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Caution is required in interpretation of mutations in the voltage sensing domain of voltage gated channels as evidence for gating mechanisms.

Kariev AM, Green ME - Int J Mol Sci (2015)

Bottom Line: Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain -OH.These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone.The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, City College of New York, 160 Convent Avenue, New York, NY 10031, USA. alisher@sci.ccny.cuny.edu.

ABSTRACT
The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R → C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is -SH (reactive form -S-); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain -OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

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The same system with two protons and one more water (total 7 water molecules), with the same color scheme as in Figure 1 and Figure 2, and corresponding labels: (A) H+ on C300 and E183; (B) H+ on E183 (in S1) and E226 (in S2) (up and middle); and (C) H+ on E226 and K306 (down and middle). Note the change in positions of the F180 and F233 side chains as well as the rotation of Y266 in going from (B) to (C).
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ijms-16-01627-f003: The same system with two protons and one more water (total 7 water molecules), with the same color scheme as in Figure 1 and Figure 2, and corresponding labels: (A) H+ on C300 and E183; (B) H+ on E183 (in S1) and E226 (in S2) (up and middle); and (C) H+ on E226 and K306 (down and middle). Note the change in positions of the F180 and F233 side chains as well as the rotation of Y266 in going from (B) to (C).

Mentions: In Figure 1C, the R300C mutation with four water molecules: There are two water molecules now bridging R297 to E183, and two in the cavity center, one of them hydrogen bonded to the cysteine sulfur and to the other water molecule, which stretches across to the glutamate (E226) on the other side of the cavity. The sulfur atom now points into the cavity, with its second hydrogen bond to tyrosine (Y266), the side chain of which has itself moved appreciably again. The cysteine appears to be available to a reagent in the cavity. The F180 ring has folded sharply out of the way, as shown by the dihedral angle defined by atoms ring C4, ring C1 (the link to the atoms toward the backbone) and the next two atoms of the side chain toward the backbone, which is +171.3°, compared to −99.1° in the two water case. The Y266 ring has moved somewhat back from where it was in Figure 1B. The two additional water molecules make a major difference in conformation of the side chains. F233 shows more limited motion in the 0 H+ case than it does with H+ present (Figure 2 and Figure 3), and is not specifically labeled in Figure 1.


Caution is required in interpretation of mutations in the voltage sensing domain of voltage gated channels as evidence for gating mechanisms.

Kariev AM, Green ME - Int J Mol Sci (2015)

The same system with two protons and one more water (total 7 water molecules), with the same color scheme as in Figure 1 and Figure 2, and corresponding labels: (A) H+ on C300 and E183; (B) H+ on E183 (in S1) and E226 (in S2) (up and middle); and (C) H+ on E226 and K306 (down and middle). Note the change in positions of the F180 and F233 side chains as well as the rotation of Y266 in going from (B) to (C).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307324&req=5

ijms-16-01627-f003: The same system with two protons and one more water (total 7 water molecules), with the same color scheme as in Figure 1 and Figure 2, and corresponding labels: (A) H+ on C300 and E183; (B) H+ on E183 (in S1) and E226 (in S2) (up and middle); and (C) H+ on E226 and K306 (down and middle). Note the change in positions of the F180 and F233 side chains as well as the rotation of Y266 in going from (B) to (C).
Mentions: In Figure 1C, the R300C mutation with four water molecules: There are two water molecules now bridging R297 to E183, and two in the cavity center, one of them hydrogen bonded to the cysteine sulfur and to the other water molecule, which stretches across to the glutamate (E226) on the other side of the cavity. The sulfur atom now points into the cavity, with its second hydrogen bond to tyrosine (Y266), the side chain of which has itself moved appreciably again. The cysteine appears to be available to a reagent in the cavity. The F180 ring has folded sharply out of the way, as shown by the dihedral angle defined by atoms ring C4, ring C1 (the link to the atoms toward the backbone) and the next two atoms of the side chain toward the backbone, which is +171.3°, compared to −99.1° in the two water case. The Y266 ring has moved somewhat back from where it was in Figure 1B. The two additional water molecules make a major difference in conformation of the side chains. F233 shows more limited motion in the 0 H+ case than it does with H+ present (Figure 2 and Figure 3), and is not specifically labeled in Figure 1.

Bottom Line: Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain -OH.These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone.The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, City College of New York, 160 Convent Avenue, New York, NY 10031, USA. alisher@sci.ccny.cuny.edu.

ABSTRACT
The gating mechanism of voltage sensitive ion channels is generally considered to be the motion of the S4 transmembrane segment of the voltage sensing domains (VSD). The primary supporting evidence came from R → C mutations on the S4 transmembrane segment of the VSD, followed by reaction with a methanethiosulfonate (MTS) reagent. The cys side chain is -SH (reactive form -S-); the arginine side chain is much larger, leaving space big enough to accommodate the MTS sulfonate head group. The cavity created by the mutation has space for up to seven more water molecules than were present in wild type, which could be displaced irreversibly by the MTS reagent. Our quantum calculations show there is major reorientation of three aromatic residues that face into the cavity in response to proton displacement within the VSD. Two phenylalanines reorient sufficiently to shield/unshield the cysteine from the intracellular and extracellular ends, depending on the proton positions, and a tyrosine forms a hydrogen bond to the cysteine sulfur with its side chain -OH. These could produce the results of the experiments that have been interpreted as evidence for physical motion of the S4 segment, without physical motion of the S4 backbone. The computations strongly suggest that the interpretation of cysteine substitution reaction experiments be re-examined in the light of these considerations.

Show MeSH