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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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Aliphatic side-chain substitutions at position 401 affect the steady state voltage dependence of activation. (A) Families of macroscopic currents from patches expressing (left to right) F401L, F401V, and F401A channels were recorded as in Fig. 1. The voltage ranges for the three families were as follows (step increment in parentheses): −85 to +25 mV (10 mV) for F401L, −80 to +60 mV (10 mV) for F401V, and −80 to +160 mV (20 mV) for F401A. Note the faster time scale for the F401A family used to resolve its kinetic features. (B) Relative conductance versus voltage relationships for the three mutants are shown. For comparison, fits to G(V) curves for the wt and F401I channels are reproduced from Fig. 1. F401L and F401V data are plotted as means ± SEM of eight and nine families, respectively. Averaged G(V) data were fitted with the fourth power of a Boltzmann function, as previously described, giving apparent z of 4.25 and 2.52 e0, and V1/2 of −69.7 and −53.1 mV for F401L and F401V, respectively. For the F401A experiment shown (representative of 11 patches), G(V) was measured either as the chord conductance assuming the reversal potential of −80 mV (denoted “pulse”), or as the isochronal tail current amplitude (denoted “tail”). The characteristic failure of the steady state conductance to reach a maximum within the attainable voltage range prevented meaningful normalization of the F401A G(V).
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Figure 5: Aliphatic side-chain substitutions at position 401 affect the steady state voltage dependence of activation. (A) Families of macroscopic currents from patches expressing (left to right) F401L, F401V, and F401A channels were recorded as in Fig. 1. The voltage ranges for the three families were as follows (step increment in parentheses): −85 to +25 mV (10 mV) for F401L, −80 to +60 mV (10 mV) for F401V, and −80 to +160 mV (20 mV) for F401A. Note the faster time scale for the F401A family used to resolve its kinetic features. (B) Relative conductance versus voltage relationships for the three mutants are shown. For comparison, fits to G(V) curves for the wt and F401I channels are reproduced from Fig. 1. F401L and F401V data are plotted as means ± SEM of eight and nine families, respectively. Averaged G(V) data were fitted with the fourth power of a Boltzmann function, as previously described, giving apparent z of 4.25 and 2.52 e0, and V1/2 of −69.7 and −53.1 mV for F401L and F401V, respectively. For the F401A experiment shown (representative of 11 patches), G(V) was measured either as the chord conductance assuming the reversal potential of −80 mV (denoted “pulse”), or as the isochronal tail current amplitude (denoted “tail”). The characteristic failure of the steady state conductance to reach a maximum within the attainable voltage range prevented meaningful normalization of the F401A G(V).

Mentions: Because of the striking effects of the F401I substitution, we substituted other amino acids for the phenylalanine at 401 to investigate the role of side chain structure on gating. We introduced individually three progressively smaller aliphatic amino acids leucine, valine, and alanine at that site. Fig. 5 A shows representative current families from these channels on different time scales to bring out the distinctive kinetic features of each channel type. In Fig. 5 B, the range of change induced by these mutations in the steady state voltage dependence of the relative open probability is shown. For comparison, previously described fits of a fourth power of the Boltzmann function to the wt and F401I data are also included. The F401V G(V) relationship is shallower than that of the wt, and similar in slope (zapp = 2.5) to the F401I mutant. However, the V1/2 in F401V is positively shifted by ∼5 mV compared with F401I. Steady state activation of the F401A mutant is the shallowest (zapp < 0.5 e0); in fact, the G(V) relationship fails to reach saturation at voltages in excess of +150 mV in five patches, and is therefore displayed on a dimensionless y axis. Unexpectedly, introduction at position 401 of a leucine, an amino acid chemically most similar to the isoleucine, carried nearly opposite consequences compared with the Sh5 replica mutation F401I. The F401L mutant has a G(V) relation as steep as that of the wt (zapp = 4.25) but with the midpoint of the activating transition shifted negatively (V1/2 = −69.7 mV), the only mutant in this study to do so.


