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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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Macroscopic activation properties of Shaker-related potassium channels. (A) Current traces in cell-attached patches in response to depolarizing voltage steps. The holding potential is −80 mV in each case except −90 for Shab. The voltage range for the depolarizations is as follows: Shaker: −58 to 26 mV in 4-mV steps; Kv1.1: −65 to 55 mV in 10-mV steps; Kv2.1: −50 to 40 mV in 5-mV steps; Shab: −70 to 50 mV in 10-mV steps. In each case, the standard pipette solution was used, which contained 60 mM K+. Recordings are from oocyte patches, except for Shab, which is from an Sf9 cell patch. Increased noise in the largest Kv2.1 currents presumably comes from internal Mg2+ block of these channels (Lopatin and Nichols 1994). (B) Voltage dependence of activation obtained from tail currents. The tail current amplitude was taken to be proportional to the open probability at the end of the voltage pulse. Values were normalized to the maximum P obtained from nonstationary noise analysis at 60–70 mV. The Pmax values are 0.79 for Shaker and 0.82 for Kv1.1. The P(V) relationship was fitted to the fourth power Boltzmann function:PV=Pmax11+e−qkTV−V04,where V is the voltage in millivolts, q is the charge per subunit in (eo), Vo is the half-activation voltage, and kT has its usual meaning. The fitted values are: Shaker, Vo = −27.5 mV and q = 2.88 eo. Kv1.1, Vo = −33.4 mV and q = 1.92 eo. (C) Voltage dependence of activation for Kv2.1 and Shab channels. Pmax values are: Kv2.1, 0.69; Shab, 0.76. The fitted parameters are: Kv2.1, Vo = −22.4 mV and q = 2.53 eo. Shab, Vo = −44.9 mV and q = 1.56 eo. (D) Voltage dependence of activation and deactivation time constants τ, and (E) delay of activation δ. The values at voltages above 50 mV, or 0 mV in the case of Kv1.1, were fitted to the functions: τ(V) = τ(0)exp(Vqτ/kT) and δ(V) = δ(0)exp(Vqδ/kT), where qτ and qδ are partial charges associated with the time constant and the delay, respectively. Fitted values are as follows: Kv2.1 (▵): qτ = 0.24 eo, qδ = 0.62 eo; Shab (○): qτ = 0.30 eo, qδ = 0.7 eo; Shaker (□): qτ = 0.51 eo, qδ = 0.22 eo; and Kv1.1 (⋄): qτ = 0.37 eo, qδ = 0.28 eo. The symbols with a dot indicate deactivation time constants derived from exponential fits of tail currents at the indicated potentials for Kv2.1 and Shab. The effective charge qδ in the most negative potential region is Kv2.1 = −0.32 eo; Shab = −0.43 eo.
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Figure 2: Macroscopic activation properties of Shaker-related potassium channels. (A) Current traces in cell-attached patches in response to depolarizing voltage steps. The holding potential is −80 mV in each case except −90 for Shab. The voltage range for the depolarizations is as follows: Shaker: −58 to 26 mV in 4-mV steps; Kv1.1: −65 to 55 mV in 10-mV steps; Kv2.1: −50 to 40 mV in 5-mV steps; Shab: −70 to 50 mV in 10-mV steps. In each case, the standard pipette solution was used, which contained 60 mM K+. Recordings are from oocyte patches, except for Shab, which is from an Sf9 cell patch. Increased noise in the largest Kv2.1 currents presumably comes from internal Mg2+ block of these channels (Lopatin and Nichols 1994). (B) Voltage dependence of activation obtained from tail currents. The tail current amplitude was taken to be proportional to the open probability at the end of the voltage pulse. Values were normalized to the maximum P obtained from nonstationary noise analysis at 60–70 mV. The Pmax values are 0.79 for Shaker and 0.82 for Kv1.1. The P(V) relationship was fitted to the fourth power Boltzmann function:PV=Pmax11+e−qkTV−V04,where V is the voltage in millivolts, q is the charge per subunit in (eo), Vo is the half-activation voltage, and kT has its usual meaning. The fitted values are: Shaker, Vo = −27.5 mV and q = 2.88 eo. Kv1.1, Vo = −33.4 mV and q = 1.92 eo. (C) Voltage dependence of activation for Kv2.1 and Shab channels. Pmax values are: Kv2.1, 0.69; Shab, 0.76. The fitted parameters are: Kv2.1, Vo = −22.4 mV and q = 2.53 eo. Shab, Vo = −44.9 mV and q = 1.56 eo. (D) Voltage dependence of activation and deactivation time constants τ, and (E) delay of activation δ. The values at voltages above 50 mV, or 0 mV in the case of Kv1.1, were fitted to the functions: τ(V) = τ(0)exp(Vqτ/kT) and δ(V) = δ(0)exp(Vqδ/kT), where qτ and qδ are partial charges associated with the time constant and the delay, respectively. Fitted values are as follows: Kv2.1 (▵): qτ = 0.24 eo, qδ = 0.62 eo; Shab (○): qτ = 0.30 eo, qδ = 0.7 eo; Shaker (□): qτ = 0.51 eo, qδ = 0.22 eo; and Kv1.1 (⋄): qτ = 0.37 eo, qδ = 0.28 eo. The symbols with a dot indicate deactivation time constants derived from exponential fits of tail currents at the indicated potentials for Kv2.1 and Shab. The effective charge qδ in the most negative potential region is Kv2.1 = −0.32 eo; Shab = −0.43 eo.

