Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 (T217/246/223/251) Antibody

What is the Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 (T217/246/223/251) Antibody and what epitope does it recognize?

The Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 (T217/246/223/251) Antibody is a polyclonal antibody produced in rabbits that specifically detects the phosphorylated forms of KCNQ2, KCNQ3, KCNQ4, and KCNQ5 potassium channel proteins at their respective threonine residues (T217, T246, T223, and T251). This antibody recognizes a conserved phosphorylation site in the S4-S5 loop of these channel proteins, a region critical for channel gating and conductance . The immunogen used for antibody production is a synthesized peptide derived from human KCNQ2/3/4/5 sequences around these phosphorylation sites, specifically in the amino acid range of 191-240 .

The specificity of this antibody is significant because it allows for the detection of these KCNQ channels only when they are phosphorylated at these specific threonine residues, making it a valuable tool for studying post-translational modifications that regulate channel function .

What are the recommended experimental applications and optimal working dilutions for this antibody?

The Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 (T217/246/223/251) Antibody has been validated for multiple experimental applications with the following recommended dilutions:

The antibody has been tested for reactivity with human, mouse, and rat samples, making it suitable for comparative studies across these species . For optimal results in immunohistochemistry applications with brain tissue, a retrieval protocol may be necessary before blocking, involving treatment with 0.3 M glycine in PBS for 30 minutes at room temperature followed by incubation with 10 mM citrate buffer (pH 6) for 30 minutes at 80°C .

How can researchers validate the specificity of this phospho-specific antibody in their experimental systems?

Validating antibody specificity is crucial for reliable research outcomes. For the Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 antibody, consider these methodological approaches:

• Phosphatase Treatment Control: Treating one sample with lambda phosphatase before immunoblotting should eliminate the signal if the antibody is truly phospho-specific.

• Mutational Analysis: Express wild-type channels alongside mutants where the threonine residues (T217/246/223/251) are substituted with alanine (preventing phosphorylation) or aspartate/glutamate (mimicking phosphorylation) in heterologous expression systems like HEK293 cells .

• Peptide Competition Assay: Pre-incubate the antibody with the phosphorylated peptide immunogen to block specific binding.

• Knockout/Knockdown Validation: As demonstrated in KCNQ4 knockout studies, using tissue from knockout animals provides an excellent negative control for antibody specificity .

• Mass Spectrometry Correlation: For advanced validation, correlate antibody-based detection with mass spectrometry identification of phosphorylated residues. This approach has been successful in identifying KCNQ phosphorylation sites, including those in the S4-S5 loop .

What is the functional significance of phosphorylation at T217/246/223/251 in KCNQ channel physiology?

Phosphorylation at T217/246/223/251 in the S4-S5 loop of KCNQ channels has substantial functional implications:

The S4-S5 loop acts as a critical structural element that couples voltage sensing (S4) to pore opening (S5), making it a key region for channel gating and conductance regulation . Mass spectrometry studies have revealed that this region undergoes phosphorylation, suggesting it serves as a regulatory mechanism for channel function .

Functionally, phosphorylation in this region may influence:

• Voltage-dependent Activation: Modification of the S4-S5 loop can alter the voltage sensitivity of the channel.

• Channel Conductance: The addition of a phosphate group with its negative charge can modulate ion flow through the channel pore.

• Subunit Interactions: Phosphorylation may affect how KCNQ2 interacts with its partners KCNQ3 and KCNQ5 in heteromeric channels .

• Neuronal Excitability: As these channels contribute to the M-current, a slowly activating and deactivating potassium conductance, their phosphorylation status directly impacts neuronal excitability and responsiveness to synaptic inputs .

Research using site-directed mutagenesis and electrophysiology has been instrumental in elucidating these functional consequences of KCNQ phosphorylation .

What experimental approaches can be used to correlate KCNQ channel phosphorylation with changes in channel function?

To establish correlations between KCNQ phosphorylation and functional changes, researchers should consider these methodological approaches:

• Electrophysiological Recordings with Phosphorylation Manipulation:

  • Express wild-type or mutant channels (T→A or T→D/E) in heterologous systems like Xenopus oocytes or HEK293 cells

  • Record currents using patch-clamp techniques while manipulating phosphorylation through:
    a) Kinase activators/inhibitors
    b) Phosphatase treatments
    c) Intracellular application of ATP/GTP analogs

• Combined Biochemical and Functional Assays:

  • Perform Western blotting with the phospho-specific antibody on samples subjected to various physiological stimuli

  • In parallel, conduct electrophysiological recordings under identical conditions

  • Correlate phosphorylation levels with functional parameters (activation kinetics, voltage dependence, etc.)

