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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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Ruling out artifacts in the estimation of charge in Kv2.1 channels. (A) Macroscopic Kv2.1 currents in response to a double-pulse protocol. The second pulse voltage was fixed at 20 mV and the potential of the 400-ms prepulse was varied from −100 to −10 mV. (B) Steady state inactivation function from the data in A. Plotted is the ratio of the current at the end of the second pulse to the current without prepulse, as a function of prepulse potential. The continuous curve is the function:II0=A1+e−V−V0qkT+1−A,where A = 0.27 is the maximum relative inactivation; q = 5.2 eo and Vo = −30 mV. (C) Dwell-time distributions at negative voltages. Histograms in the left column are the open times and those in the right show the closed times. Superimposed are the maximum-likelihood fits to single and double exponential functions of the open and closed times, respectively. (D) Voltage dependence of the time constant of the long closed state and the mean burst duration. Plotted are mean values from four patches and the error bars show the standard deviation. The parameters of the fits (lines) are given in Table .
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Figure 7: Ruling out artifacts in the estimation of charge in Kv2.1 channels. (A) Macroscopic Kv2.1 currents in response to a double-pulse protocol. The second pulse voltage was fixed at 20 mV and the potential of the 400-ms prepulse was varied from −100 to −10 mV. (B) Steady state inactivation function from the data in A. Plotted is the ratio of the current at the end of the second pulse to the current without prepulse, as a function of prepulse potential. The continuous curve is the function:II0=A1+e−V−V0qkT+1−A,where A = 0.27 is the maximum relative inactivation; q = 5.2 eo and Vo = −30 mV. (C) Dwell-time distributions at negative voltages. Histograms in the left column are the open times and those in the right show the closed times. Superimposed are the maximum-likelihood fits to single and double exponential functions of the open and closed times, respectively. (D) Voltage dependence of the time constant of the long closed state and the mean burst duration. Plotted are mean values from four patches and the error bars show the standard deviation. The parameters of the fits (lines) are given in Table .

Mentions: It is unlikely that this estimate of total charge is in error due to inactivation or missed channel events. Inactivation in the Kv2.1 channels was measured with 500-ms prepulses (Fig. 7 A) and was found to be negligible at potentials below −50 mV (Fig. 7 B). Single-channel events at negative voltages show a single open-time component with a mean duration of 8 ms (Fig. 7 C), consistent with the presence of only one open state of the channel. The duration of bursts of openings, ∼15 ms (Fig. 7 D), is essentially voltage independent, always much greater than the detection dead-time of 120 μs; thus, a very small and voltage-independent fraction of events is expected to be missed in the evaluation of NP.


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

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

Ruling out artifacts in the estimation of charge in Kv2.1 channels. (A) Macroscopic Kv2.1 currents in response to a double-pulse protocol. The second pulse voltage was fixed at 20 mV and the potential of the 400-ms prepulse was varied from −100 to −10 mV. (B) Steady state inactivation function from the data in A. Plotted is the ratio of the current at the end of the second pulse to the current without prepulse, as a function of prepulse potential. The continuous curve is the function:II0=A1+e−V−V0qkT+1−A,where A = 0.27 is the maximum relative inactivation; q = 5.2 eo and Vo = −30 mV. (C) Dwell-time distributions at negative voltages. Histograms in the left column are the open times and those in the right show the closed times. Superimposed are the maximum-likelihood fits to single and double exponential functions of the open and closed times, respectively. (D) Voltage dependence of the time constant of the long closed state and the mean burst duration. Plotted are mean values from four patches and the error bars show the standard deviation. The parameters of the fits (lines) are given in Table .
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230542&req=5

Figure 7: Ruling out artifacts in the estimation of charge in Kv2.1 channels. (A) Macroscopic Kv2.1 currents in response to a double-pulse protocol. The second pulse voltage was fixed at 20 mV and the potential of the 400-ms prepulse was varied from −100 to −10 mV. (B) Steady state inactivation function from the data in A. Plotted is the ratio of the current at the end of the second pulse to the current without prepulse, as a function of prepulse potential. The continuous curve is the function:II0=A1+e−V−V0qkT+1−A,where A = 0.27 is the maximum relative inactivation; q = 5.2 eo and Vo = −30 mV. (C) Dwell-time distributions at negative voltages. Histograms in the left column are the open times and those in the right show the closed times. Superimposed are the maximum-likelihood fits to single and double exponential functions of the open and closed times, respectively. (D) Voltage dependence of the time constant of the long closed state and the mean burst duration. Plotted are mean values from four patches and the error bars show the standard deviation. The parameters of the fits (lines) are given in Table .
Mentions: It is unlikely that this estimate of total charge is in error due to inactivation or missed channel events. Inactivation in the Kv2.1 channels was measured with 500-ms prepulses (Fig. 7 A) and was found to be negligible at potentials below −50 mV (Fig. 7 B). Single-channel events at negative voltages show a single open-time component with a mean duration of 8 ms (Fig. 7 C), consistent with the presence of only one open state of the channel. The duration of bursts of openings, ∼15 ms (Fig. 7 D), is essentially voltage independent, always much greater than the detection dead-time of 120 μs; thus, a very small and voltage-independent fraction of events is expected to be missed in the evaluation of NP.

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