Pathogenic Variants in the Polycystin Pore Helix Cause Distinct Forms of Channel Dysfunction

Biophysics Basic Medical Sciences Nephrology Cell Biology Biochemistry Genetics Life Sciences Medicine
autosomal dominant polycystic kidney disease polycystin PKD2 ion channels cryo-EM channel dysfunction protein assembly

Molecular Mechanism Analysis of ADPKD Pathogenic Variants in Ion Channels – In-depth Interpretation of PNAS 2025 Latest Original Research

I. Academic Research Background and Scientific Significance

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic disorders worldwide, affecting millions of individuals. The pathogenesis of ADPKD is closely related to genetic variants in renal polycystins PKD1 and PKD2, both of which serve as ion channel subunits playing critical roles in the primary cilia of cells. Although research on ADPKD has advanced considerably in recent years, direct evidence and systematic elucidation of how most pathogenic variants of PKD1/PKD2 affect the protein structure and function remain lacking, due to the large variety of disease-causing mutations.

Currently, there is no curative drug for ADPKD; clinical treatments are primarily symptomatic and do not directly address the root cause of channel dysfunction. ADPKD is classified both as a “channelopathy” and a “ciliopathy,” and the combined ion channel dysregulation is considered a central mechanism of disease onset. Therefore, elucidating how disease-causing mutations impact the structure and function of PKD2 ion channels, and thus cause disease, remains an urgent and significant challenge for basic and clinical research. Notably, understanding the molecular basis by which variants affect channel assembly, gating mechanisms, and ciliary targeting is highly instructive for the development of new targeted therapies.

Based on this context, the present study aims to elucidate how ADPKD pathogenic PKD2 variants—focusing on three representative missense mutations located in the Pore Helix 1 domain—cause channel dysfunction, assembly defects, or defects in ciliary trafficking through distinct mechanisms. The study investigates their pathogenicity at molecular, cellular, and functional levels, thereby providing theoretical foundations for ADPKD precision therapies.

II. Paper Source and Author Information

This study, titled “Pathogenic variants in the polycystin pore helix cause distinct forms of channel dysfunction,” was published on June 12, 2025, in volume 122, issue 24 of the Proceedings of the National Academy of Sciences of the United States of America (PNAS). The research was conducted by a team from leading North American medical and chemical research institutions. The first authors are Orhi Esarte Palomero and Eduardo Guadarrama; the corresponding author is Paul G. DeCaen. All are affiliated with the Feinberg School of Medicine and The Chemistry of Life Processes Institute at Northwestern University. The paper was a direct submission to PNAS, operates under an open-access policy, and provides comprehensive public datasets and structural coordinates, enabling reanalysis by peers.

III. Detailed Original Research Workflow

1. Study Design and Overall Approach

The study focuses on three ADPKD-causing PKD2 missense mutations (f629s, c632r, r638c, mutation notation as in the original article) located in Pore Helix 1 (PH1). It investigates the molecular impact mechanisms of each pathogenic variant across multiple levels: molecular expression and conformation, protein thermostability, channel assembly, ciliary delivery, and single-channel physiological function. Key techniques include direct cilia electrophysiology, high-resolution cryo-electron microscopy (Cryo-EM), and super-resolution 3D-structured illumination microscopy (3D-SIM), representing multidisciplinary innovation.

2. Stepwise Research Detailing

a) Protein Expression, Purification, and Assembly Detection

  • Sample Generation and Variant Design: The three PKD2 variants (f629s, c632r, r638c) and wild-type PKD2 (WT) were transiently transfected and expressed in HEK293 cells, using N-terminal Strep-tag fusion for improved protein monodispersity in a deglycosylated background.
  • Assembly Detection and Aggregation State Analysis: Following extraction with n-dodecyl-β-D-maltoside (DDM), size exclusion chromatography (SEC) was used to determine whether proteins formed functional tetramers; protein thermostability was assessed using dye-based thermal shift assays (Glomelt), recording protein folding/unfolding at various temperatures.

