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.
Highlights
The inwardly rectifying Kir7.1 K+ channel is encoded by the Kcnj13 gene in the mouse and is classified as a K+-transport type channel
Knockout mice are valuable in ascertaining the function of ion channels and, importantly, they aid in the cellular and subcellular localization by providing reliable negative controls for western blots and immunolocalisation assays
The generation by CRISPR Cas9 technology of a Kir7.1 knock-in mouse that expresses the channel tagged with an HA provides a convenient animal model to study reliably the distribution of the channel in mouse tissues using their WT littermates as negative controls
Summary
The inwardly rectifying Kir7.1 K+ channel is encoded by the Kcnj gene in the mouse and is classified as a K+-transport type channel. The Na+/K+-pump is expressed at a basolateral membrane location in these epithelia as in most transporting barriers. Kir7.1, is found at the apical membranes of retinal pigmented epithelium (RPE) and choroid plexus. These epithelia show an unusual apical membrane expression of the Na+/K+-pump (Nakamura et al, 1999; Kusaka et al, 2001; Shimura et al, 2001; Yang et al, 2003). The faithful distribution of Kir7.1 together with the Na+/K+-pump has prompted the speculation that Kir7.1’s function might be to provide a pathway to recycle K+ taken up pump in exchange for Na+ maintaining transepithelial ion transport processes without potentially deleterious intracellular K+ accumulation (Sepúlveda et al, 2015). Other roles attributed to Kir 7.1 include a participation in the mechanism of melanocortin-mediated regulation of energy homeostasis within the paraventricular nucleus (Ghamari-Langroudi et al, 2015) and the control of excitability of uterine smooth muscle in the transition to contractions in the pregnant uterus (McCloskey et al, 2014)
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