The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2.
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.
Garbarini
Nicole
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Neuroscience Graduate Program and Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, TN, USA.
Delpire
Eric
E