Rhythmic phosphorylation of the WNK-SPAK-NKCC1-KCC pathway regulates neuronal activity in the SCN

Background: The WNK-SPAK/OSR1-CCC pathway plays a critical role in regulating intracellular chloride levels through WNK proteins, which act as chloride sensors and activate the intermediate kinases SPAK and OSR1. These kinases phosphorylate cation-coupled chloride cotransporters (CCCs) such as NKCC1 and KCC2. In the suprachiasmatic nucleus (SCN), these cotransporters determine whether GABA acts excitatory (via NKCC1) or inhibitory (via KCC2). Given the SCN's role as the circadian pacemaker, synchronizing neural rhythms with the light-dark cycle, it is crucial to investigate whether the rhythmic neuronal activity of the SCN is driven by the pathway's phosphorylation. Methods: We evaluated the expression of the WNK-SPAK-NKCC1-KCC pathway in both VIP and AVP neuronal subtypes within the SCN using immunofluorescence. Also, we analyzed its phosphorylation state at ZT2 and ZT14, corresponding to day and night, respectively. To investigate whether the WNK-SPAK-CCC pathway regulates neuronal activity, we used transgenic SPAK-KI mice with inactive SPAK kinase. Furthermore, we assessed pathway phosphorylation under constant darkness and following a light pulse to explore its correlation with neuronal activity. Results We found that WNK-SPAK/OSR1 kinases are primarily expressed in AVP neurons. Pathway phosphorylation was significantly increased during ZT14 compared to ZT2. In SPAK-KI mice, we observed a notable increase in pWNK and a decrease in SCN neuronal activity at ZT2 compared to the control group. Under constant darkness, we observed that the phosphorylation of WNK kinase and the NKCC1 cotransporter follows a circadian rhythm. Furthermore, NKCC1 activity is light-inducible in wild-type mice, likely promoting an increase in SCN neuronal activity. However, SPAK-KI mice show a diminished response to the light pulse. Conclusions: Our results suggest that the WNK-SPAK/OSR1-CCC pathway regulates SCN neuronal activity through its circadian and light-responsive phosphorylation, primarily in AVP neurons. SPAK inactivation disrupts this regulation, reducing neuronal activity and light responsiveness, highlighting the pathway's role in circadian and light-driven functions. The authors are supported by grants from CONAHCyT Mexico no. 87794, and from PAPIIT UNAM no IA208522 and IN223324. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.