The Ste20 kinases Ste20-related proline-alanine-rich kinase and oxidative-stress response 1 regulate NKCC1 function in sensory neurons.
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., Méchaly, 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 cation-chloride 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.
Geng
Yang
Y
Neuroscience Graduate Program and Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Hoke
Ahmet
A
Delpire
Eric
E
D000818:Animals; D002138:Calibration; D002460:Cell Line; D002469:Cell Separation; D005727:Ganglia, Spinal / Q000201:enzymology; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D051379:Mice; D018345:Mice, Knockout; D018384:Oxidative Stress; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D011984:Sensory Receptor Cells / Q000201:enzymology*; D028021:Sodium-Potassium-Chloride Symporters / Q000235:genetics / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013793:Thallium / Q000378:metabolism
D034741:RNA, Small Interfering; C578878:Slc12a2 protein, mouse; C578879:Slc12a2 protein, rat; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013793:Thallium; D011494:Protein Kinases; C486407:Stk39 protein, mouse; C116037:PAS domain kinases; C534421:OXSR1 protein, mouse; C506604:Oxsr1 protein, rat; D017346:Protein Serine-Threonine Kinases