Characterization of CLIC2

Project Summary The proposal is focused on the physiological characterization of Chloride intracellular channel 2 [CLIC2, a channel listed in FOA: RFA-RM-21-012 (Pilot Projects Investigating Understudied Ion Channels)]. CLIC2 was identified as XAP121 in an attempt to characterize the 1200-kb telomeric region of Xq28. The region was associated with hypertrophic cardiomyopathy and X-linked intellectual disability (XLID). Patients with mutations in CLIC2 showed early onset of symptoms with death in infancy of some of the affected males and others at later age points due to cardiomegaly. Though CLIC2 has been directly associated with X-linked pathological conditions and episodic ataxia since 1997, information on its physiological roles and biophysical properties is not yet established. In our preliminary experiments, we discovered that CLIC2 is essential for cardiomyocyte differentiation. In its absence human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) differentiate at a slower rate and differentiated cardiomyocytes have abnormal function. We have discovered an unusual truncation mutation in CLIC2 in XLID patients who have reported dilated cardiomyopathy. In these patients, a mutation in the clic2 gene leads to premature truncation of CLIC2 at leucine 92 (CLIC2 Δ92). Therefore, we hypothesize that the novel mutation resulting in the truncation of C-terminus CLIC2 decreases the affinity of CLIC2 for RyR2 which worsens cardiac function in HCM patients by increasing SR Ca2+ leak through RyR2 and abnormal cardiomyocyte differentiation. We also discovered a unique peptide from CLIC2 that can rescue the function of cardiomyocytes lacking CLIC2. We have the following specific aim, 1) to establish the role of CLIC2 Δ92 in the differentiation and function of early human cardiomyocytes, and 2) to establish the role of CLIC2 in the regulation of cytosolic calcium homeostasis. In Aim 1 we use a combination of genetic and imaging approaches to elucidate the impact of CLIC2 mutations on cardiomyocyte differentiation and function. In Aim 2 we will rescue the function of hiPSC-CMs lacking CLIC2 by a unique peptide and also screen the FDA-approved small molecule library available at OSU. The proposal will enhance our understanding of CLIC2 protein by establishing its mechanism in cardiomegaly. CLIC2 mutations are also associated with autism, XLID, episodic ataxia, and learning disorders and the information obtained in our proposal could also increase our understanding of CLIC2 in other pathological conditions.