Structural and functional mapping of ion access pathways in the human K <sup>+</sup> -dependent Na <sup>+</sup> /Ca <sup>2+</sup> exchanger NCKX2 using cysteine scanning mutagenesis, thiol-modifying reagents, and homology modelling
Bibliographic record
Abstract
K+-dependent Na+/Ca2+ exchanger proteins (NCKX) are members of the CaCA superfamily with critical roles in vision, skin pigmentation, enamel formation, and neuronal functions. Despite their importance, the structural pathways governing cation transport remain unclear. To address this, we conducted a systematic study using cysteine scanning mutagenesis of human NCKX2 combined with the thiol-modifying reagents MTSET and MTSEA to probe the accessibility and functional significance of specific residues. We used homology models of outward-facing and inward-facing NCKX2 states and molecular dynamics (MD) simulations to compare and investigate residue accessibility in human NCKX2 based on the published structures of the archaeal NCK_Mj Na+/Ca2+ exchanger and the human NCX1 Na+/Ca2+ exchanger. Mutant NCKX2 proteins expressed in HEK293 cells revealed diverse effects of MTSET and MTSEA on Ca2+ transport. Of the 146 cysteine substitutions analyzed, 35 exhibited significant changes in Ca2+ transport activity upon treatment with MTSET, with 16 showing near-complete inhibition and six demonstrating increased activity. Residues within the cation binding sites and extracellular access channels were sensitive to modification, consistent with their critical role in ion transport, whereas intracellular residues showed minimal accessibility to MTSET but were inhibited by membrane-permeable MTSEA. Water accessibility maps from MD simulations corroborated these findings, providing a high-resolution view of water-accessible pathways. This study provides a comprehensive structural and functional map of NCKX2 ion access pathways, offering insights into the molecular basis of ion selectivity and transport. These findings highlight the key residues critical for cation binding and transport, advancing our understanding of the structural dynamics of NCKX2.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".