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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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Effects of replacing phenylalanine 401 with aliphatic residues on the activation kinetics and voltage dependence. (A) Representative current traces from wt, F401I, F401V, F401A, and F401L patches were superimposed and scaled to match at their peaks. Pulse voltages are indicated on the left. (B) A semilogarithmic plot of the activation time constants (mean ± SEM), determined from fitting an exponential function to the activation time course (see methods), is shown for the F401L (n = 12), F401V (n = 12), and F401A (n = 11) mutants. From the steepness of the exponential fit to the τ versus voltage relation, apparent valence associated with the forward transitions, zf, was found to be 0.41 e0 for F401L, 0.42 e0 for F401V, and 0.32 e0 for F401A. Fits are shown as thin solid and broken lines; for comparison, a fit to the wt activation τ is reproduced from Fig. 2 as a thick broken line.
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Figure 6: Effects of replacing phenylalanine 401 with aliphatic residues on the activation kinetics and voltage dependence. (A) Representative current traces from wt, F401I, F401V, F401A, and F401L patches were superimposed and scaled to match at their peaks. Pulse voltages are indicated on the left. (B) A semilogarithmic plot of the activation time constants (mean ± SEM), determined from fitting an exponential function to the activation time course (see methods), is shown for the F401L (n = 12), F401V (n = 12), and F401A (n = 11) mutants. From the steepness of the exponential fit to the τ versus voltage relation, apparent valence associated with the forward transitions, zf, was found to be 0.41 e0 for F401L, 0.42 e0 for F401V, and 0.32 e0 for F401A. Fits are shown as thin solid and broken lines; for comparison, a fit to the wt activation τ is reproduced from Fig. 2 as a thick broken line.

Mentions: A look at the activation time course on the expanded time scale in Fig. 6 A underscores that all channels bearing aliphatic substitutions for phenylalanine at position 401 activate more rapidly than the wt for a given voltage. Whereas F401L channels are least different from the wt, 401 isoleucine and valine channels are similar to each other and have faster kinetics than leucine channels; alanine channels are the fastest by far over all voltages. Quantitatively, Fig. 2 B and 6 B show that the voltage dependence of activation time constants measured late in the activation process is similarly weak no matter which of the five residues is at position 401, with the apparent valence associated with the forward transitions, zf, ranging from 0.32 to 0.42 e0. The absolute values of the time constants, τ, are comparable except for F401A, in which they are significantly diminished. Regardless of whether F401 mutations diminish steady state voltage dependence of the currents, the voltage dependence of the forward rate, zf, remains in the wt range.


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)

Effects of replacing phenylalanine 401 with aliphatic residues on the activation kinetics and voltage dependence. (A) Representative current traces from wt, F401I, F401V, F401A, and F401L patches were superimposed and scaled to match at their peaks. Pulse voltages are indicated on the left. (B) A semilogarithmic plot of the activation time constants (mean ± SEM), determined from fitting an exponential function to the activation time course (see methods), is shown for the F401L (n = 12), F401V (n = 12), and F401A (n = 11) mutants. From the steepness of the exponential fit to the τ versus voltage relation, apparent valence associated with the forward transitions, zf, was found to be 0.41 e0 for F401L, 0.42 e0 for F401V, and 0.32 e0 for F401A. Fits are shown as thin solid and broken lines; for comparison, a fit to the wt activation τ is reproduced from Fig. 2 as a thick broken line.
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Related In: Results  -  Collection

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

Figure 6: Effects of replacing phenylalanine 401 with aliphatic residues on the activation kinetics and voltage dependence. (A) Representative current traces from wt, F401I, F401V, F401A, and F401L patches were superimposed and scaled to match at their peaks. Pulse voltages are indicated on the left. (B) A semilogarithmic plot of the activation time constants (mean ± SEM), determined from fitting an exponential function to the activation time course (see methods), is shown for the F401L (n = 12), F401V (n = 12), and F401A (n = 11) mutants. From the steepness of the exponential fit to the τ versus voltage relation, apparent valence associated with the forward transitions, zf, was found to be 0.41 e0 for F401L, 0.42 e0 for F401V, and 0.32 e0 for F401A. Fits are shown as thin solid and broken lines; for comparison, a fit to the wt activation τ is reproduced from Fig. 2 as a thick broken line.
Mentions: A look at the activation time course on the expanded time scale in Fig. 6 A underscores that all channels bearing aliphatic substitutions for phenylalanine at position 401 activate more rapidly than the wt for a given voltage. Whereas F401L channels are least different from the wt, 401 isoleucine and valine channels are similar to each other and have faster kinetics than leucine channels; alanine channels are the fastest by far over all voltages. Quantitatively, Fig. 2 B and 6 B show that the voltage dependence of activation time constants measured late in the activation process is similarly weak no matter which of the five residues is at position 401, with the apparent valence associated with the forward transitions, zf, ranging from 0.32 to 0.42 e0. The absolute values of the time constants, τ, are comparable except for F401A, in which they are significantly diminished. Regardless of whether F401 mutations diminish steady state voltage dependence of the currents, the voltage dependence of the forward rate, zf, remains in the wt range.

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