The nonconducting W434F mutant adopts upon membrane depolarization an inactivated-like state that differs from wild-type Shaker-IR potassium channels

Figures

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  Fig. 1.. Shaker-IR-P475D is constitutively conductive. (A) Representative whole-cell current recordings from Shaker-IR-P475D expressed in HEK293 cells (left) and HEK293 cells transfected only with the peGFP-n1 marker (right) using the pulse protocols shown on top. For Shaker-IR-P475D, a 5-s prepulse of −100 mV was applied, thereby revealing outward currents upon subsequent depolarization. Interpulse time was 15 s, with a holding potential of −70 mV for both experiments. The horizontal bar at the start of the current traces indicates the zero-current level. (B) Current-voltage curve of the instantaneous currents in the Shaker-IR-P475D–expressing HEK293 cells and the peGFP-n1–transfected HEK293 cells. Vrev is around −50 mV for Shaker-IR-P475D (gray circles; N = 5) and around 0 mV for the peGFP-n1–transfected HEK293 cells (white circles; N = 3).

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  Fig. 2.. Activation of the channel mutants Shaker-IR-P475D and Shaker-IR-W434F-P475D. (A) Representative whole-cell current recordings in Shaker-IR-P475D and Shaker-IR-W434F-P475D, acquired with the pulse protocols shown on top. Inward currents upon hyperpolarization were observed in both Shaker-IR-P475D and Shaker-IR-W434F-P475D. The horizontal bar at the start of the current traces indicates the zero-current level. (B) The normalized conducting current as a function of membrane potential obtained by analyzing the tail current amplitudes of Shaker-IR-P475D (gray circles; N = 5) and Shaker-IR-W434F-P475D (black circles; N = 9). Vmid was −99.1 ± 3.9 mV for Shaker-IR-P475D and −113.7 ± 0.4 mV for Shaker-IR-W434F-P475D. The solid lines represent the average fit with a Boltzmann charge-voltage function. (C) Time constants of elicited currents of Shaker-IR-P475D (gray circles; N = 5) and Shaker-IR-W434F-P475D (black circles; N = 9) shown in (A), obtained by fitting the onset of inward current with a single exponential function.

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  Fig. 3.. Full activation of WT Shaker-IR and the mutant channels Shaker-IR-W434F-P475D and Shaker-IR-P475D. (A) Representative whole-cell current recordings in Shaker-IR, Shaker-IR-P475D, and Shaker-IR-W434F-P475D, elicited with a full-activation pulse protocol shown on top, to determine the ion selectivity of the channel. A horizontal bar at the beginning of the current traces indicates the zero-current level. The square inset displays the tail currents of Shaker-IR-W434F-P475D in more detail, from 20 to −20 mV, with the tail current elicited at 0 mV in red. (B) The normalized conducting current as a function of membrane potential obtained by analyzing the tail current amplitudes of Shaker-IR (white circles; N = 5), Shaker-IR-P475D (gray circles; N = 4), and Shaker-IR-W434F-P475D (black circles; N = 6). A similar Vrev was found for Shaker-IR (−76.5 ± 2.3 mV) and Shaker-IR-P475D (−60.8 ± 4.9 mV), while the Vrev of Shaker-IR-W434F-P475D displayed a large positive shift toward 0 mV. (C) Time constants of the decaying tail currents of Shaker-IR (white circles; N = 5), Shaker-IR-P475D (gray circles; N = 4), and Shaker-IR-W434F-P475D (black circles; N = 6), obtained by fitting the decaying…

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  Fig. 4.. Established pore blockers and 4-AP inhibit the Shaker-IR-W434F-P475D currents. TEA, 4-AP, and flecainide were used to prove that the observed nonselective currents recorded in standard ICS and extracellular solutions (ECS) pass through the ion-conducting pathway of the Shaker-IR-W434F-P475D channel. The black trace was recorded in control conditions, and the red trace displays steady-state inhibition (from left to right) by 100 mM TEA, 10 mM 4-AP, and 500 μM flecainide. The horizontal bar at the beginning of the whole-cell current recordings indicates the zero-current level. Currents were elicited using the pulse protocols shown on top.

