Application Methods: Immunofluorescence (Immunocytochemistry), Immunoprecipitation, Western Blotting
Background: The electroneutral cation-chloride-coupled co-transporter (SLC12) gene family comprises bumetanide-sensitive Na+/K+/Cl- (NKCC), thiazide-sensitive Na+/Cl-, and K+/Cl- (KCC) co-transporters. SLC12A1/NKCC2 and SLC12A2/NKCC1 regulate cell volume and maintain cellular homeostasis in response to osmotic and oxidative stress (1). The broadly expressed NKCC1 is thought to play roles in fluid secretion (i.e. salivary gland function), salt balance (i.e. maintenance of renin and aldosterone levels), and neuronal development and signaling (2-7). During neuronal development, NKCC1 and KCC2 maintain a fine balance between chloride influx (NKCC1) and efflux (KCC2), which regulates γ-aminobutyric acid (GABA)-mediated neurotransmission (3). Increased NKCC1 expression in immature neurons maintains high intracellular chloride levels that result in inhibitory GABAergic signaling; KCC2 maintains low intracellular chloride levels and excitatory GABAergic responses in mature neurons (4,5,8). Deletion of NKCC1 impairs NGF-mediated neurite outgrowth in PC-12D cells while inhibition of NKCC1 with bumetanide inhibits re-growth of axotomized dorsal root ganglion cells (6,7). Defective chloride homeostasis in neurons is linked to seizure disorders that are ameliorated by butemanide treatment, indicating that abnormal NKCC1 and NKCC2 expression or signaling may play a role in neonatal and adult seizures (9-12). NKCC1 is found as a homodimer or within heterooligomers with other SLC12 family members. This transport protein associates with a number of oxidative- and osmotic-responsive kinases that bind, phosphorylate, and activate NKCC1 co-transporter activity (13-16). In response to decreased intracellular chloride concentrations, Ste20-related proline-alanine-rich kinase (SPAK) phosphorylates NKCC1 to increase co-transporter activity and promote chloride influx (16-19). Oxidative stress response kinase 1 (OSR1) also phosphorylates and activates NKCC1 in response to oxidative stress (14).
|Human, Mouse, Rat|
Application Methods: Immunofluorescence (Frozen), Immunoprecipitation, Western Blotting
Background: The potassium/chloride cotransporter 2 (KCC2, SLC12A5) is a neuron-specific transport protein responsible for regulating the cotransport of potassium and chloride ions. KCC2 uses the energy of the electrochemical potassium gradient to export chloride ions from cells, therefore maintaining intracellular chloride ion concentrations in mature neurons (1,2). The intracellular concentration of chloride ions determines the neuronal response to the inhibitory neurotransmitter GABA and glycine. As a result, KCC2 can play a critical role in regulating neuronal excitability in mature central nervous system neurons (3-5). Altered KCC2 expression and reduced KCC2 activity can result in an increase in intracellular chloride ion concentrations and subsequent hyperexcitability of neuronal systems. Cases of aberrant KCC2 function are associated with neurological disorders, such as multiple forms of epilepsy, neuropathic pain, and schizophrenia (6-10).