b) Cryo-EM Structural Analysis

  • Sample Preparation: f629s and r638c, capable of forming homogeneous tetramers, were prepared for cryo-EM analysis. Other variants like c632r, due to instability and failed assembly, could not undergo structure determination.
  • Data Collection and Analysis: Over 330,000 to 660,000 protein particles were acquired and reconstructed by Cryosparc and single-particle workflow, yielding 3D structures at 2.7–2.8 Å resolution. Structure modeling and refinement utilized AlphaFold2/3, Phenix, ChimeraX, and other tools to ensure model accuracy.

c) Structural-Functional Correlation Analysis

  • Pore Domain Structural Comparison: Compared minimum pore radii (rmin) and key amino acid sidechain arrangements at the “gate” regions of the pore domain in wild-type and variant channels. The focus was on how PH1, selectivity filter, and S6 transmembrane helix interactions are perturbed by point mutations, leading to long-range allosteric coupling effects.
  • Pore Diameter and Channel State: Quantitative pore analysis (Hole Analysis) determined whether the channels were in non-conductive closed conformations, inferring the magnitude and mechanism of variant effects on internal/external gating via molecular interaction networks.

d) Super-resolution Imaging of Ciliary Trafficking

  • Cell Lines and Experimental Pipeline: Using CRISPR/Cas9-engineered dual knockout HEK cells (PKD1 and PKD2 deleted), stably expressing cilia marker ARL13b-GFP, with transient transfection of HA-tagged PKD2 variants.
  • Imaging and Quantification: 3D-SIM and co-localization analysis quantified the ability of variant channels to localize within primary cilia. Cilia length changes were measured as a functional index. Total protein expression was normalized by Western blot to avoid confounds from expression level differences.

e) Direct Cilia Single-Channel Electrophysiology

  • Electrophysiological Methods and Data Acquisition: High-resistance sealing between microelectrodes and the cilia membrane of individual cells allowed native membrane single-channel voltage-clamp recording. Critical gating parameters—activation voltage (V1/2), open probability (Po), unitary conductance (γ), and free energy requirement (ΔG°)—were analyzed for each variant under depolarization.
  • Data Analysis: Open probability–voltage relationships were fitted using the Boltzmann equation; single-channel conductance was assessed by linear regression.

f) AlphaFold3 Molecular Modeling and Structural Prediction

  • For Assembly-defective Variant (c632r): AlphaFold3 was used to generate structure prediction models. Atomic-level spatial clashes and conformational distortions were analyzed to explain assembly defects at the molecular level.

3. Data Analysis and Computational Tools

Across all stages, experimental data analysis adopted Cryosparc (cryo-EM), Igor Pro/Origin (electrophysiology), GraphPad (statistics), ChimeraX, Phenix, and ISOLDE (structure bioinformatics) for structure revision and validation.

IV. In-depth Interpretation of Major Findings

1. Protein Assembly and Thermostability Results

The three variants displayed distinct biochemical outcomes: - WT, f629s, r638c exhibited monodisperse peaks in SEC, indicating functional tetrameric assembly and molecular integrity. - c632r showed marked protein dissociation and heterogeneity, indicative of structural instability. - Thermal denaturation further confirmed that c632r denatured massively at physiological temperature (37–38°C), while the other two variants unfolded only above 50°C, showing basic thermal stability. Conclusion: Certain PH1-site variants (e.g., c632r) can fully disrupt PKD2 assembly stability, fundamentally abolishing physiological function.

2. High-resolution Cryo-EM 3D Structures

  • f629s and r638c Structures: Both maintained the primary PKD2 tetrameric architecture, each subunit containing intact S1–S6 transmembrane helices. VSD was coiled in the “deactivated” state consistent with zero-membrane potential, and the PD (pore domain) showed two restriction regions (external gate L641-N643, internal gate L677).
  • Both variants preserved the PH1 secondary helical axis, but all key local interactions were missing; e.g., f629s replaced a hydrophobic residue with hydrophilic Ser, disrupting the hydrophobic S5 pocket (L609, A612); r638c lost a positively charged group, destroying the PH1/selectivity filter triple hydrogen-bond network.
  • Strikingly, despite the mutations being local and atomic-level, they induced a 6–8 Å “internal gate collapse” and asymmetry in the channel, greatly stabilizing channel closure.
  • Mutational influence at the external gate was limited (max δrmin<0.24Å), but at the internal gate was substantial, supporting a strong allosteric coupling mechanism.

