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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 measurement of the charge in Kv2.1 channels. (A) Single Kv2.1 channel events recorded at the indicated potentials after holding the patch for 400 ms at −80 mV. The data sweeps are not consecutive. Fluctuation analysis from macroscopic currents at 70 mV yielded an estimate of N = 1,500 channels. (B) Time course of NP obtained from the same patch in A; each trace represents the average of 300–400 idealized sweeps. (C) Open probabilities from six experiments transformed according to . Superimposed in the data are two solid curves showing the fourth power of a Boltzmann function scaled to a total charge of 12 and 13 eo. The dotted curve is a simple Boltzmann function with 13 eo. The inset depicts one experiment's voltage dependence showing the extent of the P values explored. (D) Voltage dependence of the apparent gating charge (open symbols), compared with qT [1 − Q̂V], where Q̂Vis the normalized charge movement derived from gating currents and qT was 12.5 eo (filled symbols). The continuous curve is a Boltzmann function calculated with a charge of 12.5 eo.
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Figure 6: Limiting-slope measurement of the charge in Kv2.1 channels. (A) Single Kv2.1 channel events recorded at the indicated potentials after holding the patch for 400 ms at −80 mV. The data sweeps are not consecutive. Fluctuation analysis from macroscopic currents at 70 mV yielded an estimate of N = 1,500 channels. (B) Time course of NP obtained from the same patch in A; each trace represents the average of 300–400 idealized sweeps. (C) Open probabilities from six experiments transformed according to . Superimposed in the data are two solid curves showing the fourth power of a Boltzmann function scaled to a total charge of 12 and 13 eo. The dotted curve is a simple Boltzmann function with 13 eo. The inset depicts one experiment's voltage dependence showing the extent of the P values explored. (D) Voltage dependence of the apparent gating charge (open symbols), compared with qT [1 − Q̂V], where Q̂Vis the normalized charge movement derived from gating currents and qT was 12.5 eo (filled symbols). The continuous curve is a Boltzmann function calculated with a charge of 12.5 eo.

Mentions: Previous estimates of the gating charge of the rat Kv2.1 channel (Taglialatela and Stefani 1993) and the human homologue, hKv2.1 (Benndorf et al. 1994) were in the range 6–7 eo. These were obtained from model-dependent fitting, a methodology which often underestimates the gating charge. As with Shaker, we were able to record from patches containing hundreds of Kv2.1 channels in which we could measure macroscopic currents at depolarizing voltages and resolve single-channel openings at hyperpolarized voltages. Fig. 6 shows recordings from a patch containing ∼1,650 Kv2.1 channels.


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

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

Limiting-slope measurement of the charge in Kv2.1 channels. (A) Single Kv2.1 channel events recorded at the indicated potentials after holding the patch for 400 ms at −80 mV. The data sweeps are not consecutive. Fluctuation analysis from macroscopic currents at 70 mV yielded an estimate of N = 1,500 channels. (B) Time course of NP obtained from the same patch in A; each trace represents the average of 300–400 idealized sweeps. (C) Open probabilities from six experiments transformed according to . Superimposed in the data are two solid curves showing the fourth power of a Boltzmann function scaled to a total charge of 12 and 13 eo. The dotted curve is a simple Boltzmann function with 13 eo. The inset depicts one experiment's voltage dependence showing the extent of the P values explored. (D) Voltage dependence of the apparent gating charge (open symbols), compared with qT [1 − Q̂V], where Q̂Vis the normalized charge movement derived from gating currents and qT was 12.5 eo (filled symbols). The continuous curve is a Boltzmann function calculated with a charge of 12.5 eo.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230542&req=5

Figure 6: Limiting-slope measurement of the charge in Kv2.1 channels. (A) Single Kv2.1 channel events recorded at the indicated potentials after holding the patch for 400 ms at −80 mV. The data sweeps are not consecutive. Fluctuation analysis from macroscopic currents at 70 mV yielded an estimate of N = 1,500 channels. (B) Time course of NP obtained from the same patch in A; each trace represents the average of 300–400 idealized sweeps. (C) Open probabilities from six experiments transformed according to . Superimposed in the data are two solid curves showing the fourth power of a Boltzmann function scaled to a total charge of 12 and 13 eo. The dotted curve is a simple Boltzmann function with 13 eo. The inset depicts one experiment's voltage dependence showing the extent of the P values explored. (D) Voltage dependence of the apparent gating charge (open symbols), compared with qT [1 − Q̂V], where Q̂Vis the normalized charge movement derived from gating currents and qT was 12.5 eo (filled symbols). The continuous curve is a Boltzmann function calculated with a charge of 12.5 eo.
Mentions: Previous estimates of the gating charge of the rat Kv2.1 channel (Taglialatela and Stefani 1993) and the human homologue, hKv2.1 (Benndorf et al. 1994) were in the range 6–7 eo. These were obtained from model-dependent fitting, a methodology which often underestimates the gating charge. As with Shaker, we were able to record from patches containing hundreds of Kv2.1 channels in which we could measure macroscopic currents at depolarizing voltages and resolve single-channel openings at hyperpolarized voltages. Fig. 6 shows recordings from a patch containing ∼1,650 Kv2.1 channels.

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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Related in: MedlinePlus