Differential Recruitment of Kv1.4 and Kv4.2 to Lipid Rafts by PSD-95
Bibliographic record
Abstract
The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation.Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell.Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts.Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains.Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts.We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raftassociated.Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons.Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts.We then assessed the contribution of PSD-95 to the raft association of these channels.Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts.Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant.This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.Voltage-gated potassium (Kv) 1 channels are important determinants of the resting membrane potential and other electrophysiological properties of excitable cells such as neurons and myocytes.In particular, it has been suggested that Kv1.4, which is located axonally and presynaptically (1), modulates action potential propagation (2) and neurotransmitter release (3).In contrast, Kv4.2 is localized to the somatodendritic region of neurons (4), where it is poised to influence the excitability of the postsynaptic membrane (5, 6).The activity of these channels, and therefore their influence on cellular functions, can be significantly altered by protein kinases and phosphatases.The activity of Kv1.4 is known to be affected by protein kinase C (PKC) (7) and tyrosine kinases (8), while Kv4.2 can be phosphorylated by protein kinase A (PKA) (9), PKC (10), and the extracellular signal-related kinase (ERK) (6).Certain isoforms of the above kinases are found in membrane microdomains known as lipid rafts, which are enriched in cholesterol and sphingolipids and are thought to exist in a "liquid-order" state that is distinct from the more fluid, disordered state exhibited by surrounding phospholipids (11,12).This tightly ordered array of cholesterol and sphingolipids more readily incorporates molecules with saturated, unbranched side chains and tends to exclude molecules with unsaturated side chains.Thus, many proteins that reside in rafts, including several protein kinases, G proteins and other members of signal transduction cascades, are often acylated, particularly myristoylated and/or palmitoylated (13).In some cases, this acylation can serve as a targeting signal for lipid rafts (13).The enrichment of signaling molecules in lipid rafts has led to speculation that these microdomains serve to organize and concentrate signaling complexes in the cell.Kv1.4 and Kv4.2 are not known to be acylated.Thus, if they are present in lipid rafts, the mechanism by which they are recruited is not known.Therefore, we first determined if these Kv channels are found in lipid rafts.Both Kv channels interact with postsynaptic density protein 95 (PSD-95) (14, 15), a PSD-95/Dlg/ZO-1 (PDZ) domain protein, which is acylated (specifically, palmitoylated, Ref. 16).Accordingly, we also assessed whether PSD-95 can recruit these channels to lipid rafts.We show that a portion of Kv1.4,Kv4.2, and PSD-95 protein in rat brain membranes is found in lipid raft fractions.We then exploited heterologous expression to determine the role of palmitoylation in the targeting of PSD-95 to raft regions, and demonstrate that a palmitoylation-deficient PSD-95 mutant is not trafficked to rafts.Next, we assessed the contribution of PSD-95 to the lipid raft localization of Kv1.4 and Kv4.2.Coexpression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts, and substituting a palmitoylation-defi-
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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 | 0.001 |
| 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".