3. Ciliary Trafficking and Morphological Effects

  • Super-resolution imaging confirmed: c632r failed to enter cilia, remaining in the cell body and highlighting that assembly defects directly cause loss of ciliary trafficking; f629s and r638c reached cilia, demonstrating successful delivery.
  • Cilia length quantification revealed shorter cilia in mutants versus WT, most severe in c632r (from 4.7μm to 2.4μm), suggesting that functional PKD2 expression actively supports cilia formation and stability.

4. Single-channel Functional Kinetic Analysis

  • Only WT and f629s/r638c showed measurable current in cilia membranes (voltage-dependent); c632r and PKD1/2 double knockout controls had no current, confirming loss of function.
  • f629s and r638c exhibited a positive shift in activation voltage by 27–32 mV, a 133–152% increase in free energy requirement to open the channel, and significantly reduced single-channel conductance, indicating greater gating barrier and reduced ion flow.
  • In summary, c632r represents complete loss of function (assembly and trafficking); f629s and r638c exhibit partial loss due to compromised gating and conductance.

5. AlphaFold3 Modeling Reveals ‘Assembly-Eliminating’ Mechanism

  • For c632r, AI models showed atomic-level steric clash between the mutated site and S5 region Y616, effectively “jamming” assembly and fully destroying channel function—consistent with experimental protein instability and aggregation.

V. Conclusions, Scientific/Clinical Significance, and Methodological Innovations

1. Scientific Conclusions

This study systematically reveals for the first time the molecular and functional diversity among three ADPKD-related PKD2-PH1 missense variants. Different mutations lead to: A) defective protein assembly and ciliary trafficking (c632r); B) nearly normal cilia localization but elevated gating energy barrier and reduced conductivity (f629s, r638c). Even within the same structural domain, pathogenic mechanisms remain highly diverse, highlighting the importance of individualized targeted therapies for each genetic background.

2. Scientific and Applied Significance

  • Basic Scientific Value: The study elucidates, for the first time, the allosteric coupling between PH1 and S6 in PKD2’s pore domain, providing strong evidence for the “channelopathy-ciliopathy” pathological mechanism.
  • Clinical Outlook: Differentiating pathogenic variants guides the strategy for precise drug design, such as chemical channel activators for cilia-localized dysfunctional variants or molecular chaperones (“correctors”) for assembly-defective types.
  • Drug Development: Unveiling the “gene-structure-function-disease” chain offers a theoretical foundation for new ADPKD drug targets and mechanisms.

3. Methodological Highlights and Innovations

  • Direct native human cell cilia single-channel electrophysiology sets a gold standard for channelopathy studies.
  • Integration of high-resolution cryo-EM, super-resolution microscopy, and AI structural prediction forms a cross-disciplinary molecular-cellular-functional paradigm.
  • Provides raw cryo-EM datasets and structural coordinates to support global reproducibility and further research.

VI. Research Highlights and Future Directions

Key highlights of this study: - Discovery that mutations in the same structural region of ADPKD can cause radically different molecular outcomes, exemplifying the diversity of genetic channelopathies. - Revelation of the PH1 site’s long-range regulatory effects challenges the “local mutation-local effect” intuition. - Offers important cues for individualized ADPKD precision therapies. - The multidisciplinary technological route is highly replicable, applicable to mechanism research in other channelopathies and rare diseases.

Future research directions: further explore structural and functional regulation of PKD1-PKD2 heteromeric complexes in various mutation contexts; apply novel imaging to finely map protein distribution in cell compartments; and assess the targeted effects of new lead compounds in different genotypes.

VII. Supplementary Information

The research received support from the NIH, PKD Foundation, and made full use of international-scale facilities including Cryo-EM and AI computing clusters. The paper provides extensive experimental details, structure data, and analysis code (e.g., RCSB PDB codes 9DWQ/9DLI, Northwestern arch codes), facilitating global reproducibility and secondary analysis.

This study stands as a model for decoding the molecular mechanisms of ADPKD variants and opens up an entirely new vista in the era of “structure-based precision medicine.”