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Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Euden J, Mason SA, Viero C, Thomas NL, Williams AJ - J. Biol. Chem. (2013)

Bottom Line: Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function.Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels.Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.

ABSTRACT
Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

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Variation in mean WT and G4864A RyR2 open probability with changing cytosolic calcium. Experimental conditions were as described in the legend for Fig. 6. Data are displayed as mean ± S.E. for 5–10 individual channels. An unpaired t test was performed and statistical significance is indicated by asterisks. Upper, Po, open probability, Middle, To: mean open time. Lower, Tc, mean closed time. n.s., not significant.
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Figure 7: Variation in mean WT and G4864A RyR2 open probability with changing cytosolic calcium. Experimental conditions were as described in the legend for Fig. 6. Data are displayed as mean ± S.E. for 5–10 individual channels. An unpaired t test was performed and statistical significance is indicated by asterisks. Upper, Po, open probability, Middle, To: mean open time. Lower, Tc, mean closed time. n.s., not significant.

Mentions: Detailed information on the consequences of residue replacement in RyR2 can be obtained by monitoring the ion handling and gating characteristics of individual channel molecules incorporated into planar phospholipid bilayers under voltage clamp conditions. Consistent with the data obtained in the macroscopic assays described earlier, incorporation of RyR2, in which Gly4864 had been replaced with alanine, into bilayers yielded channels with ion handling and gating characteristics equivalent to those of WT RyR2. The plot in Fig. 5 shows the relationship between single channel current amplitude and holding potential for WT and G4864A channels monitored in symmetrical 210 mm KCl. We observed no significant difference in the conductance of the two species of channel (WT; 641 ± 4 picosiemens, G4864A; 637 ± 3 picosiemens). The responses of the WT and G4864A channels to changing cytosolic Ca2+ were also equivalent, as demonstrated in Fig. 6. The representative traces, monitored in symmetrical 210 mm KCl at a holding potential of +40 mV, demonstrate a dramatic decrease in single channel open probability (Po) of both WT and G4864A channels accompanying a reduction in cytosolic Ca2+ from 10 μm to 0.7 nm as the result of the addition of the EGTA, HEDTA, and nitrilotriacetic acid chelating mixture. Subsequent elevation of the cytosolic Ca2+ to 100 μm raises the Po of both species of channel and produces a small decrease in single channel current amplitude as Ca2+ competes with K+ as the charge-carrying species (32). Analysis of the gating parameters of WT and G4864A channels is shown in Fig. 7 and reveals no significant difference in the variation of WT and G4864A channel Po with changing cytosolic Ca2+. Similarly, determination of mean open and closed times demonstrates no significant difference in the mechanisms underlying altered Po in response to changing cytosolic Ca2+ in the WT and G4864A channels.


Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Euden J, Mason SA, Viero C, Thomas NL, Williams AJ - J. Biol. Chem. (2013)

Variation in mean WT and G4864A RyR2 open probability with changing cytosolic calcium. Experimental conditions were as described in the legend for Fig. 6. Data are displayed as mean ± S.E. for 5–10 individual channels. An unpaired t test was performed and statistical significance is indicated by asterisks. Upper, Po, open probability, Middle, To: mean open time. Lower, Tc, mean closed time. n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675601&req=5

Figure 7: Variation in mean WT and G4864A RyR2 open probability with changing cytosolic calcium. Experimental conditions were as described in the legend for Fig. 6. Data are displayed as mean ± S.E. for 5–10 individual channels. An unpaired t test was performed and statistical significance is indicated by asterisks. Upper, Po, open probability, Middle, To: mean open time. Lower, Tc, mean closed time. n.s., not significant.
Mentions: Detailed information on the consequences of residue replacement in RyR2 can be obtained by monitoring the ion handling and gating characteristics of individual channel molecules incorporated into planar phospholipid bilayers under voltage clamp conditions. Consistent with the data obtained in the macroscopic assays described earlier, incorporation of RyR2, in which Gly4864 had been replaced with alanine, into bilayers yielded channels with ion handling and gating characteristics equivalent to those of WT RyR2. The plot in Fig. 5 shows the relationship between single channel current amplitude and holding potential for WT and G4864A channels monitored in symmetrical 210 mm KCl. We observed no significant difference in the conductance of the two species of channel (WT; 641 ± 4 picosiemens, G4864A; 637 ± 3 picosiemens). The responses of the WT and G4864A channels to changing cytosolic Ca2+ were also equivalent, as demonstrated in Fig. 6. The representative traces, monitored in symmetrical 210 mm KCl at a holding potential of +40 mV, demonstrate a dramatic decrease in single channel open probability (Po) of both WT and G4864A channels accompanying a reduction in cytosolic Ca2+ from 10 μm to 0.7 nm as the result of the addition of the EGTA, HEDTA, and nitrilotriacetic acid chelating mixture. Subsequent elevation of the cytosolic Ca2+ to 100 μm raises the Po of both species of channel and produces a small decrease in single channel current amplitude as Ca2+ competes with K+ as the charge-carrying species (32). Analysis of the gating parameters of WT and G4864A channels is shown in Fig. 7 and reveals no significant difference in the variation of WT and G4864A channel Po with changing cytosolic Ca2+. Similarly, determination of mean open and closed times demonstrates no significant difference in the mechanisms underlying altered Po in response to changing cytosolic Ca2+ in the WT and G4864A channels.

Bottom Line: Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function.Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels.Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff CF14 4XN, Wales, United Kingdom.

ABSTRACT
Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

Show MeSH
Related in: MedlinePlus