Potassium-chloride cotransporters KCCs mediate the coupled, electroneutral cotransport of K⁺ and Cl⁻ across the membrane and are involved in important physiological processes such as cell volume regulation and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Although structures of KCCs have been reported, the identification of ions bound in KCCs awaits experimental studies. Here using the cryo-electron microscopy (cryo-EM) titration methods, we present six structures of human KCC4 in different ion conditions at 2.38–2.58 Å resolutions. These structures, along with molecular dynamic simulations, allow us to assign one K⁺ and two Cl⁻ ions in the substrate-binding pocket. The K⁺ at S1 and Cl⁻ at the S2 site are tightly coupled in the binding and dissociation, suggesting that the Cl⁻ at S2 but not at S3 is the cotransported one. The S1 site provides coordination that largely matches the K⁺ dehydration radius and therefore displays higher selectivity to K⁺ over Na⁺. This study establishes the structural basis for the K⁺ selectivity of KCCs by the cryo-EM titration.

This article takes Kunming University of Science and Technology, Ningxia University and Zhejiang University as examples, and transforms complex university physics experiments into immersive learning scenarios through video demonstrations, AI physics experiment assistants, and data online software analysis functions. It creates a three-dimensional interactive learning platform with “student self-learning, intelligent process tracking, and precise teacher guidance”, broadens practical education channels, realizes students' diversified, personalized, and deep learning needs, trains students' ability to analyze and solve complex engineering problems, and cultivates students' innovation ability. This article provides reference cases for the teaching reform and promotion of public basic courses by exploring the construction and reform of college physics experiment courses under the background of digital education, broadening practical education channels, stimulating students' innovative spirit and practical ability.