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Voltage-gated K + (K V ) channels govern K +  ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent K V channels (K V S), however, are unable to conduct currents on their own. It has been assumed that these K V S must obligatorily assemble with subunits from the K V 2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric K V 2 channels. Herein, we show that K V S subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant K V 7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of K V S and K V 7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this K V S and K V 7 interaction may occur. Our findings demonstrate that K V cross-family interaction is much more versatile than previously thought-possibly serving nature to shape potassium conductance to the needs of individual cell types., (© 2024. The Author(s).)

Background: The transepithelial transport of electrolytes, solutes, and water in the kidney is a well-orchestrated process involving numerous membrane transport systems. Basolateral potassium channels in tubular cells not only mediate potassium recycling for proper Na + ,K + -ATPase function but are also involved in potassium and pH sensing. Genetic defects in KCNJ10 cause EAST/SeSAME syndrome, characterized by renal salt wasting with hypokalemic alkalosis associated with epilepsy, ataxia, and sensorineural deafness., Methods: A candidate gene approach and whole-exome sequencing determined the underlying genetic defect in eight patients with a novel disease phenotype comprising a hypokalemic tubulopathy with renal salt wasting, disturbed acid-base homeostasis, and sensorineural deafness. Electrophysiologic studies and surface expression experiments investigated the functional consequences of newly identified gene variants., Results: We identified mutations in the KCNJ16 gene encoding KCNJ16, which along with KCNJ15 and KCNJ10, constitutes the major basolateral potassium channel of the proximal and distal tubules, respectively. Coexpression of mutant KCNJ16 together with KCNJ15 or KCNJ10 in Xenopus oocytes significantly reduced currents., Conclusions: Biallelic variants in KCNJ16 were identified in patients with a novel disease phenotype comprising a variable proximal and distal tubulopathy associated with deafness. Variants affect the function of heteromeric potassium channels, disturbing proximal tubular bicarbonate handling as well as distal tubular salt reabsorption., (Copyright © 2021 by the American Society of Nephrology.)

The two-pore domain potassium channel (K 2P -channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC 50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K 2P -channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Auditory inner hair cells (IHCs) convert sound vibrations into receptor potentials that drive synaptic transmission. For the precise encoding of sound qualities, receptor potentials are shaped by K + conductances tuning the properties of the IHC membrane. Using patch-clamp and computational modeling, we unravel this membrane specialization showing that IHCs express an exclusive repertoire of six voltage-dependent K + conductances mediated by K v 1.8, K v 7.4, K v 11.1, K v 12.1, and BK Ca channels. All channels are active at rest but are triggered differentially during sound stimulation. This enables non-saturating tuning over a far larger potential range than in IHCs expressing fewer current entities. Each conductance contributes to optimizing responses, but the combined activity of all channels synergistically improves phase locking and the dynamic range of intensities that IHCs can encode. Conversely, hypothetical simpler IHCs appear limited to encode only certain aspects (frequency or intensity). The exclusive channel repertoire of IHCs thus constitutes an evolutionary adaptation to encode complex sound through multifaceted receptor potentials., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) was previously considered to be a paracellular channelopathy caused by mutations in the claudin-16 and claudin-19 genes. Here, we provide evidence that a missense FHHNC mutation c.908C>G (p.T303R) in the claudin-16 gene interferes with the phosphorylation in the claudin-16 protein. The claudin-16 protein carrying phosphorylation at residue T303 is localized in the distal convoluted tubule (DCT) but not in the thick ascending limb (TAL) of the mouse kidney. The phosphomimetic claudin-16 protein carrying the T303E mutation but not the wildtype claudin-16 or the T303R mutant protein increases the Trpv5 channel conductance and membrane abundance in human kidney cells. Phosphorylated claudin-16 and Trpv5 are colocalized in the luminal membrane of the mouse DCT tubule; phosphomimetic claudin-16 and Trpv5 interact in the yeast and mammalian cell membranes. Knockdown of claudin-16 gene expression in transgenic mouse kidney delocalizes Trpv5 from the luminal membrane in the DCT. Unlike wildtype claudin-16, phosphomimetic claudin-16 is delocalized from the tight junction but relocated to the apical membrane in renal epithelial cells because of diminished binding affinity to ZO-1. High-Ca 2+ diet reduces the phosphorylation of claudin-16 protein at T303 in the DCT of mouse kidney via the PTH signaling cascade. Knockout of the PTH receptor, PTH1R, from the mouse kidney abrogates the claudin-16 phosphorylation at T303. Together, these results suggest a pathogenic mechanism for FHHNC involving transcellular Ca 2+ pathway in the DCT and identify a molecular component in renal Ca 2+ homeostasis under direct regulation of PTH., Competing Interests: The authors declare no conflict of interest.