• Molecular Dynamics Simulations:

  • Use structural data to model how phosphorylation at these specific residues affects S4-S5 loop conformation

  • Predict functional consequences that can be tested experimentally

• Mass Spectrometry with Functional Correlation:

  • Quantify phosphorylation stoichiometry using techniques like LC/MS/MS

  • Correlate with functional data from the same preparations

Studies have demonstrated that KCNQ2/KCNQ3 heteromeric currents can be increased by intracellular cyclic AMP through phosphorylation-dependent mechanisms, providing a model system for testing phosphorylation-function relationships .

How should researchers optimize sample preparation for detecting phosphorylated KCNQ channels in tissues and cells?

Optimal sample preparation is critical for preserving phosphorylation status and maximizing detection sensitivity:

• Rapid Sample Processing:

  • Harvest tissues quickly and flash-freeze immediately to prevent phosphatase activity

  • For cultured cells, rapidly lyse in buffer containing phosphatase inhibitors

• Phosphatase Inhibitor Cocktail Components:

  • Include sodium fluoride (50 mM), sodium orthovanadate (1 mM), and β-glycerophosphate (10 mM)

  • Add commercially available phosphatase inhibitor cocktails containing calyculin A and okadaic acid

• Lysis Buffer Optimization:

  • Use Non-idet P-40 lysis buffer as demonstrated in co-immunoprecipitation studies

  • Maintain cold temperature (4°C) throughout processing

  • Include protease inhibitors to prevent degradation

• Tissue-Specific Considerations:

  • For brain tissues, employ region-specific dissection techniques as demonstrated in studies isolating the PPN (pedunculopontine nucleus)

  • Process rapidly to maintain phosphorylation state in neuronal samples

• Fixation for Immunohistochemistry/Immunofluorescence:

  • For tissue sections, post-fix in 4% paraformaldehyde

  • Employ antigen retrieval with 0.3 M glycine in PBS (30 min at room temperature) followed by 10 mM citrate buffer pH 6 (30 min at 80°C)

  • Permeabilize with 2% Nonidet and 1% bovine serum albumin

These protocols have been validated in studies examining KCNQ channel phosphorylation in both heterologous expression systems and native tissues .

What are the best practices for validating phospho-specific antibody signals through comparative analysis?

To ensure reliable interpretation of results with phospho-specific antibodies, implement these validation strategies:

• Parallel Detection with Total Protein Antibodies:

  • Always run parallel samples with antibodies detecting total (phosphorylated and non-phosphorylated) KCNQ channels

  • Calculate phosphorylation ratios (phospho/total) for quantitative analysis

• Multiple Detection Methods:

  • Combine antibody-based detection with mass spectrometry for orthogonal validation

  • Mass spectrometry approaches have successfully identified phosphopeptides from KCNQ2/KCNQ3, including those in the S4-S5 loop

• Experimental Manipulations:

  • Include samples with manipulated phosphorylation status:
    a) Phosphatase-treated samples as negative controls
    b) Samples treated with kinase activators as positive controls
    c) Expression of phospho-mimetic mutants (T→D or T→E)

• Signal Quantification Standards:

  • Use densitometry with appropriate normalization controls

  • When possible, include calibrated phosphopeptide standards for absolute quantification

• Specificity Controls in Complex Samples:

  • For brain tissue analysis, include samples from knockout models as demonstrated with KCNQ4 knockout mice

  • Use peptide competition assays with both phosphorylated and non-phosphorylated peptides

These practices ensure that signals detected truly represent the phosphorylation status of KCNQ channels rather than artifacts or non-specific binding.

How can the Phospho-KCNQ2/KCNQ3/KCNQ4/KCNQ5 antibody be used effectively in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) experiments with phospho-specific antibodies require careful optimization:

• Recommended Protocol:

  • Lyse cells or tissues in Non-idet P-40 lysis buffer containing phosphatase inhibitors

  • Pre-clear lysates with protein A/G magnetic beads

  • Incubate Protein A/G Magnetic Beads with phospho-KCNQ antibody at 4°C for 2-3 hours

  • Incubate the antibody-bead complex with pre-cleared lysate for 3-4 hours at 4°C

  • Wash extensively with phosphatase inhibitor-containing buffer

  • Elute proteins in loading buffer for SDS-PAGE and Western blot analysis

• Verification Strategies:

