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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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Model description of the conductance–voltage relationships. Experimental data for wt, F401L, and F401A are shown plotted as in Fig. 1 and Fig. 5. Simulated G(V) relations were generated from current families taken every 2 mV. The resultant curves were then shifted negatively by 6 (wt and F401L) or 7 (F401A) mV and shown superimposed as lines. The predicted G(V) curves from pulse and tail (see methods) for F401A were normalized to match the maximum relative conductance of the data.
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Figure 18: Model description of the conductance–voltage relationships. Experimental data for wt, F401L, and F401A are shown plotted as in Fig. 1 and Fig. 5. Simulated G(V) relations were generated from current families taken every 2 mV. The resultant curves were then shifted negatively by 6 (wt and F401L) or 7 (F401A) mV and shown superimposed as lines. The predicted G(V) curves from pulse and tail (see methods) for F401A were normalized to match the maximum relative conductance of the data.

Mentions: In evaluating model predictions for the steady state open probability vs. voltage, we took notice of the observed differences in the voltage positions of G(V) curves obtained using wt cell-attached and excised patches. The former tended to be shifted positively by ∼6 mV (data not shown). When displaying the G(V) curves for wt, F401L, and F401A with their respective models in Fig. 18, we similarly offset the simulated curves by between −6 and −7 mV to compare them with the experimental results. With this correction, both the steepness and the midpoint of the voltage dependence for wt and F401L channels were well described by the model. As earlier, the shallowness of the G(V) relation in the F401A mutant precludes us from observing saturation of the open probability within the attainable voltage range. Therefore we cannot meaningfully normalize G(V) data from different patches for direct comparison. Instead, G(V) relations from a representative F401A patch obtained by two means [isochronal tail G(V) and pulse G(V)] are shown in Fig. 18. Model traces for F401A were analyzed in the analogous manner, and the resulting G(V) curves are shown scaled to match F401A curves at +100 mV after a −6-mV shift along the x axis. The model, which postulates that the extremely shallow G(V) curve results from a destabilization of the open state by greatly speeding the initial closing transition, is qualitatively supported by these data.


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)

Model description of the conductance–voltage relationships. Experimental data for wt, F401L, and F401A are shown plotted as in Fig. 1 and Fig. 5. Simulated G(V) relations were generated from current families taken every 2 mV. The resultant curves were then shifted negatively by 6 (wt and F401L) or 7 (F401A) mV and shown superimposed as lines. The predicted G(V) curves from pulse and tail (see methods) for F401A were normalized to match the maximum relative conductance of the data.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 18: Model description of the conductance–voltage relationships. Experimental data for wt, F401L, and F401A are shown plotted as in Fig. 1 and Fig. 5. Simulated G(V) relations were generated from current families taken every 2 mV. The resultant curves were then shifted negatively by 6 (wt and F401L) or 7 (F401A) mV and shown superimposed as lines. The predicted G(V) curves from pulse and tail (see methods) for F401A were normalized to match the maximum relative conductance of the data.
Mentions: In evaluating model predictions for the steady state open probability vs. voltage, we took notice of the observed differences in the voltage positions of G(V) curves obtained using wt cell-attached and excised patches. The former tended to be shifted positively by ∼6 mV (data not shown). When displaying the G(V) curves for wt, F401L, and F401A with their respective models in Fig. 18, we similarly offset the simulated curves by between −6 and −7 mV to compare them with the experimental results. With this correction, both the steepness and the midpoint of the voltage dependence for wt and F401L channels were well described by the model. As earlier, the shallowness of the G(V) relation in the F401A mutant precludes us from observing saturation of the open probability within the attainable voltage range. Therefore we cannot meaningfully normalize G(V) data from different patches for direct comparison. Instead, G(V) relations from a representative F401A patch obtained by two means [isochronal tail G(V) and pulse G(V)] are shown in Fig. 18. Model traces for F401A were analyzed in the analogous manner, and the resulting G(V) curves are shown scaled to match F401A curves at +100 mV after a −6-mV shift along the x axis. The model, which postulates that the extremely shallow G(V) curve results from a destabilization of the open state by greatly speeding the initial closing transition, is qualitatively supported by these data.

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