doi: 10.1038/nature12056.
Gating of the TrkH ion channel by its associated RCK protein TrkA
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- PMID:23598339
- PMCID: PMC3726529
- DOI: 10.1038/nature12056
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Gating of the TrkH ion channel by its associated RCK protein TrkA
Yu Cao et al. Nature..
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Abstract
TrkH belongs to a superfamily of K(+) transport proteins required for growth of bacteria in low external K(+) concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K(+) channel and may have a gating mechanism substantially different from K(+) channels. TrkH assembles with TrkA, a cytosolic protein comprising two RCK (regulate the conductance of K(+)) domains, which are found in certain K(+) channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two markedly different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA.
Figures
a. Single-channel currents through the TrkH-TrkA complex were recorded in symmetrical 200 mM KCl. Current traces are shown at a holding potential of +50 mV. The closed state is indicated as “c”. The gray rectangles show two individual bursts of channel openings.b. The open-channel current-voltage relationship. Current traces recorded at different voltages are shown in the left panel, and current amplitudes are plotted in the right panel.c. Current traces of the TrkH-TrkA (wt-wt) complex and the TrkH-TrkA (wt-R100A) complex in the absence (left) and presence (right) of 5 mM ATP or ADP. The Popen after perfusion of different ligands is plotted in the bar graph. Added ligand concentrations are as follows: ATP, 5 mM; ATPγS, 1 mM; AMP-PNP, 1 mM; ADP, 5 mM; AMP, 5 mM; NADH, 1 mM; NAD+, 1 mM. Error bars are s.e.m. of 3-9 independent measurements.
a.Structure of the complex between the TrkH dimer (light blue and teal) and the TrkA tetramer (dark blue and purple), viewed from the periplasmic side. The TrKA protomer is shown as a surface representation.b. The TrkH-TrkA complex viewed from within the plane of the membrane. Helix D3M2b in each TrkH subunit is highlighted in red. The inset on right shows homologous domains 1 and 3 of TrkH, with the intramembrane loop and helix D3M2b highlighted.c. Schematic showing the organization of RCK domains in a TrkA subunit.d-e. Cartoon representation of a TrkA protomer (d) or a protomer from the BK channel gating ring (e), colored by subdomain according to the color scheme in panelc. Schematics illustrating the organization of the tetrameric gating ring with the two-fold or four-fold symmetry axes marked are shown on the right. The green outlines delineate an individual subunit in each tetramer.f. Surface representation of the TrkA tetramer in the TrkH-TrkA complex, viewed from the periplasmic side (top) or parallel to the membrane (bottom). The green outline delineates an individual subunit.
a. Surface representation of the isolated TrkA tetramer, viewed from the periplamic side (top) or parallel to the membrane (bottom), colored by subdomain according to the color scheme in2c. b-c. Superposition of the N2-N2 interface (b) and N1-N1 interface (c) in the TrkH-TrkA (gray) and TrkA-only (blue or purple) structures. The alignment was based on the domains colored black in both structures.d. A single TrkA protomer from the TrkH-TrkA complex structure (left) and a protomer from the TrkA-only structure (right) viewed from the same orientation after superposition by their N1 subdomains. N1 is shown as a surface, while the other three lobes are shown as cartoon.e. The N1 and N2 lobes in a subunit of TrkA from the NADH-bound TrkH-TrkA complex (top) and the ATPγS-bound TrkA (bottom) structures are shown with their hinge helices highlighted. NADH or ATPγS are shown in green.f. Cartoons of the TrkA, BK, and KtrA gating rings showing only the N lobes. The hinge helices are marked with red rectangles. Description of interfaces as “fixed” or “rotating” is based on comparison of structures 2HMS and 2HMW for KtrA, and 3NAF and 3U6N for BK.
a. Surface representation of the TrkA tetramer from the TrkA-only (top) and TrkH-TrkA structures, viewed from the membrane-facing side. Residues forming the channel-gating ring interface, defined as residues with at least one atom within 4 Å of TrkH in the complex structure, are marked in yellow.b. Diagram illustrating a possible gating mechanism, with the closed channel shown on the left, and the open channel on the right. The D3M2b helix is represented as a red cylinder. For clarity, gating of only one TrkH protomer is shown.c. Left panel, single-channel currents through the TrkH-TrkA (Δ-loop-wt) complex before and after addition of ATP. The holding potential is +50 mV. Right panel, the Popen of TrkH-TrkA (wt-wt) and TrkH-TrkA (Δ-loop-wt) before and after addition of 5mM ATP. The error bars are s.e.m from 3 independent patches.
References
- Doyle DA, et al. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science. 1998;280:69–77. - PubMed
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