  • Use reciprocal Co-IP approaches (e.g., pull down with phospho-antibody and detect interacting partners, then perform the reverse)

  • Include IgG control immunoprecipitations to identify non-specific binding proteins

• Analyzing Complex Formation:

  • This approach has been successfully used to demonstrate that KCNQ2 can form heteromeric channels with both KCNQ3 and KCNQ5

  • Western blotting with specific antibodies for each KCNQ subtype allows determination of complex composition

• Quantitative Analysis:

  • Quantify protein bands using densitometry with ImageJ software

  • Calculate relative enrichment compared to input and IgG controls

This approach has been validated in studies examining both heterologously expressed channels in HEK293T cells and native channel complexes in brain tissue samples .

What kinases and signaling pathways regulate phosphorylation of KCNQ channels at the T217/246/223/251 sites?

The regulation of KCNQ channel phosphorylation involves multiple kinases and signaling cascades:

• Cyclic AMP-Dependent Pathways:

  • KCNQ2/KCNQ3 heteromeric currents can be increased by intracellular cyclic AMP, suggesting PKA involvement in phosphorylation

  • This pathway has implications for neuronal excitability modulation

• Key Regulatory Kinases:

  • While the specific kinases phosphorylating T217/246/223/251 are not explicitly identified in the search results, studies on KCNQ channels have implicated:
    a) Protein Kinase A (PKA)
    b) Protein Kinase C (PKC)
    c) Serine/threonine kinases

• Physiological Modulators:

  • Muscarinic receptor activation with agonists like oxotremorine-M can suppress KCNQ currents, potentially through altered phosphorylation

  • Beta-adrenergic stimulation influences KCNQ-mediated currents through cAMP-dependent mechanisms

• Experimental Approaches to Identify Regulatory Kinases:

  • Pharmacological inhibition/activation of specific kinases followed by Western blotting with the phospho-specific antibody

  • In vitro kinase assays with purified kinases and KCNQ peptides

  • Mass spectrometry analysis of phosphopeptides under various kinase activator/inhibitor conditions

• Cross-talk with Other Post-translational Modifications:

  • KCNQ channels are also regulated by ubiquitination through NEDD4L, which may interact with phosphorylation-dependent mechanisms

Understanding these regulatory pathways is essential for developing therapeutic strategies targeting KCNQ channels in epilepsy, cardiac arrhythmias, and other disorders.

How does phosphorylation at T217/246/223/251 affect heteromeric assembly of KCNQ channel subunits?

The impact of phosphorylation on KCNQ channel assembly reveals complex regulatory mechanisms:

• Heteromeric Channel Composition:

  • Research has demonstrated that KCNQ2 can form heteromeric channels not only with KCNQ3 (the traditional view) but also with KCNQ5, independent of KCNQ3

  • This discovery challenges the 30-year prevailing view that brain KCNQ channels consist primarily of KCNQ2/3 and possibly KCNQ3/5 heteromers

• Phosphorylation and Subunit Interactions:

  • Phosphorylation in the S4-S5 loop region (T217/246/223/251) may influence subunit compatibility and assembly preferences

  • Studies using epitope-tagged knockin mice and split-intein-mediated protein trans-splicing have revealed unexpected subunit compositions that could be regulated by phosphorylation status

• Experimental Evidence from Proteomic Analysis:

  • Mass spectrometry of immunoprecipitated KCNQ2 has identified significant spectral counts from KCNQ3 (25.1 ± 2.8% sequence coverage) and KCNQ5 (11.3 ± 1.4% sequence coverage) in cortical and hippocampal samples

  • This indicates direct binding rather than association through adaptor proteins

• Validation through Heterologous Expression:

  • Expression of FLAG-tagged KCNQ2 with KCNQ3 and KCNQ5 in HEK293T cells followed by co-immunoprecipitation confirmed that KCNQ2 and KCNQ5 interact directly, both with and without KCNQ3 coexpression

• Functional Implications:

  • The diversity of KCNQ channel subunit stoichiometry in the brain appears to be much richer than previously assumed

  • This has significant implications for understanding genotype-phenotype relationships in KCNQ-related disorders and for drug screening strategies

These findings suggest that phosphorylation may be a key regulatory mechanism controlling the composition of KCNQ channel complexes in the brain.

What methodological approaches can differentiate between phosphorylation of different KCNQ subtypes using this antibody?

