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Figure 2. Drosophila Shaker based Model Poorly Predicts AKv1 Steady State Inactivation.Best fit to AKv1(E2x) channel data is shown in black. While there is a trend in the data matching the prediction of the model, error bars for most data points do not contact the best fit line. The y-intercept of the best fit is significantly different from the activation midpoint for AKv1(Δ2-57).
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Figure 3. Comparing Effects of more Complex Activation Models on Steady State Inactivation Properties.Single-Step Activation Model for AKv1 was compared to channel activation model based on Zagotta et al.(1994), slightly modified to match AKv1 gating (see Methods).[20] A) Model Parameters were adjusted to optimally match steady state activation curve for AKv1(Δ2-57). B) Single-Step Voltage-dependent activation model was combined with a single-step Inactivation Model with varying KI. C) Steady State Inactivation midpoint matches prediction from the activation properties for the model. D) Voltage dependence for steady state inactivation matches activation ks, and is not affected by varying KI. E) Zagotta et al. (1994) based activation model [20] adjusted for AKv1, was combined with a single-step Inactivation Model with varying KI. F) Data fit well to a linear model with slope slightly less than predicted by channel activation (orange). G) Flatter slope for fit ks is caused by greater voltage dependence as inactivation is shifted to more negative potentials, as expected due to reduced occupancy of intermediate closed states at more negative potentials.
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Figure 4. Slowed Closing produced by N-type Inactivation matches the voltage-dependence for normal channel closing.A) Time course for AKv1 tail currents matches the Inactivation recovery time course. B) Kinetics for normal closing measured in AKv1(Δ2-57) and recovery tails measured in AKv1 are voltage dependent. C) Comparing tail current closing rates from single exponential fits. Voltage-dependence for AKv1(Δ2-57) closing matches the voltage-dependence for AKv1 tail currents despite the dramatically different rates. D) Ratio of tail closing rates predicts a consistent value for KI. For wild type AKv1 this value is similar to KI1 measured from the fraction of current that is not inactivated at the end of a pulse.
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Figure 5. Slowed Closing predicts steady state inactivation midpoints for AKv1(E2x) series mutants.A) Best fit to AKv1(E2x) channel data is shown in black. Prediction for the steady state inactivation midpoints based on AKv1(Δ2-57) activation gating shown in orange. AKv1(E2x) data are well fit with a linear model (r = −0.99); however, the y-intercept is more negative than expected and the slope is flatter than predicted from AKv1(Δ2-57) activation. B) Predicted value for ks from the fit is smaller than the activation curve ks, and similar to ks values measured from the inactivation curves, as expected from the more complex multi-step activation of the real channel.
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Figure 6. Identification the Inactivation Proximal (IP) Domain.A) Representative currents for N-terminal deletions. Removal of the initial 5 residues eliminates N-type inactivation. B) Activation midpoint for AKv1(Δ2-5) is shifted to a more negative midpoint compared to larger N-terminal Deletions AKv1(Δ2-14) and AKv1(Δ2-57). C) Deletion effects on activation midpoint identify the IP region between residues 5-14. D) Despite the shift in activation midpoint, AKv1(Δ2-5) closing kinetics and voltage dependence are similar to AKv1(Δ2-57). D) Using AKv1(Δ2-5) activation midpoint and slope from AKv1 inactivation accurately predicts inactivation midpoints for AKv1(E2x) mutant series (Model Prediction line).
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Figure 7. Identification of Conserved Motif in IP Domain.A) IP Domain retained in AKv1(Δ2-5) and deleted in AKv1(Δ2-14) contains a highly conserved [(A/V)-(G/S/C)-(H5)] Motif. Mutations to residue Leu7 from Drosophila ShB channel, highlighted in red, that make this residue more polar disrupt ShB N-type inactivation. B) AKv1(Δ2-5, I8Q) mutant shows expected non-inactivating currents. C) Activation Curve for AKv1(Δ2-5, I8Q) is shifted back to more positive potentials and matches AKv1(Δ2-57). D) Summary data showing deletions and mutations identifying the IP Domain and the disruption of IP Domain effects on activation by the I8Q mutation.
