Rapid characterization of the functional and pharmacological consequences of Cantú syndrome K ATP channel mutations in intact cells .

Gain-of-function (GOF) of K ATP channels, resulting from mutations in either KCNJ8 (encoding Kir6.1) or ABCC9 (encoding SUR2), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive DiBAC4(3) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the K ATP channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared to the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle K ATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A, was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. Significance Statement We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent K ATP channels, showing that Kir6.1 mutations increase sensitivity to activation by potassium channel openers, while SUR2B mutations markedly reduce KCO sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by K ATP channel activity under basal, non KCO-activated conditions.