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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.

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Models predict the deactivation kinetics in Shaker and F401 mutants. Ionic tail currents that arise during channel deactivation at negative voltages are shown as relative open probability as the function of time by scaling to match the instantaneous amplitudes after the end of the +50-mV test pulse (top). The current families for the tail voltages of −60 to −160 mV (wt, left), −60 to −180 mV (F401L, center), and −30 to −90 mV (F401A, right) are shown. Records at −100 mV are omitted because the current amplitudes are very low near the reversal potential. Note the inward current at the start of the tail at −90 mV in F401A, which is largely due to the OFF gating current component. Model predictions for the time course of the open probability decay are superimposed as thin lines. (Bottom) The data and model traces for the wt and F401L are replotted on a logarithmic time axis to enable closer comparison of the model to the data at the lowest voltages. For these simulations, λ0 was 50 and 29 s−1 for the wt and F401L models, respectively.
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Figure 19: Models predict the deactivation kinetics in Shaker and F401 mutants. Ionic tail currents that arise during channel deactivation at negative voltages are shown as relative open probability as the function of time by scaling to match the instantaneous amplitudes after the end of the +50-mV test pulse (top). The current families for the tail voltages of −60 to −160 mV (wt, left), −60 to −180 mV (F401L, center), and −30 to −90 mV (F401A, right) are shown. Records at −100 mV are omitted because the current amplitudes are very low near the reversal potential. Note the inward current at the start of the tail at −90 mV in F401A, which is largely due to the OFF gating current component. Model predictions for the time course of the open probability decay are superimposed as thin lines. (Bottom) The data and model traces for the wt and F401L are replotted on a logarithmic time axis to enable closer comparison of the model to the data at the lowest voltages. For these simulations, λ0 was 50 and 29 s−1 for the wt and F401L models, respectively.

Mentions: The proposed alterations in a backward transition leading away from the open state are expected to affect the time course of macroscopic ionic tail currents. Fig. 19 (top) shows, for the wt and the two F401 mutants, the decay in the relative open probability as a function of time when voltage is stepped from a depolarized value of +50 mV to hyperpolarized potentials. All current traces are normalized to match their initial amplitudes. In Fig. 19 (bottom), these data are re-plotted on a logarithmic time axis. These two transformations allow a closer examination of the kinetics of deactivation at the more hyperpolarized voltages at which tail currents are very small and rapid. Additionally, a logarithmic time scale would bring out the convergence of the open probability traces to an asymptotic value at very low voltages if a single closing transition were to become rate limiting. We used the models for the wt and F401L in which initial closing rates were modified somewhat to reflect the slower deactivation kinetics in excised patches. With λ0 set to 50 s−1 for the wt and 29 s−1 for F401L, the open probability decay is well described by the model predictions for both channels over the range −60 to −160 mV (−180 mV for F401L). The valence of 0.6 e0 assigned to this rate is the minimum required to produce the necessary spacing of the traces at voltages below −120 mV (Fig. 19, bottom). Less charge associated with the first closing step predicts a rate-limiting step that is not evident in the data. The analysis of F401A deactivation (Fig. 19, right) is complicated by the extreme rapidity of its tail currents over a wide voltage range. Fitting exponential functions to their time course yields time constants in the range of 100–500 μs, which are difficult to resolve well when the current amplitudes are small. Additionally, there is a prominent component of OFF gating current (note the lowermost trace in the panel which was taken at −90 mV) that is slower than the ionic tail in this mutant and significantly distorts its kinetics. Simulated F401A traces have a multi-exponential decay, with the very rapid component near the limit of resolution in our recordings (and perhaps more rapid than the excised patch data), and a slower component that at −30 and −50 mV cannot be reliably distinguished from the steady state component seen in the experimental data. Because of the nonionic components of the current decay (which the model does not take into account), more detailed comparison of the model simulations to the tail currents in F401A at hyperpolarized voltages was not undertaken.