Mentions: To extend the previous studies of Shab channel currents (Tsunoda and Salkoff 1995), we first compare Shab channel properties with those of the other channel types. For this study, we have used a Shaker construct having a deletion at the NH2 terminus to remove fast inactivation, to simplify the determination of steady state open probability. For Kv2.1, we have used a mutant, DRK1-Δ7, that was engineered to bind the pore blocker Agitoxin-1 with high affinity (Aggarwal and MacKinnon 1996; Gross et al. 1994), making possible the measurement of gating currents. Macroscopic currents were obtained in cell-attached patch recordings from mRNA-injected oocytes or in whole-cell recordings from recombinant baculovirus-infected Sf9 cells in the case of Shab channels. Representative current traces are shown in Fig. 2 A. The channel open probabilities P for each channel type approach limiting values between 0.7 and 0.9 for large depolarizations. The voltage-dependence of P for Shaker and Kv1.1 (Fig. 2 B) can be described well by the fourth power of a Boltzmann function, as would be predicted by a simple model incorporating four independently acting voltage sensors (Zagotta et al. 1994). The gating charges estimated from these fits are 11.5 and 7.7 eo for Shaker and Kv1.1, respectively; it should be emphasized, however, that such gating-charge estimates are strongly dependent on the particular model used. The corresponding fit to the P–V relationship for Shab channels yields an apparent charge of 6.3 eo, while for Kv2.1 it yields 10.1 eo (Fig. 2 C).


