Phospho-KCNC4 (S15) Antibody

What is KCNC4 and why is the phosphorylation at serine 15 significant?

KCNC4, also known as Kv3.4, is a potassium voltage-gated channel subfamily C member 4 that mediates voltage-dependent potassium ion permeability in excitable membranes. This channel belongs to the delayed rectifier class of channel proteins and plays a critical role in neuronal excitability .

The phosphorylation at serine 15 (S15) is particularly significant because it modulates the channel's N-type inactivation mechanism. Research demonstrates that PKC-dependent phosphorylation of serine residues within the Kv3.4 N-terminal inactivation domain (NTID) dramatically slows or eliminates fast inactivation . This phosphorylation triggers cooperative conformational changes that render the NTID unstructured and incapable of causing fast N-type inactivation . In pain-sensing neurons, this modulation affects action potential repolarization, representing a mechanism of neural plasticity with potentially significant implications in the transition from acute to chronic pain .

How does the Phospho-KCNC4 (S15) Antibody differ from general KCNC4 antibodies?

Phospho-KCNC4 (S15) Antibody specifically recognizes KCNC4 protein only when phosphorylated at the serine 15 position, whereas general KCNC4 antibodies detect the total protein regardless of its phosphorylation state . This specificity is achieved through careful immunogen design using synthesized phospho-peptides derived from the region surrounding the S15 phosphorylation site of human Kv3.4 .

The antibody undergoes affinity purification from rabbit antiserum using epitope-specific immunogen chromatography to ensure high specificity . This phospho-specific detection capability allows researchers to distinguish the phosphorylated form from the non-phosphorylated form, enabling precise studies of PKC-mediated signaling pathways that regulate channel function in neuronal excitability.

What are the recommended applications and dilutions for Phospho-KCNC4 (S15) Antibody?

Based on product specifications from multiple sources, the Phospho-KCNC4 (S15) Antibody has been validated for the following applications with specific recommended dilutions:

The antibody is reactive to human and mouse KCNC4 , and has been affinity-purified to ensure specificity for the phosphorylated S15 residue . For optimal results, researchers should conduct preliminary dilution tests in their specific experimental systems, as the working concentration may vary depending on sample type and detection method.

How should samples be prepared to preserve KCNC4 phosphorylation state?

Preserving the phosphorylation state of KCNC4 at S15 requires careful sample handling:

• Immediate processing: Phosphorylation states can rapidly change due to endogenous phosphatase activity. Process samples immediately after collection or flash-freeze in liquid nitrogen .

• Buffer composition: Use lysis buffers containing multiple phosphatase inhibitors including sodium fluoride, sodium orthovanadate, and β-glycerophosphate. The recommended buffer formulation typically includes PBS with 50% glycerol, 0.5% BSA, and 0.02% sodium azide .

• Temperature control: Maintain samples at cold temperatures (4°C or below) throughout processing. Store antibodies at -20°C or -80°C and avoid repeated freeze-thaw cycles .

• Fixation for immunostaining: For immunohistochemistry or immunofluorescence, rapid fixation is essential. Include phosphatase inhibitors in fixation solutions when possible.

• Electrophysiological studies: When combining with patch-clamp techniques, minimally invasive approaches like cell-attached patch-clamping can help preserve the native phosphorylation state of channels .

These methodological considerations are crucial for obtaining reliable results when studying the dynamic phosphorylation of KCNC4 channels in neuronal systems.

How does PKC-mediated phosphorylation of KCNC4 at S15 affect neuronal excitability?

PKC-mediated phosphorylation of KCNC4 at S15 has profound effects on neuronal excitability, particularly in pain-sensing neurons:

• Biophysical effects: Phosphorylation dramatically slows or eliminates fast N-type inactivation of Kv3.4 channels . This occurs through conformational changes in the N-terminal inactivation domain that render it unstructured and incapable of causing fast inactivation .

• Action potential modulation: The slowed inactivation enhances the channel's ability to influence action potential repolarization. Research demonstrates that this modulation accelerates the repolarization phase and reduces action potential duration by approximately 25% .

• Signaling mechanism: This modulation appears to occur in a membrane-delimited manner, suggesting a localized Kv3.4 channel signaling complex . G-protein coupled receptor (GPCR) agonists can activate PKC and recapitulate the effects on action potential repolarization .

• Functional significance: This phosphorylation-dependent modulation represents a novel mechanism of neural plasticity that may contribute to altered excitability in pain pathways. Researchers have concluded that "phosphorylation of the Kv3.4 channel inactivation gate is a mechanism by which pain-sensing neurons shape action potential repolarization" .

To investigate these effects, researchers can employ a combination of electrophysiological techniques (patch-clamping), pharmacological interventions (PKC activators/inhibitors), and molecular approaches (Kv3.4 siRNA) to manipulate and measure the impact of S15 phosphorylation on neuronal function .