Distinguishing phosphorylation across different KCNQ subtypes requires sophisticated experimental strategies:

• Combined Immunoprecipitation and Mass Spectrometry:

  • Immunoprecipitate with subtype-specific antibodies (e.g., anti-KCNQ2, anti-KCNQ3)

  • Analyze precipitates by Western blotting with the phospho-specific antibody

  • Confirm by mass spectrometry to identify phosphopeptides specific to each subtype

  • This approach has been successfully used to identify phosphopeptides from KCNQ2 and KCNQ3

• Subtype-Selective Expression Systems:

  • Express individual KCNQ subtypes in heterologous systems

  • Treat with phosphorylation-promoting conditions

  • Compare phospho-antibody reactivity across subtypes

  • Verify with phosphorylation site mutants

• Tissue-Specific Expression Patterns:

  • Leverage differential expression patterns (e.g., KCNQ4 is restricted to auditory brainstem nuclei, while KCNQ2/3/5 are more widely expressed in the CNS)

  • Compare phospho-antibody signals across brain regions with known subtype expression profiles

• Sequential Immunodepletion:

  • Deplete samples sequentially with subtype-specific antibodies

  • Analyze remaining phospho-signal to determine relative contribution of each subtype

• Genetic Models:

  • Utilize tissue from knockout or knockin models (e.g., FLAG-tagged KCNQ2 knockin mice)

  • Compare phospho-antibody reactivity between wild-type and genetic models

These approaches can help researchers determine the relative phosphorylation levels of different KCNQ subtypes in complex biological samples.

How can researchers investigate the role of KCNQ channel phosphorylation in neurological disorders?

Investigating KCNQ phosphorylation in neurological disorders requires multidisciplinary approaches:

• Disease Model Systems:

  • Animal models of epilepsy, a key KCNQ-related disorder

  • Patient-derived induced pluripotent stem cells (iPSCs) differentiated into neurons

  • Heterologous expression systems with disease-associated KCNQ mutations

• Comparative Phosphorylation Analysis:

  • Compare phosphorylation levels between control and disease tissues/cells using the phospho-specific antibody

  • Correlate with functional changes in M-current and neuronal excitability

  • KCNQ2 is associated with Epileptic Encephalopathy, Early Infantile, 7 and Seizures, Benign Familial Neonatal 1

• Genetic-Phosphorylation Interactions:

  • Examine how disease-associated mutations (e.g., P441L in KCNQ1 associated with long QT syndrome ) affect phosphorylation patterns

  • Test if phosphorylation status modifies the functional consequences of mutations

• Therapeutic Targeting:

  • Screen compounds that modulate KCNQ channel phosphorylation

  • Assess effects on channel function and disease phenotypes

  • This approach may reveal targets for therapeutic intervention in disorders like epilepsy

• Combined Structural-Functional Approaches:

  • Use structural biology to model how disease mutations and phosphorylation interact

  • Test predictions with electrophysiology and biochemical assays

• Spatiotemporal Regulation:

  • Investigate developmental changes in phosphorylation patterns

  • Map regional differences in brain phosphorylation using immunohistochemistry

  • Correlate with onset and progression of neurological symptoms

These strategies can provide insights into how altered KCNQ phosphorylation contributes to disease pathogenesis and identify potential therapeutic targets.

What are the challenges and solutions for imaging the spatial distribution of phosphorylated KCNQ channels in brain tissue?

Visualizing phosphorylated KCNQ channels in the brain presents several technical challenges:

• Fixation and Epitope Preservation:

  • Challenge: Phospho-epitopes are sensitive to fixation conditions

  • Solution: Optimize with 4% paraformaldehyde fixation followed by specific antigen retrieval with glycine/citrate buffer treatment

• Antibody Specificity in Complex Tissues:

  • Challenge: Distinguishing specific from non-specific signals

  • Solution: Include knockout controls as demonstrated with KCNQ4 KO mice and implement peptide competition controls with phosphorylated and non-phosphorylated peptides

• Signal Amplification:

  • Challenge: Low abundance of phosphorylated channels

  • Solution: Use tyramide signal amplification or high-sensitivity detection systems

• Co-localization with Cell Type Markers:

  • Challenge: Identifying cell types expressing phosphorylated channels

  • Solution: Combine with markers like ChAT for cholinergic neurons, as demonstrated in studies of KCNQ in the PPN

• Quantification Methods:

  • Challenge: Obtaining reproducible quantitative data

  • Solution: Use confocal microscopy with standardized acquisition parameters and automated analysis algorithms

• Spatial Resolution:

  • Challenge: Distinguishing membrane vs. intracellular phosphorylated channels

  • Solution: Employ super-resolution microscopy techniques (STORM, STED, PALM)

• Demonstrated Protocol:

  • Use 15-μm coronal brain sections

  • Post-fix in 4% paraformaldehyde

  • Permeabilize with 2% Nonidet and 1% BSA

  • Perform antigen retrieval with glycine/citrate

  • Incubate with primary antibodies for 48 hours

  • Use appropriate fluorescent secondary antibodies

  • Visualize with confocal microscopy

These approaches have been successfully implemented to map the distribution of KCNQ channels in specific brain regions.

