Inwardly rectifying K⁺(Kir) channels allow K⁺to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K⁺channels (Kir6.x) are tightly linked to cellular metabolism, and K⁺transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg²⁺and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH₂and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.

Transcellular Mg²⁺ transport across epithelia, involving both apical entry and basolateral extrusion, is essential for magnesium homeostasis, but molecules involved in basolateral extrusion have not yet been identified. Here, we show that CNNM4 is the basolaterally located Mg²⁺ extrusion molecule. CNNM4 is strongly expressed in intestinal epithelia and localizes to their basolateral membrane. CNNM4-knockout mice showed hypomagnesemia due to the intestinal malabsorption of magnesium, suggesting its role in Mg²⁺ extrusion to the inner parts of body. Imaging analyses revealed that CNNM4 can extrude Mg²⁺ by exchanging intracellular Mg²⁺ with extracellular Na⁺. Furthermore, CNNM4 mutations cause Jalili syndrome, characterized by recessive amelogenesis imperfecta with cone-rod dystrophy. CNNM4-knockout mice showed defective amelogenesis, and CNNM4 again localizes to the basolateral membrane of ameloblasts, the enamel-forming epithelial cells. Missense point mutations associated with the disease abolish the Mg²⁺ extrusion activity. These results demonstrate the crucial importance of Mg²⁺ extrusion by CNNM4 in organismal and topical regulation of magnesium. Magnesium is an essential element for living organisms. Its absorption occurs at the intestine through the barrier comprised of epithelial cells. In this process, transcellular Mg²⁺ transport across epithelia, involving both entry from one side and extrusion from the other side, is important. Previous studies have revealed the role of Mg²⁺-permeable channel protein in Mg²⁺ entry into the epithelial cells. However, the identity of proteins involved in Mg²⁺ extrusion to the inner parts of body has remained unknown. Mice genetically engineered not to express CNNM4, which localizes to the epithelial membrane facing to the inner parts of body, show hypomagnesemia due to the defect in magnesium absorption. Functional analyses using culture cells directly reveal that CNNM4 can extrude intracellular Mg²⁺ to the outside of cells. These results indicate that CNNM4 mediates transcellular Mg²⁺ transport across the intestinal epithelia. Furthermore, we also show that these CNNM4-lacking mice also have a defect in amelogenesis, which is consistent with the disease symptoms of Jalili syndrome that is known to be caused by mutations in the CNNM4 gene.

Drug interaction with target proteins including ion channels is essential for pharmacological control of various cellular functions, but the majority of its molecular mechanisms is still elusive. We recently found that a series of antidepressants preferentially block astroglial K⁺-buffering inwardly rectifying potassium channel (Kir) 4.1 channels over Kir1.1 channels. Here, using electrophysiological analyses of drug action on mutated Kir4.1 channel as well as computational analyses of three-dimensional (3D) arrangements of the ligands (i.e., bidirectional analyses), we examined the underlying mechanism for the antidepressant-Kir4.1 channel interaction. First, the effects of the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant nortriptyline on chimeric and site-directed mutants of Kir4.1 expressed in Xenopus laevis oocytes were examined using the two-electrode voltage-clamp technique. Two amino acids, Thr128 and Glu158, on pore and transmembrane domain 2 helices were critical for the drug inhibition of the current. The closed and open conformation models of the Kir4.1 pore suggested that both residues faced the central cavity, and they were positioned within a geometrical range capable of interacting with the drugs. Second, to represent molecular properties of active ligands in geometric terms, a 3D quantitative structure-activity relationship model of antidepressants was generated, which suggested that they share common features bearing a hydrogen bond acceptor and a positively charged moiety. 3D structures and physicochemical features of receptor and ligand were fitted together. Our results strongly suggest that antidepressants interact with Kir4.1 channel pore residues by hydrogen bond and ionic interactions, which account for their preferential inhibitory action on Kir4.1 current. This study may represent a possible general approach for the understanding of the mechanism of ligand-protein interactions.

Functional crosstalk between cell‐surface and intracellular ion channels plays important roles in excitable cells and is structurally supported by junctophilins (JPs) in muscle cells. Here, we report a novel form of channel crosstalk in cerebellar Purkinje cells (PCs). The generation of slow afterhyperpolarization (sAHP) following complex spikes in PCs required ryanodine receptor (RyR)‐mediated Ca2+‐induced Ca2+ release and the subsequent opening of small‐conductance Ca2+‐activated K+ (SK) channels in somatodendritic regions. Despite the normal expression levels of these channels, sAHP was abolished in PCs from mutant mice lacking neural JP subtypes (JP‐DKO), and this defect was restored by exogenously expressing JPs or enhancing SK channel activation. The stimulation paradigm for inducing long‐term depression (LTD) at parallel fiber–PC synapses adversely established long‐term potentiation in the JP‐DKO cerebellum, primarily due to the sAHP deficiency. Furthermore, JP‐DKO mice exhibited impairments of motor coordination and learning, although normal cerebellar histology was retained. Therefore, JPs support the Ca2+‐mediated communication between voltage‐gated Ca2+ channels, RyRs and SK channels, which modulates the excitability of PCs and is fundamental to cerebellar LTD and motor functions.

The HD Physiology Project is a Japanese research consortium that aimed to develop methods and a computational platform in which physiological and pathological information can be described in high-level definitions across multiple scales of time and size. During the 5 years of this project, an appropriate software platform for multilevel functional simulation was developed and a whole-heart model including pharmacokinetics for the assessment of the proarrhythmic risk of drugs was developed. In this article, we outline the description and scientific strategy of this project and present the achievements and influence on multilevel integrative systems biology and physiome research.