Related Experiment Video
Updated: Jul 2, 2026

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
Published on: December 9, 2022
The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2
Nicole Garbarini1, Eric Delpire
1Neuroscience Graduate Program and Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA.
Abstract:
GABAergic and glycinergic function is dependent on neuronal intracellular chloride. The neuron-specific electroneutral potassium (K(+)) and chloride (Cl(-)) cotransporter (KCC2), is a key regulator of neuronal Cl(-), yet little is known about KCC2 regulation. Using yeast two-hybrid, we identified Protein Associated with Myc (PAM) as a binding partner of KCC2. The RCC1 (Regulator of Chromatin Condensation) domain of PAM binds to the carboxyl terminus of KCC2, as demonstrated through yeast two-hybrid and GST-pull-down assays. RCC1/PAM and full-length KCC2 coimmunoprecipitate following heterologous co-expression in HEK293 cells. Additionally, (86)Rb/K(+) uptake assays in this model system show that RCC1/PAM causes increased KCC2-mediated flux. After narrowing down RCC1/PAM binding to a 20 amino acid region on the KCC2 carboxyl terminus, we created a point mutant in this region to eliminate interaction between the KCC2 carboxyl terminus and RCC1/PAM. This same mutation abolishes N-ethylmaleimide activation of KCC2, suggesting that PAM plays a role in modulating KCC2 function.
Related Concept Videos
Anaphase Promoting Complex
Master Transcription Regulators
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein.
Calmodulin-dependent Signaling
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
Abnormal Proliferation