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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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Steady state voltage dependence of the wild-type ShBΔ6-46 K channel and the F401I mutant. (A) Activation families were elicited from the holding potential of −100 mV with steps in 10-mV increments between −80 to +50 mV, and tail currents were recorded at −65 mV, as indicated schematically above the traces. Patch-clamp records were obtained in the inside-out excised configuration (Hamill et al. 1981), digitized at 20 kHz, and low-pass filtered at 8 (wt) or 3 (F401I) kHz. (B) Relative conductance is plotted versus voltage. Conductances were normalized to the maximal value in each family and the results from different patches were averaged to obtain the means and standard errors shown (wt: n = 5; F401I: n = 6). Averaged G(V) curves were fit with the fourth power of a Boltzmann function (see methods). The apparent gating valence per subunit from these fits is reduced from 4.18 e0 for the wild type to 2.40 e0 for F401I. V1/2 parameters from the fits are −56.3 mV for wt and −57.6 mV for F401I. (C) The amino acid sequence difference between wild-type Shaker and the neomorphic Sh5 allele is localized to a single substitution in the S5 region: phenylalanine is mutated to isoleucine at position 401 (Gautam and Tanouye 1990; Lichtinghagen et al. 1990). The sequence is shown using the standard single-letter amino acid code; dashes in the Sh5 sequence indicate amino acid identity with wt. Asterisks mark the residues that were mutated in this study.
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Figure 1: Steady state voltage dependence of the wild-type ShBΔ6-46 K channel and the F401I mutant. (A) Activation families were elicited from the holding potential of −100 mV with steps in 10-mV increments between −80 to +50 mV, and tail currents were recorded at −65 mV, as indicated schematically above the traces. Patch-clamp records were obtained in the inside-out excised configuration (Hamill et al. 1981), digitized at 20 kHz, and low-pass filtered at 8 (wt) or 3 (F401I) kHz. (B) Relative conductance is plotted versus voltage. Conductances were normalized to the maximal value in each family and the results from different patches were averaged to obtain the means and standard errors shown (wt: n = 5; F401I: n = 6). Averaged G(V) curves were fit with the fourth power of a Boltzmann function (see methods). The apparent gating valence per subunit from these fits is reduced from 4.18 e0 for the wild type to 2.40 e0 for F401I. V1/2 parameters from the fits are −56.3 mV for wt and −57.6 mV for F401I. (C) The amino acid sequence difference between wild-type Shaker and the neomorphic Sh5 allele is localized to a single substitution in the S5 region: phenylalanine is mutated to isoleucine at position 401 (Gautam and Tanouye 1990; Lichtinghagen et al. 1990). The sequence is shown using the standard single-letter amino acid code; dashes in the Sh5 sequence indicate amino acid identity with wt. Asterisks mark the residues that were mutated in this study.

Mentions: A point mutation converting the first phenylalanine of the fifth transmembrane segment to isoleucine served as a replica of the Sh5 mutation (Fig. 1 C). Families from patches expressing either wild-type or mutant F401I currents activate over a similar range of voltages and deactivate completely at the relatively depolarized tail potential of −65 mV (Fig. 1 A). Compared with the wt G(V) curve, F401I activation has a noticeably shallower voltage dependence (Fig. 1 B). The fourth power Boltzmann fits to the G(V) curves yield values of z of approximately four elementary charges (e0) per wild-type subunit, which is similar to the estimated total charge displacement per channel of 12.5–14 e0, obtained from direct gating current measurements (Schoppa et al. 1992; Aggarwal and MacKinnon 1996; Noceti et al. 1996; Seoh et al. 1996). By contrast, the value of z for F401I is decreased to 2.4 e0, which implies that the mutation either reduces the amount of charge displacement in the channel, or alters the coupling between the charge-moving transitions and the channel opening.


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)

Steady state voltage dependence of the wild-type ShBΔ6-46 K channel and the F401I mutant. (A) Activation families were elicited from the holding potential of −100 mV with steps in 10-mV increments between −80 to +50 mV, and tail currents were recorded at −65 mV, as indicated schematically above the traces. Patch-clamp records were obtained in the inside-out excised configuration (Hamill et al. 1981), digitized at 20 kHz, and low-pass filtered at 8 (wt) or 3 (F401I) kHz. (B) Relative conductance is plotted versus voltage. Conductances were normalized to the maximal value in each family and the results from different patches were averaged to obtain the means and standard errors shown (wt: n = 5; F401I: n = 6). Averaged G(V) curves were fit with the fourth power of a Boltzmann function (see methods). The apparent gating valence per subunit from these fits is reduced from 4.18 e0 for the wild type to 2.40 e0 for F401I. V1/2 parameters from the fits are −56.3 mV for wt and −57.6 mV for F401I. (C) The amino acid sequence difference between wild-type Shaker and the neomorphic Sh5 allele is localized to a single substitution in the S5 region: phenylalanine is mutated to isoleucine at position 401 (Gautam and Tanouye 1990; Lichtinghagen et al. 1990). The sequence is shown using the standard single-letter amino acid code; dashes in the Sh5 sequence indicate amino acid identity with wt. Asterisks mark the residues that were mutated in this study.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230647&req=5

Figure 1: Steady state voltage dependence of the wild-type ShBΔ6-46 K channel and the F401I mutant. (A) Activation families were elicited from the holding potential of −100 mV with steps in 10-mV increments between −80 to +50 mV, and tail currents were recorded at −65 mV, as indicated schematically above the traces. Patch-clamp records were obtained in the inside-out excised configuration (Hamill et al. 1981), digitized at 20 kHz, and low-pass filtered at 8 (wt) or 3 (F401I) kHz. (B) Relative conductance is plotted versus voltage. Conductances were normalized to the maximal value in each family and the results from different patches were averaged to obtain the means and standard errors shown (wt: n = 5; F401I: n = 6). Averaged G(V) curves were fit with the fourth power of a Boltzmann function (see methods). The apparent gating valence per subunit from these fits is reduced from 4.18 e0 for the wild type to 2.40 e0 for F401I. V1/2 parameters from the fits are −56.3 mV for wt and −57.6 mV for F401I. (C) The amino acid sequence difference between wild-type Shaker and the neomorphic Sh5 allele is localized to a single substitution in the S5 region: phenylalanine is mutated to isoleucine at position 401 (Gautam and Tanouye 1990; Lichtinghagen et al. 1990). The sequence is shown using the standard single-letter amino acid code; dashes in the Sh5 sequence indicate amino acid identity with wt. Asterisks mark the residues that were mutated in this study.
Mentions: A point mutation converting the first phenylalanine of the fifth transmembrane segment to isoleucine served as a replica of the Sh5 mutation (Fig. 1 C). Families from patches expressing either wild-type or mutant F401I currents activate over a similar range of voltages and deactivate completely at the relatively depolarized tail potential of −65 mV (Fig. 1 A). Compared with the wt G(V) curve, F401I activation has a noticeably shallower voltage dependence (Fig. 1 B). The fourth power Boltzmann fits to the G(V) curves yield values of z of approximately four elementary charges (e0) per wild-type subunit, which is similar to the estimated total charge displacement per channel of 12.5–14 e0, obtained from direct gating current measurements (Schoppa et al. 1992; Aggarwal and MacKinnon 1996; Noceti et al. 1996; Seoh et al. 1996). By contrast, the value of z for F401I is decreased to 2.4 e0, which implies that the mutation either reduces the amount of charge displacement in the channel, or alters the coupling between the charge-moving transitions and the channel opening.

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