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Figure 2. Functional effects of conservative removal of negatively charged residues in the AKv1 N-terminus.Glutamate residues at positions 2 and 9 were mutated to the uncharged but similarly sized glutamine residue. Currents recorded in response to voltage steps from â40 mV to +80 mV, with the step to +50 mV highlighted in red. A) Normal kinetics for wild type AKv1 with E2 and E9. Currents show a rapid and almost complete inactivation during strong depolarizations. A small tail current produced by N-terminal unbinding during the recovery process is evident on return of the membrane potential to â100 mV. B) The mutation E2Q, removing the negative charge from the second position greatly accelerates the inactivation decay kinetics, but leaves slightly greater levels of sustained current at the end of the pulse. Tail currents during the recovery process at â100 mV are less pronounced than with the wild type channel. C) The mutation E9Q, removing the negative charge from the 9th position slightly accelerates inactivation kinetics and slightly increases the level of block at the end of the pulse. Tail currents with this mutation are also less pronounced than wild type. D) Analysis of recovery kinetics at â100 mV shows that wild type channels recover most quickly while recovery for the uncharged mutants is dramatically slowed. All recovery kinetics are well fit by a single exponential. (N's: E2, E9(27), E2Q(8), E9Q(5)).
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Figure 3. Effects of size and charge changing mutations at position 2 on N-type inactivation.A) Typical currents recorded in response to a step depolarization to +50 mV for mutations to residue 2. Current amplitudes were normalized to the same peak rate of rise to compare the sustained currents remaining at the end of the depolarization. ÎID is a construct with the N-terminus removed, eliminating N-type inactivation. B) Analysis of inactivation properties for E2K for steps from â40 mV to +170 mV shows that the inactivation kinetic becomes more apparent as the membrane potential is more depolarized. No such effect is seen if the N-terminus is removed (ÎID). C) Average rate of recovery at â100 mV is plotted for a series of substitutions at position 2. The rate varies widely depending upon the specific substitution at residue 2, but not on the net charge at this position. Recovery for all constructs is well fit by a single exponential except for E2A where a second, smaller and slower component of recovery is evident. (N's: E2(27), E2D(6), E2N(7), E2Q(8), E2T(10), E2A(11), E2K(8)). D) Tail currents for traces shown in (a) are plotted using the same scaling factors. Tail currents become progressively smaller as the recovery kinetics become slower and the amount of block at the end of the pulse becomes larger. For most constructs the sustained current at the end of the pulse closes more slowly than ÎID, indicating a delayed closing produced by continued interactions with the N-terminus; however, for E2N, and to a lesser extent E2Q a normally closing tail current component is seen that appears to be the size expected if these unblocked channels close normally. E) Comparison of tail current kinetics with inactivation recovery kinetics at â100 mV. For charged residues E2, E2D and E2K, there is good agreement between the time constant for recovery from inactivation and the single exponential tail current decay kinetics. For uncharged substitutions, a second tail current kinetic component is seen. For E2N, E2Q and E2T, the faster component is not evident in the recovery kinetics, although for E2A both tail current decay components are evident in the recovery kinetics. F) Rapid of reopening for E2N in a two pulse protocol reveals a reduced amplitude current that can gate open and closed with no apparent inactivation kinetics. Reopening steps from â40 mV to +20 mV.
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Figure 4. Effects of size and charge changing mutations at position 9 on N-type inactivation.A) Typical currents recorded in response to a step depolarization to +50 mV for mutations to residue 9. Current amplitudes are normalized to the same maximum rate of rise as a channel lacking N-type inactivation ÎID. Note the peak amplitudes of these currents are very similar with a consistent acceleration in the decay rate as residue 9 is made more positive. B) Average rate of recovery from inactivation at â100 mV is plotted for a series of substitutions at position 9. (N's: E9(27); E9Q(5); E9A(12); E9K(6)). The recovery rate is faster for charged residues at position 9 than for uncharged substitutions.
