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. 2016 Oct 20;167(3):763-773.e11.
doi: 10.1016/j.cell.2016.09.048.

The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs

Peter S Shen  1 , Xiaoyong Yang  1 , Paul G DeCaen  2 , Xiaowen Liu  3 , David Bulkley  4 , David E Clapham  5 , Erhu Cao  6
Affiliations

The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs

Peter S Shen et al. Cell. .

Abstract

The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.

Keywords: ADPKD; PKD2; TRP channel; TRPP2; cryo-EM; ion channel; polycystic kidney disease; polycystin; single particle electron cryo-microscopy.

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Figures

Figure 1
Figure 1. PKD2 Structure Determined in Lipid Nanodisc
(A) Side and bottom-up views of PKD2 in nano-discs. Individual channel subunits are color coded. Densities of the nanodisc (gray) and well-resolved lipids (purple) are also shown. (B and C) Ribbon representations of a PKD2 sub-unit (B) with different domains denoted and the full tetrameric channel (C) with each of four identical subunits color coded as in (A). See also Figures S1, S2, and S3 and Table S1.
Figure 2
Figure 2. The PKD2-2L1 Chimera Ion Selectivity and Voltage Dependence
(A) Top, topology and alignment of PKD2s. Note that the interconnecting pores of PKD2 and PKD2-L1 are 70% identical; residues that differ are underlined. Bottom, side (only two subunits are shown) and extracellular views of the PKD2 structure indicating the residues from PKD2 that can be grafted into the PKD2-L1 channel to create the chimeric channel (red). (B) PKD2/2-L1 chimera ion selectivity and voltage-dependence. Top, a voltage ramp applied at 0.5 Hz to activate whole-cell currents from HEK293T cells. Middle, representative PKD2-L1 and chimera currents captured in the presence of 140 mM extracellular monovalent ions (divalent free: 1 mM EGTA and 0.5 mM EDTA) or 100 mM CaCl2. Internal (pipette) saline contained 90 mM NaMES, 10 mM Na4BAPTA, and 5 mM EGTA. The voltage transition is expanded in the inset green boxes to clearly show the differences in reversal potential. Right, time course of outward peak current, tail current density, and reversal potential (Erev). Color shading corresponds to the ionic conditions (black, Na+; blue, K+; gray, NMDG; red, Ca2+ shown in left traces). (C) The relationships between relative permeability, inward single-channel conductance, and tail current density of PKD2-L1 and the chimera channels. Data are summarized from the experiments shown in (A) and Figure S5 (n = 4–6 cells; error, SD). See also Figures S4, S5, and S6.
Figure 3
Figure 3. The Architecture of the Trans-membrane Core of PKD2
(A) Solvent-accessible pathway along the ion permeation pore mapped using the HOLE program. Residues located at the selectivity filter and lower gate are rendered as sticks. (B) Radius of pore calculated with the HOLE program. (C) Enrichment of negatively charged residues at the outer pore region of PKD2. Left: the PKD2 outer pore region iscolored by surface electrostatic potential. Right: negatively charged residues (glutamate and aspartate) are shown as sticks. (D) An interaction network that couples pore helix 1 and selectivity filter of one subunit with pore helix 2 and S6 of a neighboring subunit. (E) The structure of PKD2 VSD domain. Two lysine residues at the cytoplasmic end of S4 are likely stabilized by aromatic and negatively charged residues. See also Figure S6.
Figure 4
Figure 4. Role of Polycystin Domain in Channel Assembly and Its Coupling with the Channel Transmembrane Core
(A) Ribbon representation of the polycystin domain with specific structural elements denoted. (B) Channel assembly mediated by the polycystin domain. Left, a groove formed between finger 1 and β4–β5 turn of one polycystin domain (surface) interacts with β3–β4 turn and H1 helix of a neighboring polycystin domain (ribbon). Right: zoomed view indicating detailed interactions. (C) The polycystin domain (surface) interacts with the transmembrane core of the channel at two critical sites as indicated. (D and E) Zoomed views of (C) showing detailed interactions at helix turn (D) and pre-S6 loop (E). See also Figures S3 and S6.
Figure 5
Figure 5. Structural Annotation of Pathogenic PKD2 Mutations
(A) Mapping of human disease-associated missense mutations (red) onto the structure of a PKD2 subunit. Note that the polycystin domain (blue) and pore helix (purple) are mutation hotspots. (B) Missense mutations within the polycystin domain. Note that the majority of these mutated residues (labeled in red) participate in hydrogen bonding interactions denoted with black dash lines or are buried inside a hydrophobic interior.
Figure 6
Figure 6. A Hypothetical Model for PKD2 Gating
(A) Structural features that may play a role in PKD2 gating are shown, including high-energy 310- and π-helices (cyan) and a hydrogen bonding interaction that couples the S4–S5 linker (purple) with the pore-lining S6 helix. Residues that form the lower gate (L677 and N681) and lysine residues residing at the 310 helical region of S4 are also shown. (B) A hypothetical model depicting conformational changes at the 310 and π-helices, shown, respectively, as helices and stars in cyan, drive or accommodate movements of the S4–S5 linker and bending of the S6 helix to open the lower gate. See also Figure S6.
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