Hydrogen bonds as molecular timers for slow inactivation in voltage-gated potassium channels

  1. Stephan A Pless
  2. Jason D Galpin
  3. Ana P Niciforovic
  4. Harley T Kurata
  5. Christopher A Ahern  Is a corresponding author
  1. University of British Columbia, Canada
  2. University of Iowa, United States
7 figures and 1 table

Figures

The aromatic cuff is part of a highly conserved region in potassium channels.

(A) Top view of a Kv channel based on the structure of the tetrameric Kv1.2/2.1 chimera (PDB 2R9R; individual subunits are colored in gray, cyan, green and yellow, respectively). The inset shows a …

https://doi.org/10.7554/eLife.01289.003
Figure 2 with 2 supplements
Evidence for an intra-subunit H-bond between Asp447 and Trp434.

(A) Structure of a Kv1.2/2.1 chimera (2R9R) pore region demonstrating the physical proximity of Asp447 and Trp434 (Shaker residue numbering); (B) Chemical structures of side chains at position 447 …

https://doi.org/10.7554/eLife.01289.004
Figure 2—figure supplement 1
Tuning the Asp447–Trp434 intra-subunit H-bond.

(A)/(B) Recordings with Asp447Glu (A) and Trp434Tyr (B) in the absence (black) or presence of 30 mM TEA (−80 mV to +20 mV, 10 mV increments), clearly demonstrating their sensitivity to extracellular …

https://doi.org/10.7554/eLife.01289.005
Figure 2—figure supplement 2
Unlike Trp434Phe, Asp447Asn remains nonconducting on the Thr449Val background.

(A)/(B) Representative currents of Trp434Phe (A) and Asp447Asn (B) on either the WT background (black) or the Thr449Val background (red) (−80 mV to +20 mV, 10 mV increments; the vertical scale bar …

https://doi.org/10.7554/eLife.01289.006
Figure 3 with 1 supplement
Concatemers support the notion of an intra-subunit H-bond between Asp447 and Trp434.

(A) Concatemer structure and (normalized) representative currents (5 s pulses from −20 mV to +20 mV, 10 mV increments) for WT, Trp434Phe and Asp447Glu concatemers, respectively. The vertical scale …

https://doi.org/10.7554/eLife.01289.008
Figure 3—figure supplement 1
Comparison of different metrics to determine the rate of inactivation.

The decay of ionic current of WT, Trp434Phe and Asp447Glu concatemers was either fit with a single exponential (colored symbols, reproduced from Figure 3B) or quantfied by analyzing the time to …

https://doi.org/10.7554/eLife.01289.009
Figure 4 with 3 supplements
An inter-subunit H-bond connects Tyr445 with Thr439, not Trp435.

(A) Structure of a Kv1.2/2.1 chimera (2R9R) pore region demonstrating the physical proximity of Tyr445 to both Thr439 and Trp435 on the adjacent subunit (Shaker residue numbering). Note that the …

https://doi.org/10.7554/eLife.01289.010
Figure 4—figure supplement 1
Tuning the Tyr445–Thr439 inter-subunit H-bond.

(A) Recordings with Tyr445Phe in the absence (black) or presence of 30 mM TEA (green) (−80 mV to +20 mV, 10 mV increments), clearly demonstrating the sensitivity to extracellular TEA, a hallmark of …

https://doi.org/10.7554/eLife.01289.011
Figure 4—figure supplement 2
Tyr445Ala, Tyr445Val and Thr439Val remain nonconducting on the Thr449Val background.

(A)/(B) Representative currents of Tyr445Ala and Tyr445Val (A) and Thr439Val (B) on either the WT background (black) or the Thr449Val background (red) (−80 mV to +20 mV, 10 mV increments; the …

https://doi.org/10.7554/eLife.01289.012
Figure 4—figure supplement 3
The methyl moiety of Thr439 may play a minor role in inactivation.

