NKCC1 is an electroneutral cation?chloride cotransporter which belongs to SLC12A, an evolutionary ancient gene family. The transporter is found from bacteria to humans and thus has evolved to fulfill a multitude of cellula functions. NKCC1 is expressed on the basolateral membrane of Cl? secreting epithelia such as airway, intestine, salivary gland, sweat gland, etc. Or K+ secreting epithelia, e.. stria vascularis (inner ear), where it participates to the transepithelial movement of Cl? or K+, respectively. In neurons, NKCC1 is involved in modulating intracellular Cl? thereby affecting GABAergic and glycinergic neurotransmission. The cotransporter is also a key factor for cell hydration as it is activated by loss of cell water and consequently participates in the maintenance and regulation of cell volume. Recently, a de novo deletion of 11 bases was found in exon 22 of SLC12A2, the gene encoding NKCC1, in a 12?year old patient. The deletion truncates 40% of the carboxyl?terminal tail of the cotransporter. We hypothesze that the mutant cotransporter is expressed and exerts dominant?negative effects on wild?type cotransporter, or exerts toxic effects on cell metabolism. In this application, we propose to 1) examine function of the truncated cotransporter in heterologous expression systems; and 2) examine expression and function of the mutant cotransporter in cells that are isolated and cultured from the patient; and create a mouse model recapitulating this mutation.
The first aim will involve studies of expression, trafficking, function, interacton, of both truncated and wild?type cotransporter in Xenopus laevis oocytes and HEK293 cells. This study will allow us to characterize in details the mutant cotransporter and learn about the molecular role of the carboxyl?terminal tail of the cotransporter. Attempts wil be made to assess whether it is possible to rescue the truncation cotransporter functin.
The second aim will examine the impact of the mutant transporter on ions and volume homeostasis, as well as basic metabolic properties of cells isolated from the patient (e.g. fibroblasts and/or transformed lymphocytes). Using CRISPR technology, we will also create and study a mouse model recapitulating the human mutation. These studies will allow us to gain insights into NKCC1 mutant?mediated cellular dysfunction and explain the clinical presentations of the patient.

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The proposed research will study and characterize the first human mutation in the SLC12A2 (NKCC1) gene. The studies will examine expression, trafficking, function, and dominant negative effects on wild?type cotransporter of an exon 22 mutation leading to a partial carboxyl?terminal deletion of the cotransporter. Studies will also involve the study of cells isolated from the patient, assessing the impact of the mutation on cellular ions and volume homeostasis, as well as on cell metabolism. We will also create a mouse model of this mutation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Exploratory/Developmental Grants (R21)
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Special Emphasis Panel (ZHG1)
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Krasnewich, Donna M
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Vanderbilt University Medical Center
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Glykys, Joseph; Dzhala, Volodymyr; Egawa, Kiyoshi et al. (2017) Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential. Trends Neurosci 40:276-294
Delpire, Eric; Wolfe, Lynne; Flores, Bianca et al. (2016) A patient with multisystem dysfunction carries a truncation mutation in human SLC12A2, the gene encoding the Na-K-2Cl cotransporter, NKCC1. Cold Spring Harb Mol Case Stud 2:a001289