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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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Gating charge movement in the wild type and channels containing F401L and F401A mutations. Gating currents were recorded from nonconducting channels containing the indicated mutations using a cut-open oocyte voltage clamp, digitized at 20 kHz (42 kHz for wfF401A) and low-pass filtered at 10 kHz. Traces elicited by steps to the voltages indicated on the left, beginning from and returning to the holding potential of −100 mV, are shown staggered to facilitate the kinetic comparison. Chloride-free standard solutions were used (see methods).
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Figure 8: Gating charge movement in the wild type and channels containing F401L and F401A mutations. Gating currents were recorded from nonconducting channels containing the indicated mutations using a cut-open oocyte voltage clamp, digitized at 20 kHz (42 kHz for wfF401A) and low-pass filtered at 10 kHz. Traces elicited by steps to the voltages indicated on the left, beginning from and returning to the holding potential of −100 mV, are shown staggered to facilitate the kinetic comparison. Chloride-free standard solutions were used (see methods).

Mentions: Families of gating currents from the wf and the wfF401L and wfF401A mutants are shown in Fig. 8. The gating currents are shown superimposed and staggered to facilitate comparison of the development of kinetic features with changes in voltage. The wf ON gating currents (IgON) have a rising phase, appear at negative voltages, and show a slow decaying component in the voltage range where channels open. This latter component accelerates with further depolarizations. The overall time course of the IgON decay becomes faster in the order wf, wfF401L, and wfF401A, consistent with the faster time course of ionic current activation observed in the corresponding conducting species, although the F401A gating currents are accelerated to a lesser extent than the corresponding ionic currents. A prominent rising phase and slow decay appear in the wf OFF currents (IgOFF) at the voltages where there is a slow phase of the IgON decay, consistent with published observations from the cut-open oocyte clamp. (Perozo et al. 1992; Bezanilla et al. 1994; Stefani et al. 1994). The most notable change introduced by the wfF401L mutation is the profound slowing of the OFF gating charge return at voltages where the channel opens. In contrast, wfF401A all but abolishes the slowing of IgOFF. The correlation between faster OFF gating charge return and faster deactivation of ionic currents is more consistent with the slowing of OFF charge due to a stabilization of the open state rather than to channels entering a C-type inactivated state (see Chen et al. 1997).


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)

Gating charge movement in the wild type and channels containing F401L and F401A mutations. Gating currents were recorded from nonconducting channels containing the indicated mutations using a cut-open oocyte voltage clamp, digitized at 20 kHz (42 kHz for wfF401A) and low-pass filtered at 10 kHz. Traces elicited by steps to the voltages indicated on the left, beginning from and returning to the holding potential of −100 mV, are shown staggered to facilitate the kinetic comparison. Chloride-free standard solutions were used (see methods).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Gating charge movement in the wild type and channels containing F401L and F401A mutations. Gating currents were recorded from nonconducting channels containing the indicated mutations using a cut-open oocyte voltage clamp, digitized at 20 kHz (42 kHz for wfF401A) and low-pass filtered at 10 kHz. Traces elicited by steps to the voltages indicated on the left, beginning from and returning to the holding potential of −100 mV, are shown staggered to facilitate the kinetic comparison. Chloride-free standard solutions were used (see methods).
Mentions: Families of gating currents from the wf and the wfF401L and wfF401A mutants are shown in Fig. 8. The gating currents are shown superimposed and staggered to facilitate comparison of the development of kinetic features with changes in voltage. The wf ON gating currents (IgON) have a rising phase, appear at negative voltages, and show a slow decaying component in the voltage range where channels open. This latter component accelerates with further depolarizations. The overall time course of the IgON decay becomes faster in the order wf, wfF401L, and wfF401A, consistent with the faster time course of ionic current activation observed in the corresponding conducting species, although the F401A gating currents are accelerated to a lesser extent than the corresponding ionic currents. A prominent rising phase and slow decay appear in the wf OFF currents (IgOFF) at the voltages where there is a slow phase of the IgON decay, consistent with published observations from the cut-open oocyte clamp. (Perozo et al. 1992; Bezanilla et al. 1994; Stefani et al. 1994). The most notable change introduced by the wfF401L mutation is the profound slowing of the OFF gating charge return at voltages where the channel opens. In contrast, wfF401A all but abolishes the slowing of IgOFF. The correlation between faster OFF gating charge return and faster deactivation of ionic currents is more consistent with the slowing of OFF charge due to a stabilization of the open state rather than to channels entering a C-type inactivated state (see Chen et al. 1997).

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