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Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

Islas LD, Sigworth FJ - J. Gen. Physiol. (1999)

Bottom Line: We find that Shab has a relatively small gating charge, approximately 7.5 e(o).Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1.Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

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Limiting-slope measurements of Shab channels expressed in Sf9 cells. (A) Recordings of single channel openings at the indicated membrane potential in a cell-attached macro patch containing N = 230 channels, as estimated from noise analysis at 60 mV. The dotted line represents the start of the depolarization to the given voltage, from a holding potential of −90 mV. Some openings to the subconductance state are indicated as S and openings to the fully open conductance as F. (B) Time dependent (averaged) NP values obtained from the idealization of 300–400 traces such as those in A, in which only full openings were counted. (C) The complete activation curve for two patches obtained after combination of the single channel data with macroscopic [G(V)] data. The continuous curve is a fourth-power Boltzmann function with total gating charge of 7.5 eo. (•) The occupancy of the subconductance state at negative voltages, fitted with an exponential function (solid line) with a charge of 2.2 eo. (D) From the data in C, for the fully open state, the effective charge is plotted as a function of P. The continuous curve is the prediction from a fourth-power Boltzmann function with a charge of 7.5 eo. (E) The same data plotted against voltage (open symbols). The filled symbols represent the function qT [1 −Q̂V] derived from gating currents, with qT = 7.5 eo.
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Figure 9: Limiting-slope measurements of Shab channels expressed in Sf9 cells. (A) Recordings of single channel openings at the indicated membrane potential in a cell-attached macro patch containing N = 230 channels, as estimated from noise analysis at 60 mV. The dotted line represents the start of the depolarization to the given voltage, from a holding potential of −90 mV. Some openings to the subconductance state are indicated as S and openings to the fully open conductance as F. (B) Time dependent (averaged) NP values obtained from the idealization of 300–400 traces such as those in A, in which only full openings were counted. (C) The complete activation curve for two patches obtained after combination of the single channel data with macroscopic [G(V)] data. The continuous curve is a fourth-power Boltzmann function with total gating charge of 7.5 eo. (•) The occupancy of the subconductance state at negative voltages, fitted with an exponential function (solid line) with a charge of 2.2 eo. (D) From the data in C, for the fully open state, the effective charge is plotted as a function of P. The continuous curve is the prediction from a fourth-power Boltzmann function with a charge of 7.5 eo. (E) The same data plotted against voltage (open symbols). The filled symbols represent the function qT [1 −Q̂V] derived from gating currents, with qT = 7.5 eo.

Mentions: Even ignoring the sublevels, activation of Shab channels is not as voltage dependent as that of Kv2.1. At a holding potential of −90 mV it is possible to observe channel openings, and NP for the fully open state is seen to change only 10-fold for 10-mV depolarizations (Fig. 9A and Fig. B); this is about half the sensitivity seen for the other channel types. In constructing idealized traces, we took care not to confuse overlapping sublevel currents with open-state events; such overlapping events were very rare except at relatively depolarized voltages such as −70 mV. The resulting apparent charge estimate, based on P values between 10−5 and 10−3 was ql = 7.1 ± 0.3 eo (n = 2; Fig. 9 C). Plotting qs against P, the asymptotic value appears to be 7.5 eo (Fig. 9 D). Similarly, low apparent charge values were seen in recordings from Shab channels in oocytes, where ql = 7.5 ± 0.3 eo (n = 3) when evaluated over the range of P values from 10−6 to 10−4. Whole-cell recordings of gating currents in Sf9 cells allow the comparison of macroscopic charge movements with the voltage dependence of qs. The quantity superimposes fairly well on qs values with qT = 7.5 eo (Fig. 9 E) and demonstrates that very little charge movement occurs at voltages negative to −90 mV. We therefore take 7.5 e0 to be the estimate for the total charge in this channel.


Voltage sensitivity and gating charge in Shaker and Shab family potassium channels.

