Chloride homeostasis and pain: Kinase regulation of NKCC1 in sensory perception

The Na+-K+-2Cl- cotransporter 1 (NKCC1) belongs to the cation-chloride cotransporter family and serves multiple fundamental functions in a wide variety of tissues and organs. NKCC1 is involved in controlling neuronal excitability, cell volume regulation, cell proliferation and survival, and sensory perception. It is generally agreed that cotransporter regulation is mainly mediated through phosphorylation-dephosphorylation mechanisms based on the observation that the cotransporter is a phosphoprotein, and that phosphatase inhibitors affect cotransport activity.;Our laboratory has demonstrated that two Ste20p-like serine/threonine kinases, SPAK (Sterile 20 related Proline Alanine rich Kinase) and OSR1 (Oxidative Stress Response 1) mediate phosphorylation of NKCCI and lead to its activation in endogenous expression systems. In this dissertation I investigate the regulatory mechanisms of NKCCI in dorsal root ganglion neurons, where both SPAK and OSRI are expressed. Using gene silencing in an established nociceptive neuronal cell line, I show that OSRI and SPAK regulate NKCCI activity in a similar fashion and also demonstrate that their regulatory effects are additive. Bumetanide-sensitive TI+/K+ uptakes in DRG neurons isolated from SPAK-KO and control mice further confirmed these findings.;To further assess the role of SPAK in the nervous system, SPAK-KO mice were subjected to a battery of neurobehavioral tests. I show here that SPAK-KO mice display both nociceptive and locomotor phenotypes. As NKCCI activity in DRG neurons from SPAK-KO mice is reduced by 50% and NKCCI heterozygote animals with 50% NKCCI function do not exhibit motor and somatosensory phenotypes, the phenotype observed in the SPAK-KO mouse is unlikely due to the decreased NKCCI activity observed the sensory neurons. As SPAK also regulates the neuronal-specific K-Cl cotransporters, KCC2, it is likely that the observed phenotypes are related to KCC2-mediated changes in intracellular chloride concentration in central neurons.