Limits...
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.

Show MeSH

Related in: MedlinePlus

Substitutions at position 401 affect deactivation. (A) Deactivation time constants for the F401L, F401V, and F401A mutant channels were obtained from single exponential fits to currents during channel closing. The time constants, τ, are plotted as a function of tail potential and fitted with an exponential. For F401L and F401V, results are shown as the mean ± SEM of 8 and 10 experiments, respectively. Due to the bandwidth limitations on our ability to fully resolve closing kinetics in the F401A mutant, results from the two best patches are shown at selected voltages. For comparison, fits to wild-type and F401I deactivation curves from Fig. 3 are also included. The voltage dependence of deactivation τ yielded apparent zr values of 1.15 e0 for F401L, 0.74 e0 for F401V, and <0.5 e0 for F401A. (B) Differences of deactivation kinetics are illustrated with the tail families of the F401A and F401L mutants, shown on the same time scale. The F401A family (left) was recorded under standard ionic conditions (see methods) from an inside-out patch, sampled at 100 kHz and low-pass filtered at 9.5 kHz. Tail voltage ranged between −20 and −170 mV in 10-mV increments. The F401L currents (right) were recorded from an outside-out patch with symmetrical 140 mM K+ as the permeant cation. Increasing external K+ from 2 to 140 mM does not significantly affect tail kinetics of this channel (data not shown, but see Zagotta et al. 1994a. Tail voltage was stepped to between −80 and −180 mV in 10-mV increments. For both families, a 10-ms pulse to +50 mV preceded the steps to the tail potentials.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2230647&req=5

Figure 7: Substitutions at position 401 affect deactivation. (A) Deactivation time constants for the F401L, F401V, and F401A mutant channels were obtained from single exponential fits to currents during channel closing. The time constants, τ, are plotted as a function of tail potential and fitted with an exponential. For F401L and F401V, results are shown as the mean ± SEM of 8 and 10 experiments, respectively. Due to the bandwidth limitations on our ability to fully resolve closing kinetics in the F401A mutant, results from the two best patches are shown at selected voltages. For comparison, fits to wild-type and F401I deactivation curves from Fig. 3 are also included. The voltage dependence of deactivation τ yielded apparent zr values of 1.15 e0 for F401L, 0.74 e0 for F401V, and <0.5 e0 for F401A. (B) Differences of deactivation kinetics are illustrated with the tail families of the F401A and F401L mutants, shown on the same time scale. The F401A family (left) was recorded under standard ionic conditions (see methods) from an inside-out patch, sampled at 100 kHz and low-pass filtered at 9.5 kHz. Tail voltage ranged between −20 and −170 mV in 10-mV increments. The F401L currents (right) were recorded from an outside-out patch with symmetrical 140 mM K+ as the permeant cation. Increasing external K+ from 2 to 140 mM does not significantly affect tail kinetics of this channel (data not shown, but see Zagotta et al. 1994a. Tail voltage was stepped to between −80 and −180 mV in 10-mV increments. For both families, a 10-ms pulse to +50 mV preceded the steps to the tail potentials.

Mentions: We expected that, as in the case of Sh5 (F401I), the other aliphatic substitutions would preferentially perturb deactivating transitions. In Fig. 7 A, time constants from fits to tail current relaxations are plotted for the leucine, valine, and alanine mutations. For comparison, fits to the voltage dependence of the deactivation time constant, τ, from wt and F401I are also included. The tail time constants of F401L currents are slower than those of the wt but have similarly steep voltage dependence. Deactivation kinetics of F401V (zr = 0.74 e0) are nearly the same as those of F401I, and F401A deactivation appears to be nearly voltage independent to the best of our ability to analyze its very rapid kinetics. This finding provides a ready explanation for the very shallow G(V) of F401A. In wild-type Shaker, a greater proportion of the total gating charge movement occurs after the transition state (Zagotta et al. 1994a), and its loss will be reflected in the diminished voltage dependence of the steady state gating parameters. On the other hand, the notable decrease in the backward rates and modest increase in the forward rates seen in F401L imply that some of the gating equilibria for this channel are biased toward the open state compared with the wt, which is consistent with the finding of a negatively shifted G(V) relation. Fig. 7 B depicts families of current traces from the two mutants that differ the most in their tail kinetics. Currents from the F401L and F401A families are shown on the same time scale to illustrate that there is more than an order of magnitude difference in the tendency of these channels, once activated, to remain in (or near to) the open state long after the end of a depolarizing voltage pulse. Note that even at fairly depolarized tail potentials F401A channels relax to a new steady state level on a very rapid time scale.


