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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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F401 substitutions affect the steady state charge transfer and kinetics of the forward transitions. (A) Relative charge versus voltage relations were computed for the wf, wfF401L, and wfF401A channels by integrating the gating current during the −100-mV post-pulse (IgOFF). Normalized Q(V) relations from a number of experiments were averaged and plotted as a function of test voltage as mean ± SEM (wf: n = 17; wfF401L: n = 10; wfF401A: n = 12). The fitted lines through the data are the Boltzmann function predictions for channels with independent and identical subunit transitions with the following values of voltage midpoint (V1/2) and associated charge displacement (z): −43 mV and 3.73 e0, −61.6 mV and 4.14 e0, and −52.3 mV and 2.76 e0, for wf, wfF401L, and wfF401A, respectively. (B) The decay phase of the IgON was fitted with a single exponential and the time constants are plotted against pulse voltage for the wf (n = 11), wfF401L (n = 8), and wfF401A (n = 8) channels. From the exponential fits of the voltage dependence, beginning at −10, −25, and −20 mV, the estimate zf of the charge associated with the forward transitions is 0.57, 0.61, and 0.63 e0, for wf, wfF401L, and wfF401A, respectively.
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Figure 9: F401 substitutions affect the steady state charge transfer and kinetics of the forward transitions. (A) Relative charge versus voltage relations were computed for the wf, wfF401L, and wfF401A channels by integrating the gating current during the −100-mV post-pulse (IgOFF). Normalized Q(V) relations from a number of experiments were averaged and plotted as a function of test voltage as mean ± SEM (wf: n = 17; wfF401L: n = 10; wfF401A: n = 12). The fitted lines through the data are the Boltzmann function predictions for channels with independent and identical subunit transitions with the following values of voltage midpoint (V1/2) and associated charge displacement (z): −43 mV and 3.73 e0, −61.6 mV and 4.14 e0, and −52.3 mV and 2.76 e0, for wf, wfF401L, and wfF401A, respectively. (B) The decay phase of the IgON was fitted with a single exponential and the time constants are plotted against pulse voltage for the wf (n = 11), wfF401L (n = 8), and wfF401A (n = 8) channels. From the exponential fits of the voltage dependence, beginning at −10, −25, and −20 mV, the estimate zf of the charge associated with the forward transitions is 0.57, 0.61, and 0.63 e0, for wf, wfF401L, and wfF401A, respectively.

Mentions: Steady state Q(V) relationships for the three channels were computed from the integral of the IgOFF after a test pulse. The integral of the IgON, while not shown, agreed closely. Fig. 9 A shows that the wf Q(V) curve has a characteristically shallow base and steeper upper portion (see also Stefani et al. 1994). The wfF401L Q(V) relation is negatively shifted relative to the wf, similar to the relationship between the G(V) of F401L and the wt. For this mutant, channel opening seems to follow closely the displacement of the voltage-sensing charges. wfF401A has detectable charge movement at more negative voltages than the wf, but its Q(V) slope is somewhat shallower. However, we did not detect any obvious inflections reflecting the movement of an additional component of the gating charge in the wfF401A Q(V) curve at voltages above 0 mV and extending even beyond +100 mV. Therefore, we conclude that the reduction in the apparent voltage dependence of activation is not the result of altered coupling of activation pathway transitions carrying significant amounts of charge movement. It is likely that this mutation affects an activating transition that moves only a small amount of charge and thus would not perturb the overall shape of the Q(V) curve.


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)

F401 substitutions affect the steady state charge transfer and kinetics of the forward transitions. (A) Relative charge versus voltage relations were computed for the wf, wfF401L, and wfF401A channels by integrating the gating current during the −100-mV post-pulse (IgOFF). Normalized Q(V) relations from a number of experiments were averaged and plotted as a function of test voltage as mean ± SEM (wf: n = 17; wfF401L: n = 10; wfF401A: n = 12). The fitted lines through the data are the Boltzmann function predictions for channels with independent and identical subunit transitions with the following values of voltage midpoint (V1/2) and associated charge displacement (z): −43 mV and 3.73 e0, −61.6 mV and 4.14 e0, and −52.3 mV and 2.76 e0, for wf, wfF401L, and wfF401A, respectively. (B) The decay phase of the IgON was fitted with a single exponential and the time constants are plotted against pulse voltage for the wf (n = 11), wfF401L (n = 8), and wfF401A (n = 8) channels. From the exponential fits of the voltage dependence, beginning at −10, −25, and −20 mV, the estimate zf of the charge associated with the forward transitions is 0.57, 0.61, and 0.63 e0, for wf, wfF401L, and wfF401A, respectively.
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Related In: Results  -  Collection

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Figure 9: F401 substitutions affect the steady state charge transfer and kinetics of the forward transitions. (A) Relative charge versus voltage relations were computed for the wf, wfF401L, and wfF401A channels by integrating the gating current during the −100-mV post-pulse (IgOFF). Normalized Q(V) relations from a number of experiments were averaged and plotted as a function of test voltage as mean ± SEM (wf: n = 17; wfF401L: n = 10; wfF401A: n = 12). The fitted lines through the data are the Boltzmann function predictions for channels with independent and identical subunit transitions with the following values of voltage midpoint (V1/2) and associated charge displacement (z): −43 mV and 3.73 e0, −61.6 mV and 4.14 e0, and −52.3 mV and 2.76 e0, for wf, wfF401L, and wfF401A, respectively. (B) The decay phase of the IgON was fitted with a single exponential and the time constants are plotted against pulse voltage for the wf (n = 11), wfF401L (n = 8), and wfF401A (n = 8) channels. From the exponential fits of the voltage dependence, beginning at −10, −25, and −20 mV, the estimate zf of the charge associated with the forward transitions is 0.57, 0.61, and 0.63 e0, for wf, wfF401L, and wfF401A, respectively.
Mentions: Steady state Q(V) relationships for the three channels were computed from the integral of the IgOFF after a test pulse. The integral of the IgON, while not shown, agreed closely. Fig. 9 A shows that the wf Q(V) curve has a characteristically shallow base and steeper upper portion (see also Stefani et al. 1994). The wfF401L Q(V) relation is negatively shifted relative to the wf, similar to the relationship between the G(V) of F401L and the wt. For this mutant, channel opening seems to follow closely the displacement of the voltage-sensing charges. wfF401A has detectable charge movement at more negative voltages than the wf, but its Q(V) slope is somewhat shallower. However, we did not detect any obvious inflections reflecting the movement of an additional component of the gating charge in the wfF401A Q(V) curve at voltages above 0 mV and extending even beyond +100 mV. Therefore, we conclude that the reduction in the apparent voltage dependence of activation is not the result of altered coupling of activation pathway transitions carrying significant amounts of charge movement. It is likely that this mutation affects an activating transition that moves only a small amount of charge and thus would not perturb the overall shape of the Q(V) curve.

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