Sukhareva M et al. (2003), Constitutive activation of the Shaker Kv channel.

XB-ART-4564

J Gen Physiol 2003 Nov 01;1225:541-56. doi: 10.1085/jgp.200308905.

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Constitutive activation of the Shaker Kv channel.

Sukhareva M , Hackos DH , Swartz KJ .

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In different types of K+ channels the primary activation gate is thought to reside near the intracellular entrance to the ion conduction pore. In the Shaker Kv channel the gate is closed at negative membrane voltages, but can be opened with membrane depolarization. In a previous study of the S6 activation gate in Shaker (Hackos, D.H., T.H. Chang, and K.J. Swartz. 2002. J. Gen. Physiol. 119:521-532.), we found that mutation of Pro 475 to Asp results in a channel that displays a large macroscopic conductance at negative membrane voltages, with only small increases in conductance with membrane depolarization. In the present study we explore the mechanism underlying this constitutively conducting phenotype using both macroscopic and single-channel recordings, and probes that interact with the voltage sensors or the intracellular entrance to the ion conduction pore. Our results suggest that constitutive conduction results from a dramatic perturbation of the closed-open equilibrium, enabling opening of the activation gate without voltage-sensor activation. This mechanism is discussed in the context of allosteric models for activation of Kv channels and what is known about the structure of this critical region in K+ channels.

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Species referenced: Xenopus laevis
Genes referenced: kcnb1 tbx2

