1.3. The 1/τon values, (circles) and dissociation rate constant (1/τoff or koff, triangles) were plotted as a function of Cm28 concentrations. Data points were fitted with linear regression (r2 = 0.99). Error bars in C–F represent SEM and n ≥ 3. More
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Selectivity profile of Cm28. (A–H)  Representative current traces are shown...
Published: 14 June 2022
Figure 6. Selectivity profile of Cm28. (A–H) Representative current traces are shown in the absence of (indicated as control in black) and presence of 150 nM Cm28 (>100× of Kd value for KV1.3) of Cm28 (in red). The proper operation of solution exchange in a recording chamber was tested frequently using fully reversible blockers (shown in blue) or solutions (in green) as a positive control (HK: HK-150 solution with 150 mM extracellular K+ to reduce the K+ driving force or TEA+, Tram34, and ClGBI are known blockers of appropriate channels). Voltage protocols are shown above the current records in each panel. For external and internal solution composition, see Materials and methods. The traces shown in the presence of 150 nM Cm28 were recorded at equilibrium block (A) or after 8–20 pulses (2–5 min) in the presence of Cm28 (B–H). (I) Remaining current fractions for the indicated channels. Normalized RCF (I/I0) values were calculated as the ratio of the peak currents in the presence (I) or absence (I0) of 150 nM Cm28 at equilibrium block (for KV1.1) or after 8–20 repeated depolarization pulse. Bars and error bars indicate the mean ± SEM (n ≥ 3). More
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Cm28 does not compromise the cell viability of either quiescent or stimulat...
Published: 14 June 2022
Figure 7. Cm28 does not compromise the cell viability of either quiescent or stimulated CD4 + T EM lymphocytes. (A) Cell viability in the presence of 1.5 μM Cm28 or 50 nM rMgTx was assessed by staining the cells with fixable viability dye Zombie NIR after 24 h culture period using flow cytometry. Changes in FSC and positive staining with the Zombie NIR were considered indicators of dead cells (see Materials and methods). The percentage of cell viability was calculated as: 1—fraction of Zombie NIR positive cells. Cells were treated with 30% DMSO for 30 min before staining as a positive control for dead cells. Control cells (Ctrl) were not exposed to peptides. (B) The cytotoxic effect of 1.5 μM Cm28 or 50 nM rMgTx on TEM cells determined using the LDH cytotoxicity assay. LDH activity of the medium was determined following 24 h culture period and percentage of cytotoxicity was calculated (see Materials and methods for details). For positive control, 50 mM NaN3 was added to the cells. (A and B) Data from three independent experiments (two technical repeats in each) are shown as mean ± SEM. More
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K <sub> V </sub> 1.3 blockade by Cm28 inhibits the expression of CD40L and ...
Published: 14 June 2022
Figure 8. K V 1.3 blockade by Cm28 inhibits the expression of CD40L and IL2R. Isolated CD4+ TEM lymphocytes were stimulated through TCR with plate-bound anti-human CD3 antibody in the presence or absence of toxins. (A–D) After 24 h of stimulation, cells were labeled with anti-CD154 (CD40L; A and B) and anti-CD25 (IL2R) antibodies (C and D). Treatment labels: US, unstimulated (black); US + Cm28, unstimulated in the presence of Cm28 (1.5 µM, orange); S, stimulated with anti-human CD3 antibody coated wells (1 µg/well, green); S + Cm28, stimulated in the presence of Cm28 (1.5 µM, red); S + rMgTx, stimulated in the presence of rMgTx (5 nM, blue). (A and C) Fluorescence histograms were obtained from T lymphocytes gated on FSC versus SSC density plot (10,000 events were recorded) and then, peak-normalized overlay histograms were plotted for CD40L and IL2R. A–D follow the same color code. (B and D) Mean fluorescence intensities (MFIs) were determined from the histograms and normalized to that of their stimulated but not treated control (S). Bars with individual data points represent values from three independent experiments (two technical repeats in each). Error bars indicate SEM. Statistically significant change in the expression level of CD40L and IL2R is indicated with P values (all pairwise multiple comparison with Tukey’s test). More
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The anion permeation pathway of TMEM16A with the high-affinity Ca <sup> 2+ </sup>...
Published: 10 June 2022
Figure 1. The anion permeation pathway of TMEM16A with the high-affinity Ca 2+ binding site occupied. One subunit of TMEM16A with its extracellular (top) and intracellular (bottom) sides is shown. The anion permeation pathway is drawn as a teal mesh, and the α-helices 3–8 surrounding the pore are shown in gray. The green spheres are two Ca2+ ions sitting in the Ca2+ pocket of site 1. Note that residues of the hydrophobic gate (I641) and Ca2+ pocket (N650 and E654) are part of TM6. Thus, when Ca2+ binding occurs, the translational movement of TM6 is outwardly transmitted to open the hydrophobic gate. The side chains of the extracellular cluster of basic residues (olive), the hydrophobic gate (blue), two lysines at the middle of the pore (yellow), and the Ca2+ pocket of site 1 (orange) involved in anion selectivity were modeled as sticks. More