WNK-SPAK-NKCC1-KCC2 pathway in SCN: a novel regulator of neuronal activity

Abstract

Background: The suprachiasmatic nucleus (SCN) plays a vital role in regulating an organism's physiological and behavioral processes by synchronizing them with the light-dark cycle. The SCN receives GABAergic inputs that may be excitatory or inhibitory, depending on the intracellular chloride concentration ([Cl−]i) that is regulated by NKCC1 and KCC2 cotransporters, determined by the WNK-SPAK/OSR1 phosphorylation pathway. Our study aimed to investigate the role of the WNK-SPAK-NKCC1-KCC pathway in regulating SCN neuronal activity.Methods: First, we perform an in silico analysis to determine the expression of the genes involved in this pathway. Furthermore, the expression of the proteins was confirmed by an immunofluorescence assay. Next, to determine if the WNK-SPAK-NKCC1-KCC2 pathway affects neuronal activity in the SCN we performed the immunohistochemical analysis of c-Fos expression in the SCN at ZT2 and ZT14 in the SPAK-KI mice. Finally, to find out if the change in SCN neuronal activity between day and night is related to pathway activity, we performed an immunofluorescence study. Results: We observed that various genes governing the pathway's regulation exhibit oscillations not only in neurons but also in other cell types. Remarkably, SPAK-KI mice displayed a significant surge in neuronal activity at ZT2 when contrasted with the control group. Additionally, we detected increased phosphorylation levels of WNK, SPAK, NKCC1, and KCC2 isoforms at ZT14 as compared to ZT2.Conclusions: Our study highlights the role of SPAK kinase in regulating SCN neuronal activity and suggests that the pathway is active during the dark period, leading to an increase in [Cl−]i during the day, as previously reported. The authors are supported by grants from CONACyT Mexico no. 87794, and from PAPIIT UNAM no IA208522 and IN223324. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.