How can mass spectrometry complement antibody-based detection of phosphorylated KCNQ channels?

Mass spectrometry provides powerful complementary approaches to antibody-based detection:

• Direct Identification of Phosphorylation Sites:

  • Mass spectrometry can definitively identify phosphorylated residues, as demonstrated in studies that revealed phosphorylation in the S4-S5 loop of KCNQ2/KCNQ3

  • Techniques include MALDI-TOF and nano-LC/MS/MS using quadrupole-orthogonal TOF MS/MS

• Comprehensive Phosphorylation Mapping:

  • While antibodies detect specific sites, mass spectrometry can identify all phosphorylation sites simultaneously

  • This has revealed unexpected phosphorylation sites, including those in the C-terminal region important for subunit interactions

• Quantitative Analysis:

  • Approaches for quantifying phosphorylation stoichiometry:
    a) Extract ion chromatograms for phosphorylated and non-phosphorylated peptides
    b) Use isotope labeling strategies (SILAC, TMT, iTRAQ)
    c) Employ parallel reaction monitoring (PRM) for targeted quantification

• Sample Preparation Protocol:

  • Immunoprecipitate KCNQ channels

  • Separate by SDS-PAGE

  • Perform in-gel trypsin digestion

  • Extract and analyze peptides by MALDI-TOF and LC/MS/MS

• Data Analysis Strategies:

  • Submit monoisotopic masses to Protein Prospector for peptide mass mapping with ms-fit

  • Compare to calculated tryptic peptides including all possible phosphopeptides

  • Use internal calibration with trypsin autolysis products to achieve mass accuracy of ≈20 ppm

• Integration with Functional Studies:

  • Correlate mass spectrometry-identified phosphorylation sites with functional data from electrophysiology

  • Use site-directed mutagenesis to validate the functional significance of identified sites

This integrated approach provides the most comprehensive understanding of KCNQ channel phosphorylation dynamics.

What experimental design would best evaluate the temporal dynamics of KCNQ channel phosphorylation?

To investigate temporal dynamics of KCNQ phosphorylation, implement this experimental framework:

• Time-Course Stimulation Protocol:

  • Prepare neurons or heterologous cells expressing KCNQ channels

  • Stimulate with modulators known to affect KCNQ function (neurotransmitters, cAMP-elevating agents)

  • Collect samples at defined time points (seconds to hours)

  • Analyze phosphorylation using the phospho-specific antibody

• Parallel Functional Recordings:

  • Perform patch-clamp recordings of KCNQ currents during stimulation

  • Correlate changes in current with phosphorylation status

  • KCNQ2/KCNQ3 heteromeric current is known to be increased by intracellular cyclic AMP, providing a model system

• Live-Cell Imaging Approaches:

  • Develop FRET-based phosphorylation sensors for KCNQ channels

  • Express in neurons or heterologous cells

  • Monitor phosphorylation in real-time during stimulation

• Pulse-Chase Phosphorylation Analysis:

  • Label cells with radioactive phosphate

  • Chase with non-radioactive phosphate

  • Immunoprecipitate KCNQ channels at various timepoints

  • Determine phosphorylation turnover rates

• Quantitative Western Blot Analysis:

  • Use time-resolved fluorescence or chemiluminescence detection

  • Quantify phospho/total KCNQ ratios at each timepoint

  • Determine kinetics of phosphorylation and dephosphorylation

• Pharmacological Manipulation:

  • Apply kinase inhibitors at different timepoints after stimulation

  • Identify the temporal windows where phosphorylation is critical for functional effects

• Mathematical Modeling:

  • Develop kinetic models of KCNQ phosphorylation/dephosphorylation

  • Fit experimental data to determine rate constants

  • Predict effects of perturbations on temporal dynamics

This comprehensive approach would provide unprecedented insights into the temporal regulation of KCNQ channel phosphorylation and its relationship to channel function.