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	Figure 1. . The intracellular gate region of K+ channels. (A) Cartoon of a voltage-gated K+ channel showing separate voltage-sensing and pore domains and an S6 activation gate toward the intracellular side of the pore. (B) Sequence alignment between Kv channels, KcsA, MthK and KirBac. Pink highlighting marks the conserved Gly residue that is proposed to serve as the gating hinge (Jiang et al., 2002a,b). Blue highlighting marks the position of P475 in Shaker, corresponding to P406 in Kv2.1, G229 in KvAP, A108 in KcsA, E92 in MthK, and G143 in KirBac. Green highlighting marks positions equivalent to V478 in Shaker, where Trp substitutions result in nonconducting channels (Hackos et al., 2002).
	Figure 2. . Constitutive conduction for the P475D mutant of the Shaker Kv channel. (A) Families of Agitoxin-sensitive ionic currents for oocytes expressing the wild-type or P475D mutant Shaker Kv channels. For wild-type, holding voltage was −90 mV, tail voltage was −60 mV, and depolarizations were from −60 to +20 mV in 10-mV steps. For P475D the holding voltage was −110 mV and membrane voltage was stepped to −150 mV for 200 ms before recording the traces shown. Tail voltage was −150 mV, and depolarizations were from −140 to +10 mV, in 10-mV increments. Charge carrier in the external solution was 50 mM K+ and endogenous K+ is ∼100 mM. Linear capacity and background conductances were identified and subtracted by blocking the Shaker channel with Agitoxin-2. Dashed line indicates the position of zero current. (B) Conductance (G)-voltage (V) relations for wild-type and P475D. Normalized tail current amplitudes are plotted versus the voltage of the preceding depolarization. Smooth curves are single Boltzmann fits to the data with parameters as follows: Wt, V50 = −25 mV, z = 3; P475D, V50 = −100 mV, z = 2.9.
	Figure 3. . Gating properties for different substitutions at P475 in the Shaker Kv channel. (A) Families of ionic currents for three mutant Shaker channels. For P475Q and P475N, cells were held at −100 mV and stepped to −150 mV for 200 ms before the traces shown, tail voltage was −150 mV, and depolarizations were from −150 to −30 mV for Q and to −10 mV for N (each in 10-mV steps). For P475H, holding voltage was −110 mV, tail voltage was −110 mV, and depolarizations were from −110 to −10 mV (10-mV steps). Linear capacity and background conductances were identified and subtracted by blocking the Shaker channel with Agitoxin-2. Horizontal dashed lines correspond to the zero current level. (B) G-V relations for Wt and four P475 mutants displaying constitutive conduction. Normalized tail current amplitudes are plotted versus voltage of the preceding depolarization. For P475D the external solution contained 50 mM KCl and for all others 50 mM RbCl was used. Smooth curves are single Boltzmann fits to the data with parameters as follows: Wt, V50 = −27.3 mV, z = 3.6; P475D, V50 = −104 mV, z = 2.8; P475Q, V50 = −96.7 mV, z = 2.6; P475N, V50 = −73.9 mV, z = 2.9; P475H, V50 = −65.9 mV, z = 2.6. (C) G-V relations for Wt and 10 P475 mutants with undetectable constitutive conduction. Parameters for single Boltzmann fits are: P475W, V50 = −49.9 mV, z = 3.0; P475Y, V50 = −46.8 mV, z = 3.1; P475F, V50 = −19.0 mV, z = 2.4; P475T, V50 = −18.1 mV, z = 3.0; P475M: V50 = −11.1 mV, z = 2.8; P475G, V50 = +39 mV, z = 2.3; P475I, V50 = +71 mV, z = 1.1; P475V, V50 = +75 mV, z = 1.1; P475C, V50 = +141 mV, z = 1.6; P475S, V50 = +191 mV, z = 1.1.
	Figure 4. . Side chain dependence of gating phenotype for substitutions at P475 in the Shaker Kv channel. Plots of either side chain volume or solvation energy against ΔΔG0. Solvation energy is a measure of the energetics of side chain transfer from cyclohexane to water (Fersht, 1999). Both plots have the same x-axis. Mutants that exhibit constitutive conduction are indicated with red symbols and the wild-type Pro residue is indicated with a green symbol. ΔΔG0 = (ΔG0mut − ΔG0wt) where ΔG0 = zFV50, with z and V50 values from Table I and F is Faraday's constant. P475L results in undetectable ionic or gating currents and P475A results in gating currents but no ionic currents (i.e., a nonconducting mutant). In the bottom plot, dashed black lines are drawn at ΔΔG0= −2 kcal mol−1 and solvation energy = −3.5 kcal mol−1 to illustrate the clustering of mutations into two regions of the plot.
	Figure 5. . Constitutive-conducting phenotype for the P406D mutant in the Kv2.1 channel. (A) Family of ionic currents for Kv2.1 Δ7 and P406D Kv2.1 Δ7. Depolarizations were from −70 to +20 mV (10-mV increments) for Kv2.1 Δ7 and from −110 to −10 mV (10-mV increments) for P406D Kv2.1 Δ7. Charge carrier in the external solution was 50 mM Rb+. Linear capacity and background conductances were identified and subtracted by blocking the Kv2.1Δ7 channel with Agitoxin-2. Horizontal dashed lines correspond to the zero current level. (B) G-V relations obtained by plotting normalized tail current amplitudes against test voltage. Same cells as in A. Smooth curves are single Boltzmann fits to the data with parameters as follows: Kv2.1 Δ7, V50 = −1.1 mV, z = 2.7; P406D Kv2.1 Δ7, V50 = −52 mV, z = 2.7.
	Figure 6. . Effects of Hanatoxin on the P406D Kv2.1 channel. (A) Current records elicited by weak depolarization in control, Hanatoxin (4 μM) or Agitoxin-2 (100 nM). Traces are shown without subtraction of leak or capacitive currents. Charge carrier in the external solution was 50 mM Rb+. (B) G-V relations for P406D Kv2.1 Δ7 channels in the absence and presence of 4 μM Hanatoxin. Same cell as in A. Currents remaining in the presence of Agitoxin-2 have been subtracted. Smooth curves are single Boltzmann fits to the data with parameters as follows: control, V50 = −51.8 mV, z = 2.8; toxin, V50 = +5.7 mV, z = 2.3.
