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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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Predictions of the ZHA model at different levels of cooperativity. (A) Connectivity of the ZHA model is shown in the collapsed format (for expanded version showing all nondegenerate states, see Fig. 7 of Zagotta et al. 1994b). Note that the rate of the first backward transition for leaving the open state is slowed by the cooperative factor θ. Simulations were performed on the versions of the ZHA model that varied the value of the factor θ as indicated. Simulated currents were then analyzed in the manner identical to the experimental data. Shown are the simulations for the normalized steady state G(V) relationship (B), ionic tail currents at −65 mV (C), normalized steady state Q(V) relation (D), and pulse-length dependence of the OFF gating current at −30 mV (E).
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Figure 13: Predictions of the ZHA model at different levels of cooperativity. (A) Connectivity of the ZHA model is shown in the collapsed format (for expanded version showing all nondegenerate states, see Fig. 7 of Zagotta et al. 1994b). Note that the rate of the first backward transition for leaving the open state is slowed by the cooperative factor θ. Simulations were performed on the versions of the ZHA model that varied the value of the factor θ as indicated. Simulated currents were then analyzed in the manner identical to the experimental data. Shown are the simulations for the normalized steady state G(V) relationship (B), ionic tail currents at −65 mV (C), normalized steady state Q(V) relation (D), and pulse-length dependence of the OFF gating current at −30 mV (E).

Mentions: One relatively simple formalism that has been put forth to account for these features of gating is the scheme of Zagotta et al. 1994b, which will be referred as the ZHA model, which explicitly introduces a cooperative factor θ by which the first closing transition rate is divided. Otherwise, the activation pathway in this model is an independent process involving four gating subunits, each undergoing two sequential charge-moving transitions. The ZHA model is shown in an abbreviated form in Fig. 13 A, emphasizing the fourfold symmetry. We used the parameters of the original ZHA model (Zagotta et al. 1994b) to simulate the G(V) (Fig. 13 B), ionic currents for steps to +50 mV followed by a tail voltage of −65 mV (Fig. 13 C), steady state charge-voltage relation [Q(V)] (Fig. 13 D), and the dependence of the time course of the OFF gating currents on the duration of −30-mV pulses (Fig. 13 E). In each case, the variable parameter was the factor θ, which was either 1, 9.4, or 50. These were chosen to give the best overall approximations of the F401A, wt, and F401L channels' behavior, respectively. For F401A, the cooperativity factor (θ) was set to a value of 1, the equivalent of complete subunit independence. The model succeeds in correctly describing the order of relative steepness of the G(V) curves, of the deactivation kinetics, and of the duration dependence of IgOFF. However, the extremely shallow voltage dependence of F401A channel opening (Fig. 5) could not be reproduced by the model, even with the introduction of a modest amount of negative cooperativity (θ = 0.4; Fig. 13 B).


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)

Predictions of the ZHA model at different levels of cooperativity. (A) Connectivity of the ZHA model is shown in the collapsed format (for expanded version showing all nondegenerate states, see Fig. 7 of Zagotta et al. 1994b). Note that the rate of the first backward transition for leaving the open state is slowed by the cooperative factor θ. Simulations were performed on the versions of the ZHA model that varied the value of the factor θ as indicated. Simulated currents were then analyzed in the manner identical to the experimental data. Shown are the simulations for the normalized steady state G(V) relationship (B), ionic tail currents at −65 mV (C), normalized steady state Q(V) relation (D), and pulse-length dependence of the OFF gating current at −30 mV (E).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 13: Predictions of the ZHA model at different levels of cooperativity. (A) Connectivity of the ZHA model is shown in the collapsed format (for expanded version showing all nondegenerate states, see Fig. 7 of Zagotta et al. 1994b). Note that the rate of the first backward transition for leaving the open state is slowed by the cooperative factor θ. Simulations were performed on the versions of the ZHA model that varied the value of the factor θ as indicated. Simulated currents were then analyzed in the manner identical to the experimental data. Shown are the simulations for the normalized steady state G(V) relationship (B), ionic tail currents at −65 mV (C), normalized steady state Q(V) relation (D), and pulse-length dependence of the OFF gating current at −30 mV (E).
Mentions: One relatively simple formalism that has been put forth to account for these features of gating is the scheme of Zagotta et al. 1994b, which will be referred as the ZHA model, which explicitly introduces a cooperative factor θ by which the first closing transition rate is divided. Otherwise, the activation pathway in this model is an independent process involving four gating subunits, each undergoing two sequential charge-moving transitions. The ZHA model is shown in an abbreviated form in Fig. 13 A, emphasizing the fourfold symmetry. We used the parameters of the original ZHA model (Zagotta et al. 1994b) to simulate the G(V) (Fig. 13 B), ionic currents for steps to +50 mV followed by a tail voltage of −65 mV (Fig. 13 C), steady state charge-voltage relation [Q(V)] (Fig. 13 D), and the dependence of the time course of the OFF gating currents on the duration of −30-mV pulses (Fig. 13 E). In each case, the variable parameter was the factor θ, which was either 1, 9.4, or 50. These were chosen to give the best overall approximations of the F401A, wt, and F401L channels' behavior, respectively. For F401A, the cooperativity factor (θ) was set to a value of 1, the equivalent of complete subunit independence. The model succeeds in correctly describing the order of relative steepness of the G(V) curves, of the deactivation kinetics, and of the duration dependence of IgOFF. However, the extremely shallow voltage dependence of F401A channel opening (Fig. 5) could not be reproduced by the model, even with the introduction of a modest amount of negative cooperativity (θ = 0.4; Fig. 13 B).

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