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

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

Macroscopic activation properties of Shaker-related potassium channels. (A) Current traces in cell-attached patches in response to depolarizing voltage steps. The holding potential is −80 mV in each case except −90 for Shab. The voltage range for the depolarizations is as follows: Shaker: −58 to 26 mV in 4-mV steps; Kv1.1: −65 to 55 mV in 10-mV steps; Kv2.1: −50 to 40 mV in 5-mV steps; Shab: −70 to 50 mV in 10-mV steps. In each case, the standard pipette solution was used, which contained 60 mM K+. Recordings are from oocyte patches, except for Shab, which is from an Sf9 cell patch. Increased noise in the largest Kv2.1 currents presumably comes from internal Mg2+ block of these channels (Lopatin and Nichols 1994). (B) Voltage dependence of activation obtained from tail currents. The tail current amplitude was taken to be proportional to the open probability at the end of the voltage pulse. Values were normalized to the maximum P obtained from nonstationary noise analysis at 60–70 mV. The Pmax values are 0.79 for Shaker and 0.82 for Kv1.1. The P(V) relationship was fitted to the fourth power Boltzmann function:PV=Pmax11+e−qkTV−V04,where V is the voltage in millivolts, q is the charge per subunit in (eo), Vo is the half-activation voltage, and kT has its usual meaning. The fitted values are: Shaker, Vo = −27.5 mV and q = 2.88 eo. Kv1.1, Vo = −33.4 mV and q = 1.92 eo. (C) Voltage dependence of activation for Kv2.1 and Shab channels. Pmax values are: Kv2.1, 0.69; Shab, 0.76. The fitted parameters are: Kv2.1, Vo = −22.4 mV and q = 2.53 eo. Shab, Vo = −44.9 mV and q = 1.56 eo. (D) Voltage dependence of activation and deactivation time constants τ, and (E) delay of activation δ. The values at voltages above 50 mV, or 0 mV in the case of Kv1.1, were fitted to the functions: τ(V) = τ(0)exp(Vqτ/kT) and δ(V) = δ(0)exp(Vqδ/kT), where qτ and qδ are partial charges associated with the time constant and the delay, respectively. Fitted values are as follows: Kv2.1 (▵): qτ = 0.24 eo, qδ = 0.62 eo; Shab (○): qτ = 0.30 eo, qδ = 0.7 eo; Shaker (□): qτ = 0.51 eo, qδ = 0.22 eo; and Kv1.1 (⋄): qτ = 0.37 eo, qδ = 0.28 eo. The symbols with a dot indicate deactivation time constants derived from exponential fits of tail currents at the indicated potentials for Kv2.1 and Shab. The effective charge qδ in the most negative potential region is Kv2.1 = −0.32 eo; Shab = −0.43 eo.
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Figure 2: Macroscopic activation properties of Shaker-related potassium channels. (A) Current traces in cell-attached patches in response to depolarizing voltage steps. The holding potential is −80 mV in each case except −90 for Shab. The voltage range for the depolarizations is as follows: Shaker: −58 to 26 mV in 4-mV steps; Kv1.1: −65 to 55 mV in 10-mV steps; Kv2.1: −50 to 40 mV in 5-mV steps; Shab: −70 to 50 mV in 10-mV steps. In each case, the standard pipette solution was used, which contained 60 mM K+. Recordings are from oocyte patches, except for Shab, which is from an Sf9 cell patch. Increased noise in the largest Kv2.1 currents presumably comes from internal Mg2+ block of these channels (Lopatin and Nichols 1994). (B) Voltage dependence of activation obtained from tail currents. The tail current amplitude was taken to be proportional to the open probability at the end of the voltage pulse. Values were normalized to the maximum P obtained from nonstationary noise analysis at 60–70 mV. The Pmax values are 0.79 for Shaker and 0.82 for Kv1.1. The P(V) relationship was fitted to the fourth power Boltzmann function:PV=Pmax11+e−qkTV−V04,where V is the voltage in millivolts, q is the charge per subunit in (eo), Vo is the half-activation voltage, and kT has its usual meaning. The fitted values are: Shaker, Vo = −27.5 mV and q = 2.88 eo. Kv1.1, Vo = −33.4 mV and q = 1.92 eo. (C) Voltage dependence of activation for Kv2.1 and Shab channels. Pmax values are: Kv2.1, 0.69; Shab, 0.76. The fitted parameters are: Kv2.1, Vo = −22.4 mV and q = 2.53 eo. Shab, Vo = −44.9 mV and q = 1.56 eo. (D) Voltage dependence of activation and deactivation time constants τ, and (E) delay of activation δ. The values at voltages above 50 mV, or 0 mV in the case of Kv1.1, were fitted to the functions: τ(V) = τ(0)exp(Vqτ/kT) and δ(V) = δ(0)exp(Vqδ/kT), where qτ and qδ are partial charges associated with the time constant and the delay, respectively. Fitted values are as follows: Kv2.1 (▵): qτ = 0.24 eo, qδ = 0.62 eo; Shab (○): qτ = 0.30 eo, qδ = 0.7 eo; Shaker (□): qτ = 0.51 eo, qδ = 0.22 eo; and Kv1.1 (⋄): qτ = 0.37 eo, qδ = 0.28 eo. The symbols with a dot indicate deactivation time constants derived from exponential fits of tail currents at the indicated potentials for Kv2.1 and Shab. The effective charge qδ in the most negative potential region is Kv2.1 = −0.32 eo; Shab = −0.43 eo.
Mentions: To extend the previous studies of Shab channel currents (Tsunoda and Salkoff 1995), we first compare Shab channel properties with those of the other channel types. For this study, we have used a Shaker construct having a deletion at the NH2 terminus to remove fast inactivation, to simplify the determination of steady state open probability. For Kv2.1, we have used a mutant, DRK1-Δ7, that was engineered to bind the pore blocker Agitoxin-1 with high affinity (Aggarwal and MacKinnon 1996; Gross et al. 1994), making possible the measurement of gating currents. Macroscopic currents were obtained in cell-attached patch recordings from mRNA-injected oocytes or in whole-cell recordings from recombinant baculovirus-infected Sf9 cells in the case of Shab channels. Representative current traces are shown in Fig. 2 A. The channel open probabilities P for each channel type approach limiting values between 0.7 and 0.9 for large depolarizations. The voltage-dependence of P for Shaker and Kv1.1 (Fig. 2 B) can be described well by the fourth power of a Boltzmann function, as would be predicted by a simple model incorporating four independently acting voltage sensors (Zagotta et al. 1994). The gating charges estimated from these fits are 11.5 and 7.7 eo for Shaker and Kv1.1, respectively; it should be emphasized, however, that such gating-charge estimates are strongly dependent on the particular model used. The corresponding fit to the P–V relationship for Shab channels yields an apparent charge of 6.3 eo, while for Kv2.1 it yields 10.1 eo (Fig. 2 C).

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