(A) GVs for Thr439Ser (V1/2 Thr441Ser: −28.4 ± 0.3 mV; vs V1/2 for WT: −23.5 ± 0.9 mV); (B) Representative currents for WT and Thr439Ser (5 s pulses from −20 mV to +20 mV, 10 mV increments); (C) …

https://doi.org/10.7554/eLife.01289.013
Figure 5 with 2 supplements
Breaking the Tyr445–Thr439 inter-subunit H-bond results in rapid inactivation.

(A) Concatemer structure and representative currents (5 s pulses from −20 mV to +20 mV, 10 mV increments) for WT, Tyr445Ala and Thr439Val concatemers, respectively. The vertical scale bars indicate …

https://doi.org/10.7554/eLife.01289.014
Figure 5—figure supplement 1
Characterizing the Thr439Val and Tyr445Ala concatemers.

(A)/(C) Representative recordings from Thr439Val (A) and Tyr445Ala (C) concatemers in the absence (black) or presence (green) of 30 mM TEA (−20 mV to +20 mV, 10 mV increments), clearly demonstrating …

https://doi.org/10.7554/eLife.01289.015
Figure 5—figure supplement 2
Pronounced gating currents at hyperpolarized potentials in Tyr445Ala and Thr439 Val concatemers.

(A)/(B) Representative currents of Tyr445Ala concatemers (A) and Thr439Val concatemers (B) showing signicant gating currents before the onset of ionic currents at around −40 mV (50 ms …

https://doi.org/10.7554/eLife.01289.016
Figure 6 with 3 supplements
Thr441 and Thr442 are critical to channel function but not inactivation.

(A) Structure of a Kv1.2/2.1 chimera (2R9R) pore region highlighting the positions of Thr441 and Thr442 at the bottom of the selectivity filter (Asp447 and Trp434 are shown for reference; all by …

https://doi.org/10.7554/eLife.01289.017
Figure 6—figure supplement 1
The Thr441Val mutation results in a loss of potassium selectivity.

Current vs voltage plot for the Thr441Val mutant (n = 9). The inset shows that the current reverses around −40 mV.

https://doi.org/10.7554/eLife.01289.018
Figure 6—figure supplement 2
Hydroxyl moieties are critical to channel function in positions 441 and 442.

(A)/(D) GVs for Thr441Ser (A) and Thr442Ser (V1/2 Thr441Ser: −19.7 ± 1.5 mV; V1/2 Thr442Ser: −45.6 ± 0.6 mV; vs V1/2 for WT: −23.5 ± 0.9 mV); (B)/(E) Representative currents for WT and Thr441Ser (B) …

https://doi.org/10.7554/eLife.01289.019
Figure 6—figure supplement 3
Comparison of inactivation time constants using different pulse durations.

Averaged inactivation time constants obtained from WT, Thr441Val and Thr442Val concatemers by fitting the current decay during 5 s (colored symbols, reproduced from Figure 6E) or 20 s (empty …

https://doi.org/10.7554/eLife.01289.020
A network of inter- and intra-subunit H-bonds regulates slow inactivation.

(A) The left panel shows a top view of pore helix and selectivity filter (based on the Kv1.2/2.1 chimera structure (2R9R); individual subunits are colored in gray, cyan, green and yellow, …

https://doi.org/10.7554/eLife.01289.021

Tables

Table 1

Mutants that result in gating currents only show very similar gating charge-voltage (QV) relations

https://doi.org/10.7554/eLife.01289.007
ConstructV1/2 (mV)Zn
Trp434Phe−52.9 ± 0.95.2 ± 0.36
Asp447Asn−53.4 ± 0.95.3 ± 0.36
Thr439Val−51.0 ± 1.45.5 ± 0.35
Tyr445Ala−50.3 ± 2.15.3 ± 0.44
  1. Displayed are the values for the midpoints (V1/2), the amount of gating charge (Z) of the QVs (derived from the OFF gating currents) and the number of experiments conducted (n).

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