Limits...
Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH

Related in: MedlinePlus

A kinetic model for Shaker and the F401 mutants. Note that, compared with the ZHA model, independent transitions within each of the four subunits are completely symmetrical and that a concerted transition precedes channel opening. A closed state in which all four subunits are in the C2 state is implicit in the model; is it referred to as the Cn state in the text. (Bottom) The model estimates of the zero-voltage rate constants (k0, in s−1) and associated quantities of charge movement (zk, in e0) for each of the rates for the wt, F401L, and F401A channels are shown. The initial closing transition parameters are enclosed in a box to highlight the differences among the models.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230647&req=5

Figure 14: A kinetic model for Shaker and the F401 mutants. Note that, compared with the ZHA model, independent transitions within each of the four subunits are completely symmetrical and that a concerted transition precedes channel opening. A closed state in which all four subunits are in the C2 state is implicit in the model; is it referred to as the Cn state in the text. (Bottom) The model estimates of the zero-voltage rate constants (k0, in s−1) and associated quantities of charge movement (zk, in e0) for each of the rates for the wt, F401L, and F401A channels are shown. The initial closing transition parameters are enclosed in a box to highlight the differences among the models.

Mentions: A detailed kinetic model of this class has been proposed for Shaker and a mutant channel (V2) (Schoppa and Sigworth 1998c), which argues for the necessity to include a third charge-translocating step per subunit as well as two sequential concerted transitions preceding channel opening (a so-called 3+2′ scheme). We opted for the simpler (2+1′) model because of the limited experimental means to constrain a more elaborate scheme for all three channel species in this study. Our goal is to provide a robust description of the main aspects of the channels' gating while minimizing the number of transitions that differ among the models for the wt, F401L, and F401A channels. Our ability to do so supports the hypothesis that F401 mutations do not disrupt the wt gating mechanism in a global sense, but only target specific aspects of it. Our model provides reasonable fits to the wt and mutant channels, with major differences among the three species primarily limited to the first closing transition, as suggested by our data and the predictions of the original ZHA model (Fig. 13). Fig. 14 shows the connectivity of the model and illustrates that despite the large number (17) of kinetic states, only 12 free parameters are needed to constrain the mechanism up to and including the concerted Closed ↔ Open transition, compared with 9 for the ZHA model and 20 for the 3+2′ model of Schoppa and Sigworth 1998c. These are the zero-voltage rate (k0) and associated valence (zk) of the two forward rates α and γ and the two reverse rates β and δ for each of the four subunits and of the forward rate κ and the reverse rate λ for the concerted transition. The rates are assumed to be instantaneous exponential functions of voltage, according to: \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k=k_{0}e^{{z_{k}F{\mathrm{V}}}/{RT}}{\mathrm{.}}\end{equation*}\end{document}


Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

Kanevsky M, Aldrich RW - J. Gen. Physiol. (1999)

A kinetic model for Shaker and the F401 mutants. Note that, compared with the ZHA model, independent transitions within each of the four subunits are completely symmetrical and that a concerted transition precedes channel opening. A closed state in which all four subunits are in the C2 state is implicit in the model; is it referred to as the Cn state in the text. (Bottom) The model estimates of the zero-voltage rate constants (k0, in s−1) and associated quantities of charge movement (zk, in e0) for each of the rates for the wt, F401L, and F401A channels are shown. The initial closing transition parameters are enclosed in a box to highlight the differences among the models.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230647&req=5

Figure 14: A kinetic model for Shaker and the F401 mutants. Note that, compared with the ZHA model, independent transitions within each of the four subunits are completely symmetrical and that a concerted transition precedes channel opening. A closed state in which all four subunits are in the C2 state is implicit in the model; is it referred to as the Cn state in the text. (Bottom) The model estimates of the zero-voltage rate constants (k0, in s−1) and associated quantities of charge movement (zk, in e0) for each of the rates for the wt, F401L, and F401A channels are shown. The initial closing transition parameters are enclosed in a box to highlight the differences among the models.
Mentions: A detailed kinetic model of this class has been proposed for Shaker and a mutant channel (V2) (Schoppa and Sigworth 1998c), which argues for the necessity to include a third charge-translocating step per subunit as well as two sequential concerted transitions preceding channel opening (a so-called 3+2′ scheme). We opted for the simpler (2+1′) model because of the limited experimental means to constrain a more elaborate scheme for all three channel species in this study. Our goal is to provide a robust description of the main aspects of the channels' gating while minimizing the number of transitions that differ among the models for the wt, F401L, and F401A channels. Our ability to do so supports the hypothesis that F401 mutations do not disrupt the wt gating mechanism in a global sense, but only target specific aspects of it. Our model provides reasonable fits to the wt and mutant channels, with major differences among the three species primarily limited to the first closing transition, as suggested by our data and the predictions of the original ZHA model (Fig. 13). Fig. 14 shows the connectivity of the model and illustrates that despite the large number (17) of kinetic states, only 12 free parameters are needed to constrain the mechanism up to and including the concerted Closed ↔ Open transition, compared with 9 for the ZHA model and 20 for the 3+2′ model of Schoppa and Sigworth 1998c. These are the zero-voltage rate (k0) and associated valence (zk) of the two forward rates α and γ and the two reverse rates β and δ for each of the four subunits and of the forward rate κ and the reverse rate λ for the concerted transition. The rates are assumed to be instantaneous exponential functions of voltage, according to: \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage{pmc}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}k=k_{0}e^{{z_{k}F{\mathrm{V}}}/{RT}}{\mathrm{.}}\end{equation*}\end{document}

Bottom Line: We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion.Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating.These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.

ABSTRACT
The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.

Show MeSH
Related in: MedlinePlus