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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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(A) Model predictions for the activation of ionic currents. On the left, representative families of currents recorded at between −90 and +50 mV (wt), −95 to +25 mV (F401L), and −80 to +140 mV (F401A) are displayed. Voltage increments were 10 mV (20 mV for F401A). Corresponding simulated ionic currents are shown on the right. Model parameters were not altered from those in Fig. 14B and Fig. C. Comparison of the model with measured ionic current activation kinetics. Activation time constants (B) and time-to-half-maximum (C), obtained as described in the text, are plotted as a function of test voltage for wt, F401L, and F401A channels. Predictions of the models are shown as solid and dashed lines. These were obtained by the same analysis procedures applied to the simulated currents as to the experimental records.
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Figure 17: (A) Model predictions for the activation of ionic currents. On the left, representative families of currents recorded at between −90 and +50 mV (wt), −95 to +25 mV (F401L), and −80 to +140 mV (F401A) are displayed. Voltage increments were 10 mV (20 mV for F401A). Corresponding simulated ionic currents are shown on the right. Model parameters were not altered from those in Fig. 14B and Fig. C. Comparison of the model with measured ionic current activation kinetics. Activation time constants (B) and time-to-half-maximum (C), obtained as described in the text, are plotted as a function of test voltage for wt, F401L, and F401A channels. Predictions of the models are shown as solid and dashed lines. These were obtained by the same analysis procedures applied to the simulated currents as to the experimental records.

Mentions: The predictions of the models for the macroscopic ionic currents are shown in Fig. 17. Representative families of activating currents recorded from patches containing wt, F401L, and F401A channels are qualitatively comparable to model simulations at matching voltages in terms of the overall sigmoidal character and the voltage range over which activation kinetics are most noticeably changing. A consistent finding for all three channels is that the model traces appear to have a slower overall time course than the patch data. This discrepancy is quantified in Fig. 17 B, which displays model predictions for the time constant of activation derived from fitting the late phase of current time course (Zagotta et al. 1994a) and in Fig. 17 C in which the time-to-half-maximum current is plotted for the three models. The relative order of magnitudes of experimentally derived values for the three channels are preserved in the model simulations. While a shift of approximately −10 mV between the model and data results in closer agreement, these kinetic measurements obtained in excised inside- and outside-out patches are not easily superimposable on the model simulations by a simple voltage offset. We wondered if the model parameters that are mostly determined using gating current recordings obtained with cut-open oocyte clamp systematically predict slower ionic currents as the result of differences inherent in the two recording techniques. Stefani et al. 1994 demonstrated that for the kinetics of gating currents, the cut-open oocyte clamp technique produced very similar results to those obtained with cell-attached macropatches. In our experience, activation kinetics of Shaker in cell-attached patches are somewhat slower (and deactivation is faster) than the excised patches we used. We elected not to alter the models extensively to try to accommodate both the cut-open oocyte clamp and excised patch data sets, but instead focused on qualitative agreement between experimental ionic current results and model simulations.


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) Model predictions for the activation of ionic currents. On the left, representative families of currents recorded at between −90 and +50 mV (wt), −95 to +25 mV (F401L), and −80 to +140 mV (F401A) are displayed. Voltage increments were 10 mV (20 mV for F401A). Corresponding simulated ionic currents are shown on the right. Model parameters were not altered from those in Fig. 14B and Fig. C. Comparison of the model with measured ionic current activation kinetics. Activation time constants (B) and time-to-half-maximum (C), obtained as described in the text, are plotted as a function of test voltage for wt, F401L, and F401A channels. Predictions of the models are shown as solid and dashed lines. These were obtained by the same analysis procedures applied to the simulated currents as to the experimental records.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 17: (A) Model predictions for the activation of ionic currents. On the left, representative families of currents recorded at between −90 and +50 mV (wt), −95 to +25 mV (F401L), and −80 to +140 mV (F401A) are displayed. Voltage increments were 10 mV (20 mV for F401A). Corresponding simulated ionic currents are shown on the right. Model parameters were not altered from those in Fig. 14B and Fig. C. Comparison of the model with measured ionic current activation kinetics. Activation time constants (B) and time-to-half-maximum (C), obtained as described in the text, are plotted as a function of test voltage for wt, F401L, and F401A channels. Predictions of the models are shown as solid and dashed lines. These were obtained by the same analysis procedures applied to the simulated currents as to the experimental records.
Mentions: The predictions of the models for the macroscopic ionic currents are shown in Fig. 17. Representative families of activating currents recorded from patches containing wt, F401L, and F401A channels are qualitatively comparable to model simulations at matching voltages in terms of the overall sigmoidal character and the voltage range over which activation kinetics are most noticeably changing. A consistent finding for all three channels is that the model traces appear to have a slower overall time course than the patch data. This discrepancy is quantified in Fig. 17 B, which displays model predictions for the time constant of activation derived from fitting the late phase of current time course (Zagotta et al. 1994a) and in Fig. 17 C in which the time-to-half-maximum current is plotted for the three models. The relative order of magnitudes of experimentally derived values for the three channels are preserved in the model simulations. While a shift of approximately −10 mV between the model and data results in closer agreement, these kinetic measurements obtained in excised inside- and outside-out patches are not easily superimposable on the model simulations by a simple voltage offset. We wondered if the model parameters that are mostly determined using gating current recordings obtained with cut-open oocyte clamp systematically predict slower ionic currents as the result of differences inherent in the two recording techniques. Stefani et al. 1994 demonstrated that for the kinetics of gating currents, the cut-open oocyte clamp technique produced very similar results to those obtained with cell-attached macropatches. In our experience, activation kinetics of Shaker in cell-attached patches are somewhat slower (and deactivation is faster) than the excised patches we used. We elected not to alter the models extensively to try to accommodate both the cut-open oocyte clamp and excised patch data sets, but instead focused on qualitative agreement between experimental ionic current results and model simulations.

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