g-Aminobutyric Acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain, and plays a prominent inhibitory role in the brainstem and spinal cord as well. One mechanism through which GABA produces its inhibitory action is via GABAA receptors which produce fast synaptic inhibition of neurons by activation of intrinsic CI- channels. GABA opening of CI- channels produces an inward movement of CI-, driven by a low intracellular CI- concentration which is maintained by an active CI- extrusion mechanism: presumed to be the neuronal-specific isoform of the K-CI cotransporter, KCC2. We have disrupted Sic12a5, the gene encoding this isoform of the K-CI cotransporter, and the homozygous mutant mice die shortly after birth of repeated seizures. Epileptic seizure activity in the KCC2 knockout brain suggests hyper-excitability, in agreement with the putative role of KCC2 in controlling hyperpolarizing GABA responses. This proposal is aimed at understanding the role of KCC2 in controlling CNS excitability and epilepsy. We will 1) investigate the developmental role of KCC2 in regulating intracellular CI- and controlling the maturation of GABA hyperpolarization, 2) investigate the role of KCC2 in preventing hyper-excitability and the participation of the cotransporter in depolarizing GABA responses during high frequency synaptic activation. This will be achieved through detailed electrophysiological measurements in hippocampal slices and isolated cortical neurons, 3) develop brain-region-specific and inducible knockout of KCC2 to study the knockout phenotype in the adult and examine the effect of graded reduction in KCC2 expression, and 4) examine the determinants of KCC2 function and regulation, by focusing mainly on phosphorylation/dephosphorylation of the protein. The epileptic seizure phenotype of the KCC2 knockout mouse demonstrates the importance of KCC2 in preventing hyper-excitability and controlling CNS function. Results of these molecular, physiological, and behavioral studies will lead to a better understanding of the relationship between cation-chloride cotransporters, ion homeostasis, synaptic transmission and brain excitability.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS036758-06
Application #
6806029
Study Section
Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
Program Officer
Stewart, Randall
Project Start
1999-07-30
Project End
2007-08-31
Budget Start
2004-09-01
Budget End
2005-08-31
Support Year
6
Fiscal Year
2004
Total Cost
$349,188
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Anesthesiology
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Lee, Hanmi; Bach, Eva; Noh, Jihyun et al. (2016) Hyperpolarization-independent maturation and refinement of GABA/glycinergic connections in the auditory brain stem. J Neurophysiol 115:1170-82
Ding, Jinlong; Delpire, Eric (2014) Deletion of KCC3 in parvalbumin neurons leads to locomotor deficit in a conditional mouse model of peripheral neuropathy associated with agenesis of the corpus callosum. Behav Brain Res 274:128-36
Gagnon, Kenneth B; Delpire, Eric (2013) Physiology of SLC12 transporters: lessons from inherited human genetic mutations and genetically engineered mouse knockouts. Am J Physiol Cell Physiol 304:C693-714
Gagnon, Kenneth B; Delpire, Eric (2012) Molecular physiology of SPAK and OSR1: two Ste20-related protein kinases regulating ion transport. Physiol Rev 92:1577-617
Stil, Aurélie; Jean-Xavier, Céline; Liabeuf, Sylvie et al. (2011) Contribution of the potassium-chloride co-transporter KCC2 to the modulation of lumbar spinal networks in mice. Eur J Neurosci 33:1212-22
Garbarini, Nicole; Delpire, Eric (2008) The RCC1 domain of protein associated with Myc (PAM) interacts with and regulates KCC2. Cell Physiol Biochem 22:31-44
Zhu, Lei; Polley, Nathan; Mathews, Gregory C et al. (2008) NKCC1 and KCC2 prevent hyperexcitability in the mouse hippocampus. Epilepsy Res 79:201-12
Zhang, Ling-Li; Delpire, Eric; Vardi, Noga (2007) NKCC1 does not accumulate chloride in developing retinal neurons. J Neurophysiol 98:266-77
Byun, Nellie; Delpire, Eric (2007) Axonal and periaxonal swelling precede peripheral neurodegeneration in KCC3 knockout mice. Neurobiol Dis 28:39-51
Pieraut, Simon; Laurent-Matha, Valerie; Sar, Chamroeun et al. (2007) NKCC1 phosphorylation stimulates neurite growth of injured adult sensory neurons. J Neurosci 27:6751-9

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