1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
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Atomic mutagenesis in ion channels with engineered stoichiometry

  1. John D Lueck
  2. Adam L Mackey
  3. Daniel T Infield
  4. Jason D Galpin
  5. Jing Li
  6. Benoît Roux
  7. Christopher A Ahern  Is a corresponding author
  1. The University of Iowa, United States
  2. University of Chicago, United States
Research Advance
Cite this article as: eLife 2016;5:e18976 doi: 10.7554/eLife.18976
5 figures, 2 videos and 1 table

Figures

Conformational dynamics of wild type and Trp434Ind Shaker channel.

(A) Position of the Trp434 –Asp447 H-bond. (B) Time series of dihedral angles χ1 (N–Cα–Cβ–Cγ) of Arp447 for wild type (left) and Trp434Ind (right) demonstrating the flipping of the Arp447 side-chains during the simulations. (C) Time series of distance between Asp447 and Trp434 in four subunits characterizing the local conformational dynamics during equilibrium simulations for wild type (left) and Trp434Ind (right) of Shaker. The distance is measured between Asp447 (Cγ) and Trp434 (Nε1) or equivalent atom in the same position in Trp434Ind. (D) Top panels, typical conformations for protein and water molecules and occupancy (20%) map of water molecules around selectivity filter of subunit C. Bottom panels, 2D average occupancy map for all four subunits during 500ns MD simulations. The x-axis describes the radius to the center of the selectivity filter, and the y-axis is the z-coordinate of water molecules. In all simulations the selectivity filter in the conductive state. Videos 1 and 2 indicate 150 ns vignettes of the simulation for the W434-D447 and W434Ind-D447 in the presence shown water molecules. In each case, a subunit of Shaker (residues 433 to 447) is shown in new-cartoon representation, and side chains of W434 and D447 are shown in sticks, and water molecules are in VDW representations.

https://doi.org/10.7554/eLife.18976.002
Attachment of ER retention/retrieval motifs paired with split inteins results in co-dependent surface membrane expression of Shaker channels.

(A) Expression of either Shaker channel with C-terminal appended Kir6.2 ER retention/retrieval motif (ERret; yellow) with N-intein (red) (A, left) or Shaker channel with N-terminal appended Kir6.2 ER retention/retrieval motif (yellow) with C-intein (green) (A, right), resulted in negligible ionic current (A, lower panels).Representative currents in lower panel A, are raw capacitance and ionic currents that are blanked above and below 25 and −5µA, respectively. P/4 subtracted currents are shown in lower panel B to display normal channel kinetics observed following expression of engineered ERret-intein Shaker channels. See Materials and methods for ER-retention/retrieval and intein sequences. (B) Co-expression of Shaker N- and C-terminal ERret-intein channels resulted in surface membrane trafficking of Shaker channels and robust ionic current (lower panel) measured by TEVC. (C) Injection of increased amounts of C-terminal (filled circles) or N-terminal (open circles) ERret-intein-tagged Shaker cRNA resulted in insignificant surface expression of Shaker channels as measured with TEVC. Co-injection of increased amounts of N- and C-terminal ERret-intein cRNA resulted in saturated ionic current (filled triangles). Number of observations where two Shaker monomers are expressed are indicated in parentheses above symbols. Average values where only one Shaker monomer was expressed were composed of 2–10 observations.

https://doi.org/10.7554/eLife.18976.005
Use of ERret-intein tagged Shaker monomers allow for stoichiometric expression of Ind.

(A) Normalized representative currents for Trp434Trp (WT) (left) and Trp434Ind (right). A schematic of the stoichiometry of the incorporated amino acids Trp (white) and Ind (black) is shown below the traces. (B) Average voltage dependence of normalized currents for Trp434Trp (WT) (open circles; n = 5) and Trp434Ind (filled circles, n = 9). (C) Average inactivation rates at +20 mV of WT Shaker, Trp434Phe (first monomer) four monomer concatemer, and Trp434Ind ERret-intein constructs.

https://doi.org/10.7554/eLife.18976.006
Figure 4 with 1 supplement
ERret-intein construction allows for expression of functional split WT Nav1.4.

(A) Schematic of Nav1.4 protein with ERret-intein sequence engineered on the C- (blue) and N-terminus (orange) of domains I-II and III-IV, respectively. (B) Western blot shows reconstitution and covalent linking of split-intein Nav1.4 proteins following expression in HEK293 cells. Lane 1, full-length WT Nav1.4; Lane 2, front half WT Nav1.4 I-II N-intein; Lane 3, Back half WT Nav1.4 III-IV C-intein; Lane 4 front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein expressed at 1:1; Lane 5, front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein expressed at 2:1, respectively. (C) Representative Nav1.4 currents 12 hr following co-injection of front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein. (D) Quantification of peak Na+ current @ −20 mV following injection of full-length WT Nav1.4 (circles; n = 10), front half WT Nav1.4 I-II N-intein (triangles; n = 6), back half WT Nav1.4 III-IV C-intein (squares; n = 7) and both front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein (squares; n = 10).

https://doi.org/10.7554/eLife.18976.008
Figure 4—figure supplement 1
Assembly of split-intein Nav1.4 constructs occurs within the cells.

