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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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Mutations of F401 and cooperative gating: return of the gating charge before and after channel opening. (A) Families of gating currents recorded in the cut-open oocyte clamp are shown. In all families, steps were given from the holding voltage of −100 mV. For the wf (left) and wfF401L (center) channels, pulse voltages were (top to bottom) −50, −30, and 0 mV. For wfF401A (right), pulse voltages were −50, 0, and +100 mV. Voltage electrode traces are included above each family to indicate the voltage protocol used. In each family, gating currents resulting from different durations of the pulse are overlaid such that the changes with the pulse length can be observed as the envelope of OFF gating currents. Data were filtered at 8–10 kHz. (B) Voltage and pulse-length dependence of charge return kinetics. For the wf (left), wfF401L (center), and wfF401A (right) channels, records obtained as in Fig. 8 were fitted with an exponential function to describe the time course of IgOFF decay. The time constants from the fits for the different levels of pulse voltage are plotted as a function of duration of the pulse.
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Figure 12: Mutations of F401 and cooperative gating: return of the gating charge before and after channel opening. (A) Families of gating currents recorded in the cut-open oocyte clamp are shown. In all families, steps were given from the holding voltage of −100 mV. For the wf (left) and wfF401L (center) channels, pulse voltages were (top to bottom) −50, −30, and 0 mV. For wfF401A (right), pulse voltages were −50, 0, and +100 mV. Voltage electrode traces are included above each family to indicate the voltage protocol used. In each family, gating currents resulting from different durations of the pulse are overlaid such that the changes with the pulse length can be observed as the envelope of OFF gating currents. Data were filtered at 8–10 kHz. (B) Voltage and pulse-length dependence of charge return kinetics. For the wf (left), wfF401L (center), and wfF401A (right) channels, records obtained as in Fig. 8 were fitted with an exponential function to describe the time course of IgOFF decay. The time constants from the fits for the different levels of pulse voltage are plotted as a function of duration of the pulse.

Mentions: Fig. 12 A contrasts the effect of test-pulse duration at three voltage levels on IgOFF in wf, wfF401L, and wfF401A channels. With pulses to −50 mV, charge return upon repolarization to −100 mV remains rapid in wf and wfF401A for pulse durations between 1 and 57 ms (wf) and 41 ms (wfF401A). F401L channels are significantly open at this voltage, however, and the OFF gating currents in wfF401L accordingly display progressively diminished amplitude and a prolonged declining phase as pulse length exceeds ∼3 ms. A pulse amplitude of −30 mV (Fig. 12 A, middle left) marks a transition zone for the kinetics of the wf IgOFF. Pulses of a few milliseconds duration do not impede subsequent rapid charge return, those longer than ∼10 ms give rise to OFF currents with complex time courses in which at least three kinetic components can be recognized, and those >25 ms produce a rising phase and exponential decay. wfF401L OFF gating currents with all but the shortest −30 mV pulses are notable for the greatly slowed charge return that is incomplete after up to 30 ms at −100 mV (Fig. 12 A, middle center). The families of wf and wfF401L channels show progression of the same trends when the pulse amplitude is 0 mV. In fact, IgOFF becomes nearly “immobilized” in wfF401L, displaying protracted decay after pulses lasting longer than 3–4 ms.


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)

Mutations of F401 and cooperative gating: return of the gating charge before and after channel opening. (A) Families of gating currents recorded in the cut-open oocyte clamp are shown. In all families, steps were given from the holding voltage of −100 mV. For the wf (left) and wfF401L (center) channels, pulse voltages were (top to bottom) −50, −30, and 0 mV. For wfF401A (right), pulse voltages were −50, 0, and +100 mV. Voltage electrode traces are included above each family to indicate the voltage protocol used. In each family, gating currents resulting from different durations of the pulse are overlaid such that the changes with the pulse length can be observed as the envelope of OFF gating currents. Data were filtered at 8–10 kHz. (B) Voltage and pulse-length dependence of charge return kinetics. For the wf (left), wfF401L (center), and wfF401A (right) channels, records obtained as in Fig. 8 were fitted with an exponential function to describe the time course of IgOFF decay. The time constants from the fits for the different levels of pulse voltage are plotted as a function of duration of the pulse.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230647&req=5

Figure 12: Mutations of F401 and cooperative gating: return of the gating charge before and after channel opening. (A) Families of gating currents recorded in the cut-open oocyte clamp are shown. In all families, steps were given from the holding voltage of −100 mV. For the wf (left) and wfF401L (center) channels, pulse voltages were (top to bottom) −50, −30, and 0 mV. For wfF401A (right), pulse voltages were −50, 0, and +100 mV. Voltage electrode traces are included above each family to indicate the voltage protocol used. In each family, gating currents resulting from different durations of the pulse are overlaid such that the changes with the pulse length can be observed as the envelope of OFF gating currents. Data were filtered at 8–10 kHz. (B) Voltage and pulse-length dependence of charge return kinetics. For the wf (left), wfF401L (center), and wfF401A (right) channels, records obtained as in Fig. 8 were fitted with an exponential function to describe the time course of IgOFF decay. The time constants from the fits for the different levels of pulse voltage are plotted as a function of duration of the pulse.
Mentions: Fig. 12 A contrasts the effect of test-pulse duration at three voltage levels on IgOFF in wf, wfF401L, and wfF401A channels. With pulses to −50 mV, charge return upon repolarization to −100 mV remains rapid in wf and wfF401A for pulse durations between 1 and 57 ms (wf) and 41 ms (wfF401A). F401L channels are significantly open at this voltage, however, and the OFF gating currents in wfF401L accordingly display progressively diminished amplitude and a prolonged declining phase as pulse length exceeds ∼3 ms. A pulse amplitude of −30 mV (Fig. 12 A, middle left) marks a transition zone for the kinetics of the wf IgOFF. Pulses of a few milliseconds duration do not impede subsequent rapid charge return, those longer than ∼10 ms give rise to OFF currents with complex time courses in which at least three kinetic components can be recognized, and those >25 ms produce a rising phase and exponential decay. wfF401L OFF gating currents with all but the shortest −30 mV pulses are notable for the greatly slowed charge return that is incomplete after up to 30 ms at −100 mV (Fig. 12 A, middle center). The families of wf and wfF401L channels show progression of the same trends when the pulse amplitude is 0 mV. In fact, IgOFF becomes nearly “immobilized” in wfF401L, displaying protracted decay after pulses lasting longer than 3–4 ms.

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