How can Phospho-KCNC4 (S15) Antibody be integrated with other techniques for comprehensive signaling studies?

Integrating Phospho-KCNC4 (S15) Antibody with complementary techniques enables comprehensive analysis of channel phosphorylation in the context of broader signaling networks:

• Electrophysiology-immunostaining correlation: Combine patch-clamp recording with subsequent immunostaining using Phospho-KCNC4 (S15) Antibody to directly correlate functional changes with phosphorylation status at the single-cell level .

• Single-cell analysis: Employ minimally invasive cell-attached patch-clamping followed by single-cell qPCR and immunofluorescence to link electrophysiological properties, gene expression, and phosphorylation states .

• Multi-modal profiling: Consider emerging technologies like Phospho-seq for integrated analysis of cytoplasmic and nuclear proteins with post-translational modifications alongside other molecular modalities . This approach can connect signaling activity with cis-regulatory elements and transcriptional targets.

• Pharmacological manipulation: Use PKC activators (PDBu, GPCR agonists) and inhibitors (bisindolylmaleimide II) in conjunction with Phospho-KCNC4 (S15) immunodetection to demonstrate pathway specificity .

• Genetic approaches: Combine siRNA knockdown or CRISPR-Cas9 editing of KCNC4 with rescue experiments using wild-type or phospho-mutant constructs, verifying expression and phosphorylation with the antibody.

This integrated approach provides a more complete understanding of how S15 phosphorylation fits within broader cellular signaling networks that regulate neuronal excitability in normal physiology and pathological conditions.

What controls should be included when using Phospho-KCNC4 (S15) Antibody?

To ensure reliable and interpretable results with Phospho-KCNC4 (S15) Antibody, researchers should include several critical controls:

• Phosphorylation state controls:

  • Positive control: Samples treated with PKC activators (PDBu or GPCR agonists) to induce phosphorylation at S15

  • Negative control: Samples treated with phosphatases or PKC inhibitors (bisindolylmaleimide II) to prevent or reverse phosphorylation

• Antibody specificity controls:

  • Peptide competition assay: Pre-incubate the antibody with the phospho-peptide immunogen to block specific binding

  • Phosphatase-treated samples: Demonstrate that signal is lost when phosphorylation is removed

• Technical controls:

  • No primary antibody control: Assess secondary antibody non-specific binding

  • Total KCNC4 detection: Use parallel samples or sequential probing to compare phosphorylated fraction to total protein

  • Loading controls: Include housekeeping proteins (β-actin, GAPDH) for normalization

• Validation controls:

  • siRNA knockdown of KCNC4: Confirm signal specificity by eliminating the target protein

  • Cross-species validation: Test reactivity in both human and mouse samples as specified in product information

How can I troubleshoot weak or absent signals when using Phospho-KCNC4 (S15) Antibody?

When encountering weak or absent signals with Phospho-KCNC4 (S15) Antibody, consider these methodological troubleshooting approaches:

• Sample preparation issues:

  • Verify phosphorylation preservation: Ensure phosphatase inhibitors were included in all buffers

  • Check protein extraction efficiency: KCNC4 is membrane-associated and may require appropriate detergents for solubilization

  • Confirm protein integrity: Avoid proteolytic degradation by including protease inhibitors

• Antibody handling:

  • Verify storage conditions: Antibody should be stored at -20°C or -80°C and aliquoted to avoid freeze-thaw cycles

  • Check antibody concentration: Dilution may need optimization beyond the recommended ranges

  • Consider fresh antibody: Reactivity may decrease over time or with improper handling

• Detection optimization:

  • For Western blotting: Try longer exposure times, more sensitive detection systems, or signal enhancement methods

  • For immunostaining: Test different antigen retrieval methods, extend primary antibody incubation time, or use signal amplification systems

  • For ELISA: Optimize coating conditions, blocking agents, and detection system

• Biological considerations:

  • Verify phosphorylation status: Include positive controls (PKC activator-treated samples) to confirm phosphorylation induction

  • Check expression levels: Low KCNC4 expression may require enrichment steps like immunoprecipitation

  • Consider timing: Phosphorylation may be transient and missed without careful experimental timing

• Application-specific approaches:

  • IHC/IF (1:100-1:300/1:200-1:1000): Try signal amplification systems or decrease dilution

  • ELISA (1:5000): Decrease dilution significantly if signal is weak

  • Consider alternative validated applications if the chosen method consistently fails

Systematic evaluation of these factors will help identify and resolve issues with Phospho-KCNC4 (S15) Antibody signal detection in various experimental contexts.

How can I quantitatively analyze KCNC4 phosphorylation changes across different experimental conditions?