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)

Substitutions at position 401 affect deactivation. (A) Deactivation time constants for the F401L, F401V, and F401A mutant channels were obtained from single exponential fits to currents during channel closing. The time constants, τ, are plotted as a function of tail potential and fitted with an exponential. For F401L and F401V, results are shown as the mean ± SEM of 8 and 10 experiments, respectively. Due to the bandwidth limitations on our ability to fully resolve closing kinetics in the F401A mutant, results from the two best patches are shown at selected voltages. For comparison, fits to wild-type and F401I deactivation curves from Fig. 3 are also included. The voltage dependence of deactivation τ yielded apparent zr values of 1.15 e0 for F401L, 0.74 e0 for F401V, and <0.5 e0 for F401A. (B) Differences of deactivation kinetics are illustrated with the tail families of the F401A and F401L mutants, shown on the same time scale. The F401A family (left) was recorded under standard ionic conditions (see methods) from an inside-out patch, sampled at 100 kHz and low-pass filtered at 9.5 kHz. Tail voltage ranged between −20 and −170 mV in 10-mV increments. The F401L currents (right) were recorded from an outside-out patch with symmetrical 140 mM K+ as the permeant cation. Increasing external K+ from 2 to 140 mM does not significantly affect tail kinetics of this channel (data not shown, but see Zagotta et al. 1994a. Tail voltage was stepped to between −80 and −180 mV in 10-mV increments. For both families, a 10-ms pulse to +50 mV preceded the steps to the tail potentials.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Substitutions at position 401 affect deactivation. (A) Deactivation time constants for the F401L, F401V, and F401A mutant channels were obtained from single exponential fits to currents during channel closing. The time constants, τ, are plotted as a function of tail potential and fitted with an exponential. For F401L and F401V, results are shown as the mean ± SEM of 8 and 10 experiments, respectively. Due to the bandwidth limitations on our ability to fully resolve closing kinetics in the F401A mutant, results from the two best patches are shown at selected voltages. For comparison, fits to wild-type and F401I deactivation curves from Fig. 3 are also included. The voltage dependence of deactivation τ yielded apparent zr values of 1.15 e0 for F401L, 0.74 e0 for F401V, and <0.5 e0 for F401A. (B) Differences of deactivation kinetics are illustrated with the tail families of the F401A and F401L mutants, shown on the same time scale. The F401A family (left) was recorded under standard ionic conditions (see methods) from an inside-out patch, sampled at 100 kHz and low-pass filtered at 9.5 kHz. Tail voltage ranged between −20 and −170 mV in 10-mV increments. The F401L currents (right) were recorded from an outside-out patch with symmetrical 140 mM K+ as the permeant cation. Increasing external K+ from 2 to 140 mM does not significantly affect tail kinetics of this channel (data not shown, but see Zagotta et al. 1994a. Tail voltage was stepped to between −80 and −180 mV in 10-mV increments. For both families, a 10-ms pulse to +50 mV preceded the steps to the tail potentials.
Mentions: We expected that, as in the case of Sh5 (F401I), the other aliphatic substitutions would preferentially perturb deactivating transitions. In Fig. 7 A, time constants from fits to tail current relaxations are plotted for the leucine, valine, and alanine mutations. For comparison, fits to the voltage dependence of the deactivation time constant, τ, from wt and F401I are also included. The tail time constants of F401L currents are slower than those of the wt but have similarly steep voltage dependence. Deactivation kinetics of F401V (zr = 0.74 e0) are nearly the same as those of F401I, and F401A deactivation appears to be nearly voltage independent to the best of our ability to analyze its very rapid kinetics. This finding provides a ready explanation for the very shallow G(V) of F401A. In wild-type Shaker, a greater proportion of the total gating charge movement occurs after the transition state (Zagotta et al. 1994a), and its loss will be reflected in the diminished voltage dependence of the steady state gating parameters. On the other hand, the notable decrease in the backward rates and modest increase in the forward rates seen in F401L imply that some of the gating equilibria for this channel are biased toward the open state compared with the wt, which is consistent with the finding of a negatively shifted G(V) relation. Fig. 7 B depicts families of current traces from the two mutants that differ the most in their tail kinetics. Currents from the F401L and F401A families are shown on the same time scale to illustrate that there is more than an order of magnitude difference in the tendency of these channels, once activated, to remain in (or near to) the open state long after the end of a depolarizing voltage pulse. Note that even at fairly depolarized tail potentials F401A channels relax to a new steady state level on a very rapid time scale.

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