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  Fig. 5.. Shaker-IR-W434F-P475D conducts NMDG+. (A) Representative whole-cell current recordings in Shaker-IR-W434F-P475D, elicited with the pulse protocol on top, using ICS and ECS that contained no divalent cations and with the monovalent cations replaced by NMDG+. The square inset displays the tail currents in more detail. (B) Conductance versus voltage relation obtained in NMDG+ solutions (N = 5). The solid line represents the average fit with a Boltzmann charge-voltage function. (C) Time constants of hyperpolarization-induced current increases in standard solutions (white circles; N = 6) and NMDG+ solutions (black circles; N = 5). Note that NMDG+ slowed down the time constants.

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  Fig. 6.. Activation of WT Shaker-IR and the mutant channels Shaker-IR-T449V and Shaker-IR-W434F-T449V. (A) Representative whole-cell current recordings from Shaker-IR, Shaker-IR-T449V, and Shaker-IR-W434F-T449V, acquired with the activation protocol shown on top. A horizontal bar at the beginning of the current traces indicates the zero-current level. (B) The normalized conducting current as a function of membrane potential obtained by analyzing the tail current amplitudes of Shaker-IR (white circles; N = 4), Shaker-IR-T449V (gray circles; N = 10), and Shaker-IR-T449V-W434F (black circles; N = 3). Vmid was −25.2 ± 1.5 mV for WT Shaker-IR, −27.8 ± 1.4 mV for Shaker-IR-T449V, and −15.3 ± 3.1 mV for Shaker-IR-W434F-T449V. (C) Time constants of activation, obtained by fitting the onset of activation with a single exponential function, of Shaker-IR (white circles; N = 4), Shaker-IR-T449V (gray circles; N = 5), and Shaker-IR-T449V-W434F (black circles; N = 10).

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  Fig. 7.. Ion selectivity and deactivation kinetics of WT Shaker-IR and the mutant channels Shaker-IR-W434F-P475D and Shaker-IR-P475D. (A) Representative whole-cell current recordings in Shaker-IR, Shaker-IR-T449V, and Shaker-IR-W434F-T449V, acquired using the full activation protocol shown on top. The zero-current level is indicated by a horizontal bar at the beginning of the current traces. (B) The normalized conducting current as a function of membrane potential obtained by analyzing the tail current amplitudes of Shaker-IR (white circles; N = 5), Shaker-IR-T449V (gray circles; N = 7), and Shaker-IR-W434F-T449V (black circles; N = 7). This shows that all three channels have a similar degree of K+ selectivity with a Vrev of −76.5 ± 2.3 mV for Shaker-IR, −76.8 ± 1.7 mV, for Shaker-IR-T449V and −64.0 ± 2.4 mV for Shaker-IR-W434F-T449V. (C) Time constants of deactivation of Shaker-IR (white circles; N = 5), Shaker-IR-T449V (gray circles; N = 7), and Shaker-IR-W434F-T449V (black circles; N = 7), obtained by fitting the deactivating tail currents with a single exponential function.

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  Fig. 8.. Crystal structure of the KcsA-W67F mutant and WT KcsA pore helix and SF. A structural alignment superimposing the WT KcsA (light blue sticks) and the KcsA-W67F mutant (yellow sticks) pore helix and SF is shown. The amino acid side chains of the Tyr82 and the phenylalanine substitution at the position 67 are indicated with black arrows to highlight the structural changes at the aromatic cuff caused by the W67F mutant. On the left, an inset zooming at the G77 carbonyl group highlights its structural changes, which underlies the ion vacancy at the second ion binding site.

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  Fig. 9.. Ion occupancy in the SF of the KcsA-W67F mutant with its AG closed. The SF of the KcsA-W67F mutant in the closed conformation displaying only two diagonal subunits for clarity. The Fo-Fc electron density map is displayed in light blue mesh (contoured at 7σ), validating the ions bound to the channel’s SF. Ions were bound at the first, third, and fourth K+ binding sites. The W67F’s SF remains conductive and adopted a WT-like structure, albeit with no or reduced K+ occupancy at the second binding site.