Biophysical and Pharmacological Characterization of Atp-Sensitive Potassium Channels in Mice Kir6.1WT/V65M Mirroring the Human Cantu′ Syndrome

Abstract

Cantu syndrome(CS) arises from GOF mutations in KCNJ8 or ABCC9, the genes encoding for Kir6.1 and SUR2 subunits of ATP-sensitive potassium(KATP) channels. Kir6.1 is prominent in vascular and skeletal muscle, but absent from cardiac muscle. Heterozygous Kir6.1[V65M] mice, in which the CS mutation was knocked in to the endogenous locus by CRISPR/Cas9 gene editing, exhibit KATP over-activity and reduced contractility in vascular smooth muscle, but not cardiomyocite. No information is available for other tissues. We measured the forelimb strength and ultrasonography of diaphragm and skeletal muscle, and investigated the KATP channel properties in native flexor digitorum brevis (FDB) and soleus (SOL) fibers, skin fibroblasts and long bones/calvaria-derived osteoblasts from WT and Kir6.1wt/V65M mice, by patch clamp technique. Forelimb forces were lower, and ultrasonography evaluation of the hind-limb showed significant enhancement of echodensity, in Kir6.1wt/V65M mice with respect to WT. Increased organ/tissue weights were observed in Kir6.1wt/V65M mice with respect to WT. In patch clamp experiments, higher KATP amplitude, reflecting the GOF associated with the mutation was observed in FDB fibers, skin fibroblasts and long bones/calvaria- derived osteoblasts from Kir6.1wt/V65M mice with respect to WT. No differences in KATP amplitude were detected between WT vs Kir6.1wt/V65M SOL fibers. A marked loss of channel sensitivity to Glib, also characteristic of the mutation was observed in all the mutant cells vs controls. Finally, a lower FDB fiber survival was observed in Kir6.1wt/V65M mice respect to the WT mice within 24 hours of isolation. The CS mutation Kir6.1wt/V65M has significant effects on KATP currents in multiple skeletal muscle and other tissues, reducing skeletal muscle contractility and fiber viability. This suggests that skeletal muscle insufficiency could be a significant issue in CS patients suffering from Kir6.1 GOF mutations.