HEK293 cell lysates expressing either front half WT Nav1.4 I-II N-intein and back half WT Nav1.4 III-IV C-intein were mixed on ice for 10, 30 and 60 min and separated by acrylamide gels, transferred and immunoblotted. No higher order band was detected following incubation of split-intein constructs following cell-lysis indicating that assembly and intein-dependent peptide bond formation occurred within the cell.

https://doi.org/10.7554/eLife.18976.009
Figure 5 with 1 supplement
ERret-intein construction allows for expression of functional split Nav1.4 with two suppressed codons.

(A) Co-injection of 25 ng of cRNA encoding Nav1.4 I-II N-intein Tyr401TAG + Nav1.4 III-IV C-intein WT + tRNA(Phe) (left), Nav1.4 I-II N-intein WT + Nav1.4 III-IV C-intein F1304TAG + tRNA(Phe) (center) and Nav1.4 I-II N-intein Tyr401TAG + Nav1.4 III-IV C-intein Phe1304TAG + tRNA(Phe) (right), resulted in robust currents at 24 hr (lower panels). (B) Quantification of peak Na+ current @ −20 mV following injection of 25 ng of cRNA of constructs in A with tRNA(Phe) (open symbols) and uncharged tRNA (closed symbols). (C) Representative Nav1.4 current following co-injection of 50 ng of cRNA encoding Nav1.4 I-II N-intein Y401TAG and Nav1.4 III-IV C-intein Phe1304TAG constructs. (D) Quantification of peak Na+ current @ −20 mV following injection of 50 ng of cRNA encoding Nav1.4 I-II N-intein Tyr401TAG and Nav1.4 III-IV C-intein Phe1304TAG (orange) constructs with tRNA(Phe) (open symbols) and uncharged tRNA (closed symbols).

https://doi.org/10.7554/eLife.18976.010
Figure 5—figure supplement 1
Voltage dependence of Nav1.4 activation is unaltered by engineered split inteins.

(A) Conductance-Voltage relationships of full-length WT Nav1.4 (solid line, circles) and front half WT Nav1.4 I-II N-intein + back half WT Nav1.4 III-IV C-intein (dashed line, diamonds). (B) Conductance-Voltage relationships of Nav1.4 I-II N-intein Y401TAG + Nav1.4 III-IV C-intein WT + tRNA(Phe) (circles), Nav1.4 I-II N-intein WT + Nav1.4 III-IV C-intein Phe1304TAG + tRNA(Phe) (triangles) and Nav1.4 I-II N-intein Tyr401TAG + Nav1.4 III-IV C-intein Phe1304TAG + tRNA(Phe) (squares).

https://doi.org/10.7554/eLife.18976.011

Videos

Video 1
In the WT a hydrogen bond is formed between Trp434 and Asp447 during a large fraction of MD simulation, which blocks the exchange of water molecules between extracellular bulk and the water binding pocket behind selectivity filter.

Upon rare occasion the D447 H-bond is broken spontaneously but quickly reforms.

https://doi.org/10.7554/eLife.18976.003
Video 2
In the Trp434Ind Shaker, there is a flipping of the Asp447 side-chain toward the extracellular bulk solution, promoting the formation of a continuous connection between the bulk solution and the water molecules in the buried pocket.
https://doi.org/10.7554/eLife.18976.004

Tables

Table 1

Shaker and Nav1.4 Channel activation parameters.

https://doi.org/10.7554/eLife.18976.007

Injected cRNA

V ½ (mV)

K (mV)

Shaker Inteins

WT-WT (0.25 ng cRNA)

−20.74 ± 2.27 (7)

11.42 ± 0.55 (7)

WT-Trp434Trp (0.25 ng cRNA)

−23.29 ± 1.76 (9)

9.38 ± 0.85 (9)

WT-Trp434Ind (0.25 ng cRNA)

−17.84 ± 2.17 (5)

7.75 ± 0.74 (5)

Nav1.4

Full-length WT Nav1.4 (2.5 ng cRNA)

−25.63 ± 0.71 (5)

2.70 ± 0.24 (5)

Split WT Nav1.4 (2.5 ng cRNA)

−25.97 ± 1.21 (6)

2.86 ± 0.23 (6)

Y401TAG (FH) + WT (BH) + tRNAPhe (25 ng cRNA)

−22.78 ± 0.54 (8)

3.99 ± 0.13 (8)

WT (FH) + F1304TAG (BH) + tRNA(Phe) (25 ng cRNA)

−25.22 ± 1.12 (7)

4.09 ± 0.19 (7)

Y401TAG (FH) + F1304TAG (BH) + tRNA(Phe) (50 ng cRNA)

−24.22 ± 0.55 (7)

3.78 ± 0.18 (7)

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