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Figure 5. Analysis of Energetic effects of Charge Changing mutations on N-type inactivation.A) Relative energy for the rate limiting step to inactivation at +50 mV (Transition) and the difference between this energy and the relative energy for the rate limiting step for recovery from inactivation at â100 mV (Bound) are plotted versus the charge at residue 2. Transition energy lowers as the charge on residue 2 is made more positive, equivalent to a net â15.9 mV electrostatic effect on residue 2 at Transition. There is almost no-consistent electrostatic effect evident when comparing the effects of residue 2 charge on the stability of the Bound state. Uncharged residues show an additional lowering of energy suggesting they are able to make additional favorable interactions in these states that are not available to charged residues. (N's: E2(12), E2D(6), E2N(7), E2Q(6), E2T(7), E2A(12), E2K(10)). B) Similar analysis for residue 9 substitutions shows a consistent linear electrostatic effect for residue 9 at Transition that is equivalent to a â11.6 mV electrostatic effect on residue 9. A â18.8 mV electrostatic effect is apparent on residue 9 at the Bound state with additional stability noted for uncharged residues, similar to residue 2 in the transition state. (N's: E9(12); E9Q(4); E9A(5); E9K(6)). C) Summary plot of electrostatic effects on residues from 2â55 in the AKv1 N-terminus. Green arrows indicate electrostatic environment changes for residues that are energetically favorable between Transition and Bound, whereas red arrows are changes that are energetically unfavorable. Unfavorable energy changes as inactivation proceeds are seen for most N-terminal negative charges, except for E2, which shows a favorable energetic environmental change between Transition and Bound. D) Linear Brønsted plots comparing the energetic impact of mutations on the Transition and Bound states. N-terminal mutational effects, âChain Mutants,â track the Φâ=â1 line, indicating a primary impact prior to Transition, until the most N-terminal negative charges are reached. Then, the curves become much flatter Φ<0.2 indicating the largest impact of mutations at these sites are occurring later in the inactivation cycle near the Bound state. For residue 2 mutations there is an additional vertical shift down from the origin indicative of effects on intermediate steps in the inactivation/recovery cycle.
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Figure 6. Electrostatic Coupling of residues 2 and 9 with the Channel Core Resides regulates the time constant to inactivate at +50 mV.Energy plots looking at the interactions between charge changing mutations to residues 2 and 9 and charge changing mutations in residues 161-3 and 135 on the time constant to inactivate. A) As the charge on position 2 becomes more positive, the effect of mutating 161â3 to more positively charged residues becomes more favorable. This suggests that a stronger interaction between residue 2 and 161â3 slows the rate limiting step in the ON inactivation pathway. (N's: 161-3EDE: E2(17), E2A(8), E2K(5); 161-3AAA: E2(9), E2A(5), E2K(4); 161-3KKK: E2(16), E2A(7)). B) If residue 9 is made more positive, the effect of mutating 161â3 to more positively charged residues has an increasingly larger energetic cost for the time constant to inactivate, opposite to the effects seen at position 2. (N's: 161-3EDE: E9(17), E9A(12), E9K(5); 161-3AAA: E9(9), E9A(5), E9K(6); 161-3KKK: E9(16), E9A(8), E9K(5)). C) If instead we examine residue 135, at the top of the T1 domain just below the transmembrane pore, then making position 2 more positive increases the energetic cost of placing more positive charges at position 135. (N's: V135D: E2(3), E2A(3); V135: E2(17), E2A(5); V135K: E2(12), E2A(7)). D) Adding positive charge at 135 shows an increasingly larger energetic cost when residue 9 is also made more positive. (N's: V135D: E9(3), E9A(4), E9K(8); V135: E9(17), E9A(12), E9K(5); V135K: E9(12), E9A(4), E9K(3)).
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Figure 7. Electrostatic Coupling of residues 2 and 9 with the Channel Core Resides regulates the time constant to recover from inactivation at â100 mV.Energy plots looking at the interactions between charge changing mutations to residues 2 and 9 and charge changing mutations in residues 161â3 and 135 on the time constant to recover from inactivation. A) The energetic effects of charge changing mutations at 161â3 on the recovery from inactivation are small and show little evidence for a significant interaction with the charge placed at position 2. (N's: 161-3EDE: E2(27), E2A(7), E2K(8) ; 161-3AAA: E2(6), E2A(6), E2K(4); 161-3KKK: E2(10), E2A(7)). B) Similar results are seen with residue 9, where there is little impact of the charge at residue 9 on the small slowing of recovery seen when residues 161â3 are made more positive. (N's: 161-3EDE: E9(27), E9A(12), E9K(6); 161-3AAA: E9(6), E9A(5), E9K(7); 161-3KKK: E9(10), E9A(7), E9K(6)). C) Recovery from inactivation also shows little sensitivity to the charge placed at 135, with little impact of the charge at position 2 on this sensitivity. (N's: V135D: E2(3), E2A(3); V135: E2(27), E2A(5); V135K: E2(12), E2A(7)). D) Charge on residue 9 shows little effect on the relative impact of charge at position 135. (N's: V135D: E9(3), E9A(3), E9K(4); V135: E9(27), E9A(12), E9K(5); V135K: E9(12), E9A(4), E9K(4)).
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Figure 8. Summary Plots of Electrostatic Coupling of Residues 2 and 9 with the Channel Core Resides 161-3 and 135.Slopes of linear fits to curves in Figs. 6, 7 are plotted versus the charge at positions 2 and 9. A) Residue 2 shows a large counterintuitive effect where making 161â3 more positive changes the potential experienced by residue 2 during the rate limiting ON transition by â21 mV. This suggests that residue 2 is net moving away from 161â3 to reach Threshold. Recovery shows only a small coupling of â4 mV suggesting that more positive charges at 161â3 stabilize a negative charge at position 2 in the pore Binding site by 4 mV relative to an uncharged residue. B) Residue 2 experiences a 12.8 mV more positive potential during the rate limiting transition as residue 135 is made more positive. There is a small â2 mV coupling of 135 charge to residue 2 in the pore binding site, suggesting a small stabilization is residue 2 is negatively charged. C) For residue 9, more positive charges at residue 161â3 increase the electrostatic potential experienced by residue 2 at the Transition state by 9 mV. There is little impact of changing the charge at 161â3 on recovery that is transmitted through the charge at position 9. D) More positive charge at 135 similarly increases the electrostatic potential experienced by residue 2 at the Transition state by 8 mV. There is little impact of changing the charge at 135 on recovery that is transmitted through the charge at position 9.
