NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., MĂ©chaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751-6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cationchloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.
The regulation of intracellular ClÏȘ in neurons is a critical determinant of inhibitory synaptic neurotransmission. Sensory or peripheral neurons express, in abundance, an inwardly poised Na Ï© -and K Ï© -dependent Cl ÏȘ transport mechanism, NKCC1, 2 whose activity drives the uphill accumulation of Cl ÏȘ ions (2, 3). High intracellular Cl ÏȘ concentration in dorsal root ganglion (DRG) neurons permits depolarizing â„-aminobutyric acid responses, which mediate presynaptic inhibition and filtration of sensory noise (4). Consequently, the knockout of NKCC1 exhibits a redistribution of internal Cl ÏȘ in DRG neurons and a pain perception phenotype (3, 5). In addition, peripheral inflammation or nerve injury results in increased NKCC1 function in primary afferents (6, 7). Using a phosphopeptide-specific NKCC1 antibody, it was recently shown that NKCC1 phosphorylation instead of expression level increased in DRG neurons upon nerve injury (1). This observation points to the importance of NKCC1 regulation in the neuropathic pain pathway.In recent work, our laboratory identified two Ste20-like kinases that directly bind to the cytosolic N-terminal tail of NKCC1 (8). The binding is a pre-requisite for NKCC1 phosphorylation and activation (9 -13). The kinases, named SPAK (Ste20-related proline-alanine-rich kinase) and OSR1 (oxidative-stress response 1), share high homology in b...