Islas LD, Sigworth FJ - J. Gen. Physiol. (1999)

Limiting-slope measurements of Shab channels expressed in Sf9 cells. (A) Recordings of single channel openings at the indicated membrane potential in a cell-attached macro patch containing N = 230 channels, as estimated from noise analysis at 60 mV. The dotted line represents the start of the depolarization to the given voltage, from a holding potential of −90 mV. Some openings to the subconductance state are indicated as S and openings to the fully open conductance as F. (B) Time dependent (averaged) NP values obtained from the idealization of 300–400 traces such as those in A, in which only full openings were counted. (C) The complete activation curve for two patches obtained after combination of the single channel data with macroscopic [G(V)] data. The continuous curve is a fourth-power Boltzmann function with total gating charge of 7.5 eo. (•) The occupancy of the subconductance state at negative voltages, fitted with an exponential function (solid line) with a charge of 2.2 eo. (D) From the data in C, for the fully open state, the effective charge is plotted as a function of P. The continuous curve is the prediction from a fourth-power Boltzmann function with a charge of 7.5 eo. (E) The same data plotted against voltage (open symbols). The filled symbols represent the function qT [1 −Q̂V] derived from gating currents, with qT = 7.5 eo.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230542&req=5

Figure 9: Limiting-slope measurements of Shab channels expressed in Sf9 cells. (A) Recordings of single channel openings at the indicated membrane potential in a cell-attached macro patch containing N = 230 channels, as estimated from noise analysis at 60 mV. The dotted line represents the start of the depolarization to the given voltage, from a holding potential of −90 mV. Some openings to the subconductance state are indicated as S and openings to the fully open conductance as F. (B) Time dependent (averaged) NP values obtained from the idealization of 300–400 traces such as those in A, in which only full openings were counted. (C) The complete activation curve for two patches obtained after combination of the single channel data with macroscopic [G(V)] data. The continuous curve is a fourth-power Boltzmann function with total gating charge of 7.5 eo. (•) The occupancy of the subconductance state at negative voltages, fitted with an exponential function (solid line) with a charge of 2.2 eo. (D) From the data in C, for the fully open state, the effective charge is plotted as a function of P. The continuous curve is the prediction from a fourth-power Boltzmann function with a charge of 7.5 eo. (E) The same data plotted against voltage (open symbols). The filled symbols represent the function qT [1 −Q̂V] derived from gating currents, with qT = 7.5 eo.
Mentions: Even ignoring the sublevels, activation of Shab channels is not as voltage dependent as that of Kv2.1. At a holding potential of −90 mV it is possible to observe channel openings, and NP for the fully open state is seen to change only 10-fold for 10-mV depolarizations (Fig. 9A and Fig. B); this is about half the sensitivity seen for the other channel types. In constructing idealized traces, we took care not to confuse overlapping sublevel currents with open-state events; such overlapping events were very rare except at relatively depolarized voltages such as −70 mV. The resulting apparent charge estimate, based on P values between 10−5 and 10−3 was ql = 7.1 ± 0.3 eo (n = 2; Fig. 9 C). Plotting qs against P, the asymptotic value appears to be 7.5 eo (Fig. 9 D). Similarly, low apparent charge values were seen in recordings from Shab channels in oocytes, where ql = 7.5 ± 0.3 eo (n = 3) when evaluated over the range of P values from 10−6 to 10−4. Whole-cell recordings of gating currents in Sf9 cells allow the comparison of macroscopic charge movements with the voltage dependence of qs. The quantity superimposes fairly well on qs values with qT = 7.5 eo (Fig. 9 E) and demonstrates that very little charge movement occurs at voltages negative to −90 mV. We therefore take 7.5 e0 to be the estimate for the total charge in this channel.

Bottom Line: We find that Shab has a relatively small gating charge, approximately 7.5 e(o).Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1.Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
The members of the voltage-dependent potassium channel family subserve a variety of functions and are expected to have voltage sensors with different sensitivities. The Shaker channel of Drosophila, which underlies a transient potassium current, has a high voltage sensitivity that is conferred by a large gating charge movement, approximately 13 elementary charges. A Shaker subunit's primary voltage-sensing (S4) region has seven positively charged residues. The Shab channel and its homologue Kv2.1 both carry a delayed-rectifier current, and their subunits have only five positively charged residues in S4; they would be expected to have smaller gating-charge movements and voltage sensitivities. We have characterized the gating currents and single-channel behavior of Shab channels and have estimated the charge movement in Shaker, Shab, and their rat homologues Kv1.1 and Kv2.1 by measuring the voltage dependence of open probability at very negative voltages and comparing this with the charge-voltage relationships. We find that Shab has a relatively small gating charge, approximately 7.5 e(o). Surprisingly, the corresponding mammalian delayed rectifier Kv2.1, which has the same complement of charged residues in the S2, S3, and S4 segments, has a gating charge of 12.5 e(o), essentially equal to that of Shaker and Kv1.1. Evidence for very strong coupling between charge movement and channel opening is seen in two channel types, with the probability of voltage-independent channel openings measured to be below 10(-9) in Shaker and below 4 x 10(-8) in Kv2.1.

Show MeSH
Related in: MedlinePlus