DEX effects on TREK-1 currents in human primary TM cells

PRIMARY OUTCOME

TREK-1 current amplitude and membrane potential

EXPECTED OUTCOMES

- 1. Control TM cells will exhibit resting membrane potential of -40 to -60 mV with input resistance of 500-1500 MΩ - 2. DEX-treated cells will show depolarized membrane potential (-25 to -40 mV) compared to vehicle controls (p<0.01) - 3. ML-402 (30 μM) will evoke outward K+ currents of 200-500 pA amplitude in control cells within 2-3 minutes - 4. Chronic DEX treatment will reduce ML-402-evoked current amplitude by 40-60% compared to control cells - 5. DEX-induced current reduction will be dose-dependent, with 1 μM showing greater suppression than 100 nM treatment - 6. ML-402 currents will show partial reversibility (60-80% recovery) within 5-10 minutes of washout - 7. Vehicle control applications will produce minimal current changes (<10% of ML-402 response) confirming drug specificity

SUCCESS CRITERIA

- • Achieve successful whole-cell recordings in >70% of attempted cells with stable access resistance throughout experiment - • Demonstrate statistically significant membrane potential depolarization in DEX-treated cells (p<0.05, n≥12 cells per group) - • Document significant reduction in ML-402-evoked currents following chronic DEX treatment (p<0.05) with effect size >30% - • Show dose-dependent DEX effects with 1 μM treatment producing greater current suppression than 100 nM - • Maintain recording stability with <20% change in series resistance during ML-402 application and washout - • Complete recordings from cells derived from at least 3 different donor tissues to ensure generalizability - • Vehicle control experiments must show minimal current responses (<50 pA) to validate ML-402 specificity

PROTOCOL

**Phase 1: Primary Cell Isolation and Culture** -- Days 1-10 Human trabecular meshwork cells isolated from donor eyes (age 45-75 years, <48h post-mortem) obtained through eye banks with IRB approval. Tissue dissected under sterile conditions and cells cultured in DMEM with 10% FBS, 1% P/S. Validate TM cell identity by immunocytochemistry for myocilin, α-SMA, and fibronectin. Use cells between passages 2-5. Power calculation: n=15 cells per condition for 80% power to detect 30% change in current amplitude. **Phase 2: Chronic Dexamethasone Treatment** -- Days 11-18 Seed cells on glass coverslips in 35mm dishes. Chronic DEX treatment protocol: Control (vehicle, 0.1% ethanol), DEX 100 nM, DEX 1 μM for 72-96 hours. Replace medium and drugs every 48 hours. Assess cell viability by calcein-AM staining (>95% viability required). Document morphological changes by phase-contrast microscopy. **Phase 3: Patch-Clamp Setup and Calibration** -- Days 19-20 Patch-clamp amplifier (Axopatch 200B) and digitizer (Digidata 1550) calibrated. Borosilicate pipettes pulled to 3-5 MΩ resistance. External solution: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4, 300 mOsm. Internal solution: 140 mM KCl, 1 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 3 mM Na2ATP, pH 7.2, 290 mOsm. **Phase 4: Whole-Cell Current Recordings** -- Days 21-28 Whole-cell patch-clamp recordings at room temperature. Establish gigaseals (>2 GΩ) and break into whole-cell mode. Series resistance <15 MΩ with >70% compensation. Measure resting membrane potential immediately after break-in. Record baseline K+ currents using voltage steps (-80 to +60 mV, 10 mV increments, 200 ms duration). **Phase 5: ML-402 Response Testing** -- Days 21-28 After stable baseline recording, apply ML-402 (TREK-1 agonist, 30 μM) via perfusion system. Record current responses for 5-10 minutes to assess activation kinetics and steady-state amplitude. Measure ML-402-evoked current as difference between peak and baseline. Test reversibility with washout. Include vehicle control applications (0.1% DMSO). **Phase 6: Data Analysis and Statistical Testing** -- Days 29-32 Data analysis using pClamp 11 and GraphPad Prism. Measure: resting membrane potential, input resistance, ML-402-evoked current amplitude and kinetics. Current density calculated (pA/pF) using cell capacitance. Statistics: unpaired t-tests for between-group comparisons, paired t-tests for before/after drug applications. One-way ANOVA with Tukey's post-hoc for multiple group comparisons. Significance threshold p<0.05.

LINKED HYPOTHESES

HTML iframe

<iframe src="http://scidex.ai/artifact/exp-167553a1-f3c4-49d4-88ee-206df2fd0471?embed=1" width="100%" height="600" style="border:0;border-radius:8px"></iframe>

Markdown link

[DEX effects on TREK-1 currents in human primary TM cells](http://scidex.ai/artifact/exp-167553a1-f3c4-49d4-88ee-206df2fd0471)

Direct URL

http://scidex.ai/artifact/exp-167553a1-f3c4-49d4-88ee-206df2fd0471

Preview