Structural determinants regulating surface expression and function of Kv-related ion channels
Bibliographic record
Abstract
To date, the mechanisms and structural determinants which contribute to the regulation of ion channel trafficking, surface expression and function have only been limitedly explored. Through biophysical and molecular characterization of the hyperpolarization-activated cyclic-nucleotide gated channel 2 (HCN2), we have identified a four-amino acid motif (EEYP) in the B-helix of the cyclic-nucleotide binding domain (CNBD) that strongly promotes channel export from the endoplasmic reticulum (ER) and cell surface expression but does not contribute to the inhibition of channel opening. We further demonstrate that this motif augments a step in the trafficking pathway and/or the efficiency of correct folding and assembly. The role of post-translational modifications, specifically N-linked glycosylation, has also been investigated in two different HCN isoforms. All four mammalian HCN channel isoforms have been shown to undergo N-linked glycosylation in the brain. HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. The evolutionary significance of this isoforms specific regulation is also examined. Finally, the role of palmitoylation in the regulation of Kv4 channels is examined. Using acylbiotin exchange (ABE) chemistry we are able demonstrate that Kv4.2 is present as a palmitoylated protein in both rat cortical neurons and COS-7 cells. Through mutational analysis of the twelve intracellular cysteine residues within Kv4.2, we were able to localize the site of palmitoylation to the intracellular COOH-terminus. Palmitoylation of Kv4.2 does not contribute to the regulation of activation and inactivation gating parameters. Rather, inhibition of palmitoylation through either mutation of COOH-terminal cysteine residues or the pharmacological agent 2-bromopalmitate results in significant reductions in overall current density measurements.
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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".