Garbarini N, Delpire E. The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 22(22). 31-44. PMID: 18769030 [PubMed] PMCID: PMC2535904 NIHMSID: NIHMS48160.
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.