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)

Models predict the deactivation kinetics in Shaker and F401 mutants. Ionic tail currents that arise during channel deactivation at negative voltages are shown as relative open probability as the function of time by scaling to match the instantaneous amplitudes after the end of the +50-mV test pulse (top). The current families for the tail voltages of −60 to −160 mV (wt, left), −60 to −180 mV (F401L, center), and −30 to −90 mV (F401A, right) are shown. Records at −100 mV are omitted because the current amplitudes are very low near the reversal potential. Note the inward current at the start of the tail at −90 mV in F401A, which is largely due to the OFF gating current component. Model predictions for the time course of the open probability decay are superimposed as thin lines. (Bottom) The data and model traces for the wt and F401L are replotted on a logarithmic time axis to enable closer comparison of the model to the data at the lowest voltages. For these simulations, λ0 was 50 and 29 s−1 for the wt and F401L models, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 19: Models predict the deactivation kinetics in Shaker and F401 mutants. Ionic tail currents that arise during channel deactivation at negative voltages are shown as relative open probability as the function of time by scaling to match the instantaneous amplitudes after the end of the +50-mV test pulse (top). The current families for the tail voltages of −60 to −160 mV (wt, left), −60 to −180 mV (F401L, center), and −30 to −90 mV (F401A, right) are shown. Records at −100 mV are omitted because the current amplitudes are very low near the reversal potential. Note the inward current at the start of the tail at −90 mV in F401A, which is largely due to the OFF gating current component. Model predictions for the time course of the open probability decay are superimposed as thin lines. (Bottom) The data and model traces for the wt and F401L are replotted on a logarithmic time axis to enable closer comparison of the model to the data at the lowest voltages. For these simulations, λ0 was 50 and 29 s−1 for the wt and F401L models, respectively.
Mentions: The proposed alterations in a backward transition leading away from the open state are expected to affect the time course of macroscopic ionic tail currents. Fig. 19 (top) shows, for the wt and the two F401 mutants, the decay in the relative open probability as a function of time when voltage is stepped from a depolarized value of +50 mV to hyperpolarized potentials. All current traces are normalized to match their initial amplitudes. In Fig. 19 (bottom), these data are re-plotted on a logarithmic time axis. These two transformations allow a closer examination of the kinetics of deactivation at the more hyperpolarized voltages at which tail currents are very small and rapid. Additionally, a logarithmic time scale would bring out the convergence of the open probability traces to an asymptotic value at very low voltages if a single closing transition were to become rate limiting. We used the models for the wt and F401L in which initial closing rates were modified somewhat to reflect the slower deactivation kinetics in excised patches. With λ0 set to 50 s−1 for the wt and 29 s−1 for F401L, the open probability decay is well described by the model predictions for both channels over the range −60 to −160 mV (−180 mV for F401L). The valence of 0.6 e0 assigned to this rate is the minimum required to produce the necessary spacing of the traces at voltages below −120 mV (Fig. 19, bottom). Less charge associated with the first closing step predicts a rate-limiting step that is not evident in the data. The analysis of F401A deactivation (Fig. 19, right) is complicated by the extreme rapidity of its tail currents over a wide voltage range. Fitting exponential functions to their time course yields time constants in the range of 100–500 μs, which are difficult to resolve well when the current amplitudes are small. Additionally, there is a prominent component of OFF gating current (note the lowermost trace in the panel which was taken at −90 mV) that is slower than the ionic tail in this mutant and significantly distorts its kinetics. Simulated F401A traces have a multi-exponential decay, with the very rapid component near the limit of resolution in our recordings (and perhaps more rapid than the excised patch data), and a slower component that at −30 and −50 mV cannot be reliably distinguished from the steady state component seen in the experimental data. Because of the nonionic components of the current decay (which the model does not take into account), more detailed comparison of the model simulations to the tail currents in F401A at hyperpolarized voltages was not undertaken.

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