Tissue Distribution of Kir7.1 Inwardly Rectifying K+ Channel Probed in a Knock-in Mouse Expressing a Haemagglutinin-Tagged Protein

Abstract

Kir7.1 encoded by the Kcnj13 gene in the mouse is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. Further investigations of the localisation and function of Kir7.1 would benefit from the availability of a knockout mouse, but perinatal mortality attributed to cleft palate in the neonate has thwarted this research. To facilitate localisation studies we now use CRISPR/Cas9 technology to generate a knock-in mouse, the Kir7.1-HA that expresses the channel tagged with a haemagglutinin (HA) epitope. The availability of antibodies for the HA epitope allows for application of western blot and immunolocalisation methods using widely available anti-HA antibodies with WT tissues providing unambiguous negative control. We demonstrate that Kir7.1-HA cloned from the choroid plexus of the knock-in mouse has the electrophysiological properties of the native channel, including characteristically large Rb+ currents. These large Kir7.1-mediated currents are accompanied by abundant apical membrane Kir7.1-HA immunoreactivity. WT-controlled western blots demonstrate the presence of Kir7.1-HA in the eye and the choroid plexus, trachea and lung, and intestinal epithelium but exclusively in the ileum. In the kidney, and at variance with previous reports in the rat and guinea-pig, Kir7.1-HA is expressed in the inner medulla but not in the cortex or outer medulla. In isolated tubules immunoreactivity was associated with inner medulla collecting ducts but not thin limbs of the loop of Henle. Kir7.1-HA shows basolateral expression in the respiratory tract epithelium from trachea to bronchioli. The channel also appears basolateral in the epithelium of the nasal cavity and nasopharynx in newborn animals. We show that HA-tagged Kir7.1 channel introduced in the mouse by a knock-in procedure has functional properties similar to the native protein and the animal thus generated has clear advantages in localisation studies. It might therefore become a useful tool to unravel Kir7.1 function in the different organs where it is expressed.

G protein–coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1

“…Within tissues where Kir7.1 is highly expressed, large variations in glycosylation have been recently observed using a knockin mouse expressing an HA-tagged Kir7.1 channel (38). Notably, the study showed predominantly immature forms of the channel in the lung, whereas full complex glycosylation was observed in the trachea (38). No explanation for variation in glycosylation patterns between these two tissues was given, but our data may suggest a possible mechanism by which such changes could occur.…”

“…The data shown here provide evidence for molecular regulation of Kir7.1 structure and function by GPCRs. Within tissues where Kir7.1 is highly expressed, large variations in glycosylation have been recently observed using a knockin mouse expressing an HA-tagged Kir7.1 channel (38). Notably, the study showed predominantly immature forms of the channel in the lung, whereas full complex glycosylation was observed in the trachea (38).…”

Constitutive activation of canonical Wnt signaling disrupts choroid plexus epithelial fate

“…Key features of β-catenin GOF in the CP include dysregulation of factors that are essential for the CP to produce the CSF. These include several anion cation exchangers, ion channels, and solute transporters each of which acts as regulators of CSF composition: a sodium-potassium ATPase, Atp1a1 (Ernst et al, 1986); an epithelial sodium channel, Scnn1a (Amin et al, 2009); an inward rectifying potassium channel, Kcnj13 (Cornejo et al, 2018); a voltage-gated potassium channel, Kcna1 (Speake et al, 2003); and a sodium-potassium-chloride cotransporter 1, Nkcc1 , that modulates water transport underlying CSF production (Steffensen et al, 2018) and is also associated with Blood CSF barrier disruption (Kim & Jung., 2012). Furthermore, the expression of several genes encoding transporters for glucose, chloride, and amino acids, is also downregulated in the β-catenin GOF CP.…”

Altered phosphatidylinositol regulation of mutant inwardly rectifying K+Kir7.1 channels associated with inherited retinal degeneration disease

“…HEK‐293 cells seeded in 6‐well plates were transfected with 1 μg of plasmid DNA for WT Kir7.1‐HA (2 μg for the case of the concatemer) (Cornejo et al . 2018) or mutants thereof (2 μg for the case of the concatemer) using Lipofectamine 3000. After 24 h the cells were biotinylated with 1 mg ml −1 of sulfo‐NHS‐SS‐biotin twice for 20 min each time on ice.…”

“…Fig. 2B shows an experiment using WT Kir7.1 where a large current (in red) present at the onset of the pulse decays slowly to a value that corresponds to patch leak as inhibition by Ba 2+ takes place (Shimura et al 2001;Cornejo et al 2018). The difference between the initial current and that at the end of the pulse is defined as the specific Kir7.1-mediated current.…”