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Figure 8. I8Q mutation has distinct effects on AKv1 Inactivation.A) Inactivation of AKv1(I8Q) is much less complete that AKv1, similar to the IB region mutant AKv1(E2D). B) Inactivation midpoint for AKv1(I8Q) is predicted by the activation midpoint for AKv1(Δ2-5, I8Q) further showing that the regulation of activation by the IP region is retained during N-type inactivation of AKv1(E2x) channels. C) Despite less efficient inactivation block, the kinetics for inactivation of AKv1(I8Q) and AKv1(E2D) are not significantly different from AKv1. D) Despite similar inactivation levels and inactivation and recovery time courses, tail currents for AKv1(I8Q) show 2-exponential decay whereas AKv1(E2D) tail currents are single exponential.
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Figure 9. Modeling the Pre-Block Interaction.A) Minimal AKv1 N-type Inactivation gating scheme. AKv1 gating model is only slightly more complicated than the Single Step Drosophila Model (red states) because in addition to the C, O and I labels to indicate the pore state, a subscript is needed to indicate : P- the P site bound states (black states) that shift activation and enhances inactivation and, W- a separate Withheld state (blue state), from which the IB region cannot directly access the pore block state. Rate constants kc(v) = 40(−0.015) and ko(v) = 1500(0.32) as described previously. Equilibrium constants and cooperativity factor α determined as described in the text. The model produces a reasonable fit for the steady state properties for AKv1, AKv1(I8Q), and AKv1(E2D) (with KI changed to 0.1). Accurate activation and inactivation kinetics at strong depolarizations requires additional steps along the red pathway to rate limit the kinetics. If direct closing from OP and OW open states is included using the same kc(v) value then closing and recovery kinetics for AKv1, AKv1(I8Q), and AKv1(E2D) channels at strong negative potentials can be reproduced with this model if the KI equilibrium is made rapid. B) Structural model of key regions involved in N-type inactivation based on the 3LUT structure of Kv1.2 [23]. A tilted perspective (see inset upper right) of the channel showing the inner aqueous volume of the channel in gray. Residue Tyr417 is shown in red and the S4-S5 linker in green. Selectivity filter is marked by the locations of the potassium ions in purple. C) Internal aqueous volume of the channel seen from a side perspective divided into the pore inner vestibule (white) and the side window vestibule (green). Volumes of these regions are given in the matching color. A single subunit P region backbone trace from the S4-S5 linker to the end of the determined S6 structure is shown along with its residue Tyr417 (red) and S4-S5 linker (brown) highlighted. The S4-S5 linker from the adjacent subunit is close to Tyr417 and therefore is also shown. D) Same picture only rotated 90o to show the locations of Tyr417 and the S4-S5 linkers from 2 subunits relative to the pore inner vestibule and the side window vestibule. Both Tyr417 and the S4-S5 linkers are side window vestibule lining residues, not pore inner vestibule lining residues.
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Figure 1.
Drosophila Shaker based Single-Step Model for N-type Inactivation.Model has 3 basic states: Closed (C), Open (O) and Inactivated (I). Relative occupancy of these three states at equilibrium is given by the Equilibrium constants: Voltage-Dependent Activation- KO(v), and Inactivation- KI. The rate limiting step for inactivation at strong positive potentials is the binding of the N-terminus into the pore, the kon rate, highlighted in red. The rate limiting step for recovery at strong negative potentials is the unbinding of the N-terminus from the pore block site, the koff rate, highlighted in orange. These same rate limiting steps are proposed to control Ball Peptide inactivation (Dashed), or for that matter pore block by long chain quaternary ammonium derivatives like C9. Note that the rate limiting steps are not inherently voltage dependent; however, some voltage dependence to the recovery rate could be due to electric field effects mediated in the pore by K+ ions or charges on the ball peptide that accelerate unblocking, or re-blocking before closing that becomes less likely as kc(v) becomes faster.
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