	Figure 7. . N-type inactivation in the P475Q Shaker Kv channel. (A) Family of ionic currents for P475Q with deletion of residues 6–46. The cell was held at −100 mV, stepped to −150 mV for 200 ms before the traces shown, tail voltage was −150 mV, and depolarizations were between −140 and −40 mV in 20-mV steps. Linear capacity and background conductances were identified and subtracted by blocking the Shaker channel with Agitoxin-2. Horizontal dashed lines correspond to the zero current level. Charge carrier in the external solution was 50 mM Rb+. (B) Family of ionic currents for P475Q with an intact NH2-terminal inactivation ball. Same protocol and conditions as in A except that depolarizations were to between −140 and 0 mV in 20-mV steps. (C) Family of ionic currents for the wild-type Shaker channel with an intact NH2-terminal inactivation domain. Same conditions as in A except that depolarizations were from −80 to +20 mV (10-mV steps). (D) Kinetics of inactivation for wild-type and P475Q channels. Inactivation during the test pulses in B and C was fit with single exponential functions and the resulting time constants (τ) plotted against test voltage. n = 5 for both wild-type and P475Q. (E) Voltage dependence for activation and N-type inactivation. Open triangles, normalized tail current amplitude (measured in A) plotted against test voltage. Smooth black curve is a single Boltzmann fit to the G-V data with V50 = −97 mV, z = 2.5 with a fractional constitutive conductance of 0.31. Dashed black curve is a simulated G-V normalized to remove the constitutive conductance. Open squares, normalized fractional inactivation of the constitutive current at −150 mV plotted against voltage of the preceding test depolarization. Inactivation of constitutive current was measured at the black arrow in B, at which time deactivation in Δ6–46 (gray arrow in A) is nearly complete. Smooth curve is a single Boltzmann fit to the inactivation data with V50 = −92 mV, z = 2.3. Open diamonds, normalized fractional inactivation during the test depolarization (measured as the difference between the peak and steady-state currents in B) plotted against voltage of the test depolarization. Smooth black curve is a single Boltzmann fit to the inactivation data with V50 = −85 mV, z = 2.7.
	Figure 8. . Single-channel properties of the Shaker P475Q mutant channel recorded in symmetrical Rb+. (A) Single-channel currents recorded for P475Q in symmetrical 140 mM RbCl. The single-channel patch shown was held at −100 mV and stepped to −150 mV for 350 ms before the traces shown. Filter frequency was 2 kHz. Horizontal dashed lines correspond to the zero current level. (B) Ensemble of 15 single channel sweeps from the same patch shown in A and elicited using the same protocol as in A.
	Figure 9. . Permeant ion dependence of constitutive conduction for P475Q. (A) Families of ionic currents for Shaker P475Q with either 50 mM Rb+ or 50 mM K+ as the external permeant ion. The recordings for external Rb+ and K+ are from different cells. In each case the membrane was held at −100 mV and stepped to −150 mV for 200 ms before the traces shown and tail voltage was −150 mV. Depolarizations were from −150 to −20 mV in 10-mV steps for both left and right families. For these families both linear capacity and background conductances were identified and subtracted by blocking the Shaker channel with Agitoxin-2. Horizontal dashed lines correspond to the zero current level. Middle two traces are uncorrected currents elicited by depolarization to −50 mV in either control or Agitoxin-2 with K+ as the external permeant ion. (B) G-V relations obtained by plotting normalized tail current amplitudes against test voltage for Wt and P475Q in external Rb+ and K+. Smooth curves are single Boltzmann fits to the data with parameters as follows: Wt (Rb+ external), V50 = −27.3 mV, z = 3.6; P475Q (Rb+ external), V50 = −96.7 mV, z = 2.6; P475Q (K+ external), V50 = −80.5 mV, z = 2.4.
	Figure 10. . Single-channel properties of the Shaker P475Q mutant channel recorded in symmetrical K+. (A) Representative current traces (left) and corresponding all points amplitude histograms (right) recorded in symmetrical 140 mM KCl. The single channel patch shown was held at −100 mV and stepped to −150 mV for 350 ms before the traces shown. Filter frequency was 2 kHz. Horizontal dashed lines correspond to the zero current level. Y-axes for histograms are number of events and each histogram contains data from 6–10 sweeps. For inset histograms, y-axes range from 0 to (from top to bottom) 103, 5 × 102, and 103. (B) Ensemble of 30 single-channel sweeps from the same patch shown in A and elicited using the same protocol as in A. (C) i-V relationship for the highest conducting state. Each data point represents the mean ± SD (n = 6). Linear regression of the data shown yields a conductance of 60 pS. (D) Probability (P) for the closed and highest conducting open state as a function of voltage. All point histograms in A were fit with multiple Gaussians and their relative areas used to estimate probabilities.
	Figure 11. . Single-channel properties of the Shaker P475D mutant channel recorded in symmetrical K+. (A) Representative current traces (left) and corresponding all points amplitude histograms (right) recorded in 140 mM symmetrical KCl. The single channel patch shown was held at −100 mV and stepped to −150 mV for 350 ms before the traces shown. Filter frequency was 1 kHz. Horizontal dashed lines correspond to the zero current level. Y-axes for histograms are number of events and each histogram contains data from 6–10 sweeps. (B) Ensemble of 11 single-channel sweeps elicited using the same protocol as in A. (C) i-V relationship for highest conducting open state. Each data point represents the mean ± SD (n = 4). Linear regression of the data shown yields a conductance of 100 pS. (D) Probability (P) for the closed and highest conducting open state as a function of voltage. All point histograms in A were fit with multiple Gaussians and their relative areas used to estimate probabilities.
	SCHEME I.
	SCHEME II.
	Figure 12. . Structures of the inner helices in four types of K+ channels. (A) Inner helices (TM2) of KcsA from an intracellular view with the atoms of A108 represented as CPK. PDB accession code 1J95. (B) Inner helices (TM2) of KirBac from an intracellular view with the atoms of G143 represented as CPK. PDB coordinates generously provided by Declan A. Doyle. (C) Similar representation of KcsA as in A, except surrounding hydrophobic residues (L105, L110, A111, and F114) are represented as CPK and colored green. (D) Inner helices (TM2) of MthK from an intracellular view with the atoms of E92 (modeling as Ala) represented as CPK. PDB accession code 1LNQ. (E) Inner helices (S6) of KvAP from an intracellular view with the atoms of G229 represented as CPK. PDB accession code 1ORQ. Structures were generated using DS Viewer Pro (Accelrys).

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase