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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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Calculation of the apparent gating charge content from the limiting slope in Shaker channels. (A) Representative traces of channel activity in a multichannel patch at the indicated membrane potentials. This patch contained 2,250 channels, as estimated from fluctuation analysis at +70 mV. From a holding potential of −80 mV, pulses of duration 300–400 ms to the indicated potential were applied once per second. (B) The time course of the open probability, reconstructed from sets of 200–300 sweeps at each potential. Note the very steep voltage dependence of steady state open probability (∼10-fold/5 mV). During the total of 1,500 sweeps, no channel openings were detected at −80 mV. (C) The P(V) relationships from three experiments are shown with fits (lines) to exponential functions () over the range P = 10−7 to 10−3. The apparent gating charges ql from these fits are 12.9, 12.4, and 12.8 eo. (D) The logarithmic slope qs () is plotted as a function of P. The solid curve is the predicted relationship for a fourth-power Boltzmann function having charge 3.25 eo per subunit, yielding a total charge 13 eo. The dashed curve is the relationship for a single Boltzmann function with charge 13 eo. Different symbols indicate individual patches. (E) Values of qs plotted against voltage. (•) The macroscopic charge movement 1 − Q̂V of the W434F mutant recorded under the same conditions in a patch from a different oocyte; it has been scaled to a total charge qT = 13 eo.
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Figure 4: Calculation of the apparent gating charge content from the limiting slope in Shaker channels. (A) Representative traces of channel activity in a multichannel patch at the indicated membrane potentials. This patch contained 2,250 channels, as estimated from fluctuation analysis at +70 mV. From a holding potential of −80 mV, pulses of duration 300–400 ms to the indicated potential were applied once per second. (B) The time course of the open probability, reconstructed from sets of 200–300 sweeps at each potential. Note the very steep voltage dependence of steady state open probability (∼10-fold/5 mV). During the total of 1,500 sweeps, no channel openings were detected at −80 mV. (C) The P(V) relationships from three experiments are shown with fits (lines) to exponential functions () over the range P = 10−7 to 10−3. The apparent gating charges ql from these fits are 12.9, 12.4, and 12.8 eo. (D) The logarithmic slope qs () is plotted as a function of P. The solid curve is the predicted relationship for a fourth-power Boltzmann function having charge 3.25 eo per subunit, yielding a total charge 13 eo. The dashed curve is the relationship for a single Boltzmann function with charge 13 eo. Different symbols indicate individual patches. (E) Values of qs plotted against voltage. (•) The macroscopic charge movement 1 − Q̂V of the W434F mutant recorded under the same conditions in a patch from a different oocyte; it has been scaled to a total charge qT = 13 eo.

Mentions: With the high expression obtained with Shaker in oocytes, it was possible to record from patches containing hundreds or thousands of channels. The number N of channels in a patch was estimated by nonstationary fluctuation analysis, using depolarizations to +70 mV. Subsequent recordings using small depolarizing pulses allowed NP to be estimated from the statistics of single-channel events in the same patch. Recordings from a representative patch containing 2,250 channels are shown in Fig. 4 A. The channel openings observed during the small depolarizations were identified as Shaker channels based on their conductance and kinetics.


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

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

Calculation of the apparent gating charge content from the limiting slope in Shaker channels. (A) Representative traces of channel activity in a multichannel patch at the indicated membrane potentials. This patch contained 2,250 channels, as estimated from fluctuation analysis at +70 mV. From a holding potential of −80 mV, pulses of duration 300–400 ms to the indicated potential were applied once per second. (B) The time course of the open probability, reconstructed from sets of 200–300 sweeps at each potential. Note the very steep voltage dependence of steady state open probability (∼10-fold/5 mV). During the total of 1,500 sweeps, no channel openings were detected at −80 mV. (C) The P(V) relationships from three experiments are shown with fits (lines) to exponential functions () over the range P = 10−7 to 10−3. The apparent gating charges ql from these fits are 12.9, 12.4, and 12.8 eo. (D) The logarithmic slope qs () is plotted as a function of P. The solid curve is the predicted relationship for a fourth-power Boltzmann function having charge 3.25 eo per subunit, yielding a total charge 13 eo. The dashed curve is the relationship for a single Boltzmann function with charge 13 eo. Different symbols indicate individual patches. (E) Values of qs plotted against voltage. (•) The macroscopic charge movement 1 − Q̂V of the W434F mutant recorded under the same conditions in a patch from a different oocyte; it has been scaled to a total charge qT = 13 eo.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230542&req=5

Figure 4: Calculation of the apparent gating charge content from the limiting slope in Shaker channels. (A) Representative traces of channel activity in a multichannel patch at the indicated membrane potentials. This patch contained 2,250 channels, as estimated from fluctuation analysis at +70 mV. From a holding potential of −80 mV, pulses of duration 300–400 ms to the indicated potential were applied once per second. (B) The time course of the open probability, reconstructed from sets of 200–300 sweeps at each potential. Note the very steep voltage dependence of steady state open probability (∼10-fold/5 mV). During the total of 1,500 sweeps, no channel openings were detected at −80 mV. (C) The P(V) relationships from three experiments are shown with fits (lines) to exponential functions () over the range P = 10−7 to 10−3. The apparent gating charges ql from these fits are 12.9, 12.4, and 12.8 eo. (D) The logarithmic slope qs () is plotted as a function of P. The solid curve is the predicted relationship for a fourth-power Boltzmann function having charge 3.25 eo per subunit, yielding a total charge 13 eo. The dashed curve is the relationship for a single Boltzmann function with charge 13 eo. Different symbols indicate individual patches. (E) Values of qs plotted against voltage. (•) The macroscopic charge movement 1 − Q̂V of the W434F mutant recorded under the same conditions in a patch from a different oocyte; it has been scaled to a total charge qT = 13 eo.
Mentions: With the high expression obtained with Shaker in oocytes, it was possible to record from patches containing hundreds or thousands of channels. The number N of channels in a patch was estimated by nonstationary fluctuation analysis, using depolarizations to +70 mV. Subsequent recordings using small depolarizing pulses allowed NP to be estimated from the statistics of single-channel events in the same patch. Recordings from a representative patch containing 2,250 channels are shown in Fig. 4 A. The channel openings observed during the small depolarizations were identified as Shaker channels based on their conductance and kinetics.

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