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Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack−/−) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6–P14) Slack−/− lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack−/−, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack−/−neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack−/−. We additionally detect a lack of mGluR-induced LTD in infant Slack−/−, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack−/−. This together with normalized expression levels of GluN2B and Rab4 hints to developmental “restoration” of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities.

The tumor microenvironment (TME) consists of different cell types that secrete proteins and also control the extracellular concentration of ions and metabolites. Changes in these intra-tumoral analytes and conditions, including K+, glucose, and pH, have been described to alter the metabolic activity of cancer cells, promote tumor cell growth, and impair anti-tumor immunity. However, the mechanisms regulating ion and metabolite levels and their effects on certain characteristics of the TME are still poorly understood. Therefore, accurate determination and visualization of analyte or state changes in real time within the TME is desired. In this study, we genetically combined FRET-based fluorescent biosensors with nanobodies (Nbs) and used them for targeted visualization and monitoring of extracellular changes in K+, pH, and glucose on cell surfaces. We demonstrated that these recombinant biosensors quantitatively visualize extracellular K+ alterations on multiple cancer and non-cancer cell lines and primary neurons. By implementing a HER2 specific Nb, we generated K+ and pH sensors, which retain their functionality and specifically stained HER2 positive breast cancer cells. Based on the successful technical development of several Nb-biosensor combinations, we anticipate that this approach can be easily extended to design other targeted biosensors. Such versatile probes will open new possibilities for the reliable study of extracellular analytes in advanced 3D cell models or even in vivo systems.

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