Abstract
ABSTRACTApproximately 15% of autosomal dominant polycystic kidney disease (ADPKD) is caused by variants in PKD2. PKD2 encodes polycystin-2, which forms an ion channel in primary cilia and endoplasmic reticulum (ER) membranes of renal collecting duct cells. Elevated internal Ca2+ modulates polycystin-2 voltage-dependent gating and subsequent desensitization – two biophysical regulatory mechanisms that control its function at physiological membrane potentials. Here, we refute the hypothesis that Ca2+ occupancy of the polycystin-2 intracellular EF hand is responsible for these forms of channel regulation, and, if disrupted, results in ADPKD. We identify and introduce mutations that attenuate Ca2+-EF hand affinity but find channel function is unaltered in the primary cilia and ER membranes. We generated two new mouse strains that harbor distinct mutations that abolish Ca2+-EF hand association but do not result in a PKD phenotype. Our findings suggest that additional Ca2+-binding sites within polycystin-2 or Ca2+-dependent modifiers are responsible for regulating channel activity.
Highlights
PKD2 encodes for polycystin-2, a member of the polycystin subfamily of transient receptor potential ion channels (TRPP) (Venkatachalam and Montell, 2007)
We found that disrupting Ca2+ occupancy of the EF hand did not alter Ca2+-dependent modulation’ (CDM) or Ca2+-dependent desensitization’ (CDD) of the mouse and human orthologs of polycystin-2 in the primary cilia
Our findings demonstrate that disruption of Ca2+-EF hand affinity does not lead to impaired in vitro or in vivo function of polycystin-2, which suggests that autosomal dominant polycystic kidney disease (ADPKD)-causing truncating variants found in the C-terminal domain (CTD) likely affect other motifs that have a greater impact on channel regulation
Summary
PKD2 encodes for polycystin-2, a member of the polycystin subfamily of transient receptor potential ion channels (TRPP) (Venkatachalam and Montell, 2007). Mouse models of attenuating Pkd expression by conditional genetic repression faithfully recapitulate the polycystic kidney phenotype (Happé and Peters, 2014; Ma et al, 2013; Wilson, 2008; Wu et al, 1998), whereas complete genetic deletion of Pkd causes embryonic lethality and kidney cyst development in utero (Menezes and Germino, 2013; Wu et al, 2000) These studies in mice suggest that most ADPKDcausing variants are loss of function, but our understanding of their mechanistic impact remains largely undetermined owing to their subcellular localization
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