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Figure 9. Electrostatic effects of the applied membrane potential on charges present on residues 2 and 9.A) Voltage dependence of the time constant to inactivate for different residue 2 mutants at strong depolarizations. As residue 2 becomes more positive, inactivation becomes progressively faster as the membrane becomes more depolarized. (N's: E2(12), E2D(6), E2N(7), E2Q(5), E2T(4), E2A(8), E2K(10)). B) Summary plot of the apparent charge dependence for inactivation time constant voltage dependence at residue 2 suggests Threshold occurs when residue 2 is at a position within the pore that experiences 14% of the applied electric field. C) Voltage dependence for the blocking affinity of the N-terminus in the pore at large depolarizations. As the charge at position 2 is made more positive the apparent affinity of the N-terminus becomes increasingly better as the membrane potential becomes more depolarized. (N's: E2D(3), E2N(7), E2T(7), E2A(8), E2K(5)) D) Summary plot of charge at position 2 versus the voltage dependence of the N-terminus affinity for the pore suggests that residue 2 experiences 35% of the applied electric field at the pore block site. E) Charge at residue 9 shows little impact on the voltage dependence for the time constant to inactivate as measured at strong depolarizations. (N's: E9(12); E9Q(4); E9A(5); E9K(6)). F) Summary plots show no significant coupling of membrane potential with the charge at position 9, would be expected if residue 9 is outside the transmembrane pore at the Transition state.
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Figure 10. Dependence of Efficiency of Pore block on charges present on residues 2 and 9.A) Based on the time constant for channel closing at â100 mV in the absence of N-type inactivation (ÎID), recovery should be slowed by a predictable amount based on the fraction of channels that are unblocked by the N-type inactivation domain (Line:). The wild type residue E2 falls on this predicted line, however, most substitutions lie significantly above the line indicating additional interactions slowing recovery that do not produce channel pore block. (N's: E2(27); E2D(6); E2N(7); E2Q(8); E2T(10); E2A(11); E2K(8)). B) The amount of inefficiency in the pore block process produced by different residue 2 mutants can be estimated by measuring how energetically far recovery is from the value expected if recovery were only dependent on the amount of pore block. The charged residues, and faster kinetic components in uncharged residues, fall near the high efficiency 0 kT value. There is a slight systematic effect depending upon charge at position 2, equivalent to 7.8 mV. The slower components of recovery for uncharged residues have large energetic binding components that are slowing recovery without producing additional pore block. C) Efficiency analysis for residue 9 substitutions shows again that charged residues are highly efficient at producing pore block with the expected recovery, whereas uncharged substitutions are again shifted above the line. (N's: E9(27); E9Q(5); E9A(5); E9K(6)). D) Summary plots show that the charged residues fall near the high efficiency 0 kT value with a slight systematic shift depending upon charge at position 9 of 2.5 mV. Uncharged residues again have interactions with the channel core that are not efficiency being converted into increased pore block.
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Figure 11. Model of Important Sites and Reaction Steps during N-type Inactivation.Structural model of the AKv1 channel showing the approximate locations of Sites 1, 2, and 3 along the internal aqueous pathway leading to the selectivity filter. Channel profile is taken from a slab cut from an AKv1 structural model showing a side window opening and the internal vestibule of the transmembrane pore. The 1.4 Ã
accessible surface map is colored according to electrostatic potential with red negative, white neutral, and blue positive. Schematic to the right lists the hypothesized locations of residues E2, E9 and D19 during different phases of the N-type inactivation cycle. The intensity of red indicates the approximate level of negative charge along the pathway relative to residues 161â3. Dashed vertical line indicates approximate location of the pore entry beyond which chain occupancy produces pore block. This picture provides an approximate explanation for the electrostatic coupling seen between these residues and 161â3 during the inactivation cycle. Precisely how this electrostatic field is actually experienced by any residue along the path will depend on the microenvironment of the N-terminal residue and the direction the residue is pointing relative to residues 161â3.
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Figure 1. Alignment of Kv1 N-terminal sequences showing the highly conserved negative charges in the N-terminal inactivation domain.Green Arrows show locations of highly conserved negatively charged amino acids at positions 2 and 9. Sequence homology is evident throughout this region of the N-terminus as indicated by the high level of conservation and quality of the alignment. The consensus sequence at the bottom gives an indication of residues that have likely been conserved from the hypothesized Kv1AnID.
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