The molecular nature of tight junction architecture and permeability is a long-standing mystery. Here, by comprehensive biochemical, biophysical, genetic, and electron microscopic analyses of claudin-16 and -19 interactions--two claudins that play key polygenic roles in fatal human renal disease, FHHNC--we found that 1) claudin-16 and -19 form a stable dimer through cis association of transmembrane domains 3 and 4; 2) mutations disrupting the claudin-16 and -19 cis interaction increase tight junction ultrastructural complexity but reduce tight junction permeability; and 3) no claudin hemichannel or heterotypic channel made of claudin-16 and -19 trans interaction can exist. These principles can be used to artificially alter tight junction permeabilities in various epithelia by manipulating selective claudin interactions. Our study also emphasizes the use of a novel recording approach based on scanning ion conductance microscopy to resolve tight junction permeabilities with submicrometer precision., (© 2015 Gong, Renigunta, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The intracellular transport of membrane proteins is controlled by trafficking signals: Short peptide motifs that mediate the contact with COPI, COPII or various clathrin-associated coat proteins. In addition, many membrane proteins interact with accessory proteins that are involved in the sorting of these proteins to different intracellular compartments. In the K2P channels, TASK-1 and TASK-3, the influence of protein-protein interactions on sorting decisions has been studied in some detail. Both TASK paralogues interact with the adaptor protein 14-3-3; TASK-1 interacts, in addition, with the adaptor protein p11 (S100A10) and the endosomal SNARE protein syntaxin-8. The role of these interacting proteins in controlling the intracellular traffic of the channels and the underlying molecular mechanisms are summarised in this review. In the case of 14-3-3, the interacting protein masks a retention signal in the C-terminus of the channel; in the case of p11, the interacting protein carries a retention signal that localises the channel to the endoplasmic reticulum; and in the case of syntaxin-8, the interacting protein carries an endocytosis signal that complements an endocytosis signal of the channel. These examples illustrate some of the mechanisms by which interacting proteins may determine the itinerary of a membrane protein within a cell and suggest that the intracellular traffic of membrane proteins may be adapted to the specific functions of that protein by multiple protein-protein interactions.

Over the last decade, we have seen an enormous increase in the number of experimental studies on two-pore-domain potassium channels (K2P-channels). The collection of reviews and original articles compiled for this special issue of Pflügers Archiv aims to give an up-to-date summary of what is known about the physiology and pathophysiology of K2P-channels. This introductory overview briefly describes the structure of K2P-channels and their function in different organs. Its main aim is to provide some background information for the 19 reviews and original articles of this special issue of Pflügers Archiv. It is not intended to be a comprehensive review; instead, this introductory overview focuses on some unresolved questions and controversial issues, such as: Do K2P-channels display voltage-dependent gating? Do K2P-channels contribute to the generation of action potentials? What is the functional role of alternative translation initiation? Do K2P-channels have one or two or more gates? We come to the conclusion that we are just beginning to understand the extremely complex regulation of these fascinating channels, which are often inadequately described as 'leak channels'.