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Updated: Jul 16, 2026

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting
Published on: December 9, 2022
Sex differences in the chloride cotransporters, NKCC1 and KCC2, in the developing hypothalamus
T S Perrot-Sinal1, C J Sinal, J C Reader
1Department of Psychology and Neuroscience Institute, Dalhousie University, Halifax, Nova Scotia, Canada. tperrots@dal.ca
Abstract:
In immature neurones, high basal [Cl(-)](i) results in membrane depolarisation following GABA(A) receptor activation, which is critical for various developmental processes including steroid-mediated sexual differentiation of the hypothalamus. Previously, we demonstrated that oestradiol enhances GABA-mediated Ca(2+) influx in neonate hypothalamus and that Ca(2+) induced activation of the transcription factor, cyclicAMP response element binding protein (CREB), was higher in male (high oestradiol) relative to female neonate hypothalamus. Based on these results, we hypothesised that expression of developmentally regulated chloride cotransporters may be sexually dimorphic. Here, we investigate the expression of the chloride cotransporters, NKCC1 (Na-K-2Cl(-)) and KCC2 (K-Cl(-)) in neonate mediobasal hypothalamus of male and female rats. The NKCC1 transporter moves Cl(-) into cells and helps maintain depolarising GABA action while the KCC2 transporter has the opposite effect by moving Cl(-) out of cells. NKCC1 mRNA levels were higher in males than females on the day of birth (postnatal day 0; PND 0) and total NKCC1 protein levels were significantly higher in males than females on embryonic day (ED) 20 and PND0. Levels of activated phosphorylated NKCC1 (pNKCC1) were not sexually dimorphic. Females were treated with a masculinising dose of oestradiol benzoate (EB; 100 microg; EB-females) on PND0. Total NKCC1 protein levels in tissue processed on PND1 and PND2 were similar in EB-females and oil-treated PND0 males and females. However, pNKCC1 protein levels measured on PND2 (but not PND1) were significantly higher in EB-treated females relative to oil-treated males and females. By contrast, KCC2 mRNA levels were significantly lower in males relative to females on PND0. KCC2 protein was not detectable on ED20 or PND0 but was significantly lower in males relative to females on PND5. These results suggest a complex relationship between KCC2 and NKCC1 mRNA and protein in developing brain that is not easily linked to regulation by oestradiol.
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