Quantitative analysis of KCNC4 phosphorylation requires methodical approaches that account for both total protein levels and phosphorylation-specific signals:

This quantitative approach provides objective measurement of phosphorylation changes, enabling reliable comparison across experimental conditions and correlation with functional outcomes like changes in electrophysiological properties.

What insights can changes in KCNC4 S15 phosphorylation provide about neuronal function and pathophysiology?

Changes in KCNC4 S15 phosphorylation can provide significant insights into both normal neuronal function and pathophysiological states:

• Action potential modulation: Increased S15 phosphorylation correlates with accelerated action potential repolarization and shorter duration . This directly impacts neuronal firing patterns and information coding.

• Calcium signaling regulation: By altering action potential duration, S15 phosphorylation indirectly influences calcium entry through voltage-gated calcium channels, affecting Ca²⁺-dependent processes that play vital roles in nociception .

• Pain processing: The phosphorylation-dependent modulation of Kv3.4 represents a mechanism by which pain-sensing neurons shape action potential repolarization . This may become dysregulated in chronic pain states, making it a potential biomarker or therapeutic target.

• Neuronal excitability: Tracking changes in S15 phosphorylation can provide insights into shifts in excitability across different physiological states or in response to pharmacological interventions.

• Signaling pathway integration: Because S15 phosphorylation is mediated by PKC, changes in its level reflect the activation state of upstream G-protein coupled receptor pathways, connecting channel modulation to broader signaling networks .

• Therapeutic implications: Monitoring S15 phosphorylation could help evaluate the efficacy of treatments targeting PKC pathways or Kv3.4 channel function in conditions characterized by aberrant neuronal excitability.

These insights highlight how phosphorylation at a single residue can have cascading effects on neuronal function, making Phospho-KCNC4 (S15) Antibody a valuable tool for understanding both physiological signaling and pathophysiological alterations in excitable cells.

How might KCNC4 S15 phosphorylation be relevant in non-nociceptive neuronal populations?

While KCNC4 S15 phosphorylation has been extensively studied in pain-sensing neurons, its relevance extends to other neuronal populations:

• Fast-spiking neurons: Kv3.4 channels contribute to the rapid repolarization necessary in neurons with high-frequency firing patterns. Phosphorylation at S15 could modulate firing frequency capabilities in interneurons and other fast-spiking cells .

• Neuromodulation: PKC activation occurs downstream of numerous neurotransmitter systems, suggesting that S15 phosphorylation may participate in broader neuromodulatory processes across diverse brain regions.

• Synaptic plasticity: By regulating action potential duration and consequently calcium influx, S15 phosphorylation could influence synaptic strength and plasticity mechanisms in learning and memory circuits.

• Neurodevelopment: The Phospho-seq approach mentioned in the search results integrates profiling of post-translational modifications with other molecular modalities to explore cell signaling in neurodevelopment , suggesting potential developmental roles for KCNC4 phosphorylation.

• Neuromuscular function: KCNC4 has been localized to the neuromuscular junction and axon terminals , indicating potential roles in motor control and neuromuscular signaling that could be modulated by S15 phosphorylation.

Investigating KCNC4 S15 phosphorylation across these diverse contexts requires careful experimental design with the Phospho-KCNC4 (S15) Antibody in combination with neuronal subtype markers and functional assays appropriate to each system.

What emerging technologies could enhance the study of KCNC4 phosphorylation dynamics?

Several cutting-edge technologies show promise for advancing our understanding of KCNC4 phosphorylation dynamics:

• Phospho-seq: This integrated approach allows quantification of cytoplasmic and nuclear proteins with post-translational modifications alongside other molecular modalities . Applied to KCNC4, it could connect S15 phosphorylation with transcriptional networks and chromatin accessibility.

• Live-cell phosphorylation sensors: Genetically-encoded FRET-based sensors designed around the S15 site could enable real-time visualization of KCNC4 phosphorylation dynamics in living neurons during activity.

• Mass spectrometry-based phosphoproteomics: Advanced mass spectrometry techniques can quantify multiple phosphorylation sites simultaneously, potentially revealing interplay between S15 and other post-translational modifications on KCNC4.

• Spatial transcriptomics with protein detection: Emerging spatial multi-omics approaches could map KCNC4 phosphorylation patterns across tissue sections while simultaneously capturing transcriptional states.

• Optogenetic control of kinase activity: Combining light-activated PKC variants with electrophysiological recording and Phospho-KCNC4 (S15) immunodetection could establish precise temporal relationships between kinase activation, channel phosphorylation, and functional consequences.

• Cryo-electron microscopy: Structural studies comparing non-phosphorylated and phosphorylated KCNC4 channels could reveal the precise molecular mechanisms by which S15 phosphorylation alters channel gating.

These technologies, used in combination with established approaches and the Phospho-KCNC4 (S15) Antibody, can provide unprecedented insights into the spatial, temporal, and functional dimensions of channel phosphorylation in complex neural systems.