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)

Aliphatic side-chain substitutions at position 401 affect the steady state voltage dependence of activation. (A) Families of macroscopic currents from patches expressing (left to right) F401L, F401V, and F401A channels were recorded as in Fig. 1. The voltage ranges for the three families were as follows (step increment in parentheses): −85 to +25 mV (10 mV) for F401L, −80 to +60 mV (10 mV) for F401V, and −80 to +160 mV (20 mV) for F401A. Note the faster time scale for the F401A family used to resolve its kinetic features. (B) Relative conductance versus voltage relationships for the three mutants are shown. For comparison, fits to G(V) curves for the wt and F401I channels are reproduced from Fig. 1. F401L and F401V data are plotted as means ± SEM of eight and nine families, respectively. Averaged G(V) data were fitted with the fourth power of a Boltzmann function, as previously described, giving apparent z of 4.25 and 2.52 e0, and V1/2 of −69.7 and −53.1 mV for F401L and F401V, respectively. For the F401A experiment shown (representative of 11 patches), G(V) was measured either as the chord conductance assuming the reversal potential of −80 mV (denoted “pulse”), or as the isochronal tail current amplitude (denoted “tail”). The characteristic failure of the steady state conductance to reach a maximum within the attainable voltage range prevented meaningful normalization of the F401A G(V).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230647&req=5

Figure 5: Aliphatic side-chain substitutions at position 401 affect the steady state voltage dependence of activation. (A) Families of macroscopic currents from patches expressing (left to right) F401L, F401V, and F401A channels were recorded as in Fig. 1. The voltage ranges for the three families were as follows (step increment in parentheses): −85 to +25 mV (10 mV) for F401L, −80 to +60 mV (10 mV) for F401V, and −80 to +160 mV (20 mV) for F401A. Note the faster time scale for the F401A family used to resolve its kinetic features. (B) Relative conductance versus voltage relationships for the three mutants are shown. For comparison, fits to G(V) curves for the wt and F401I channels are reproduced from Fig. 1. F401L and F401V data are plotted as means ± SEM of eight and nine families, respectively. Averaged G(V) data were fitted with the fourth power of a Boltzmann function, as previously described, giving apparent z of 4.25 and 2.52 e0, and V1/2 of −69.7 and −53.1 mV for F401L and F401V, respectively. For the F401A experiment shown (representative of 11 patches), G(V) was measured either as the chord conductance assuming the reversal potential of −80 mV (denoted “pulse”), or as the isochronal tail current amplitude (denoted “tail”). The characteristic failure of the steady state conductance to reach a maximum within the attainable voltage range prevented meaningful normalization of the F401A G(V).
Mentions: Because of the striking effects of the F401I substitution, we substituted other amino acids for the phenylalanine at 401 to investigate the role of side chain structure on gating. We introduced individually three progressively smaller aliphatic amino acids leucine, valine, and alanine at that site. Fig. 5 A shows representative current families from these channels on different time scales to bring out the distinctive kinetic features of each channel type. In Fig. 5 B, the range of change induced by these mutations in the steady state voltage dependence of the relative open probability is shown. For comparison, previously described fits of a fourth power of the Boltzmann function to the wt and F401I data are also included. The F401V G(V) relationship is shallower than that of the wt, and similar in slope (zapp = 2.5) to the F401I mutant. However, the V1/2 in F401V is positively shifted by ∼5 mV compared with F401I. Steady state activation of the F401A mutant is the shallowest (zapp < 0.5 e0); in fact, the G(V) relationship fails to reach saturation at voltages in excess of +150 mV in five patches, and is therefore displayed on a dimensionless y axis. Unexpectedly, introduction at position 401 of a leucine, an amino acid chemically most similar to the isoleucine, carried nearly opposite consequences compared with the Sh5 replica mutation F401I. The F401L mutant has a G(V) relation as steep as that of the wt (zapp = 4.25) but with the midpoint of the activating transition shifted negatively (V1/2 = −69.7 mV), the only mutant in this study to do so.

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