More than 20 recurrent missense gain-of-function (GOF) mutations have been identified in the sodium-activated potassium (K_Na) channel gene KCNT1 in patients with severe developmental and epileptic encephalopathies (DEEs), most of which are resistant to current therapies. Defining the neuron types most vulnerable to KCNT1 GOF will advance our understanding of disease mechanisms and provide refined targets for precision therapy efforts. Here, we assessed the effects of heterozygous expression of a Kcnt1 GOF variant (Kcnt1^Y777H) on K_Na currents and neuronal physiology among cortical glutamatergic and GABAergic neurons in mice, including those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), to identify and model the pathogenic mechanisms of autosomal dominant KCNT1 GOF variants in DEEs. Although the Kcnt1^Y777H variant had no effects on glutamatergic or VIP neuron function, it increased subthreshold K_Na currents in both SST and PV neurons but with opposite effects on neuronal output; SST neurons became hypoexcitable with a higher rheobase current and lower action potential (AP) firing frequency, whereas PV neurons became hyperexcitable with a lower rheobase current and higher AP firing frequency. Further neurophysiological and computational modeling experiments showed that the differential effects of the Kcnt1^Y777H variant on SST and PV neurons are not likely due to inherent differences in these neuron types, but to an increased persistent sodium current in PV, but not SST, neurons. The Kcnt1^Y777H variant also increased excitatory input onto, and chemical and electrical synaptic connectivity between, SST neurons. Together, these data suggest differential pathogenic mechanisms, both direct and compensatory, contribute to disease phenotypes, and provide a salient example of how a pathogenic ion channel variant can cause opposite functional effects in closely related neuron subtypes due to interactions with other ionic conductances.

Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB₂FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB₂FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB₂FG and LptB₂FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB₂FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.