Functional modulation of human BEST1 channel by a proline metabolic enzyme, PYCR3

초록

Bestrophin (BEST) Bestrophin (BEST) channels, including human BEST1 (hBEST1), are calcium-activated chloride (Cl⁻) channels that respond to intracellular [Ca²⁺] elevations. Recently, we identified human PYCR3, an enzyme in proline metabolism, as a novel binding partner of hBEST1 through immunoprecipitation-mass spectrometry (IP-MS). The interaction between hBEST1 and hPYCR3 was confirmed via co-immunoprecipitation (Co-IP) and size-exclusion chromatography (SEC), where the two proteins co-migrated, indicating a direct interaction. To investigate effect of hPYCR3 on hBEST1 channels, we performed whole-cell patch clamp recordings. We found that hPYCR3 overexpression increased hBEST1 current density in HEK293T cells, whereas PYCR3 knockdown significantly reduced it. Furthermore, co-expression of hBEST1 and hPYCR3 in hPYCR3 knockout HEK293T cells augmented hBEST1 current levels, underscoring hPYCR3’s critical role in modulating hBEST1 activity. Functional analysis of the enzymatically inactive hPYCR3 G252V mutant revealed no enhancement of hBEST1 current density. This result highlights that hPYCR3 enzymatic activity is essential for hBEST1 modulation. Additionally, treatments with proline and cofactors further supported a mechanistic link between hPYCR3-mediated proline metabolism and hBEST1 activity. These findings identify hPYCR3 as a key enzymatic regulator of hBEST1. We aim to investigate whether the effects of hBEST1 and hPYCR3 observed in vitro are recapitulated in vivo using retinal pigment epithelial (RPE) cells, where hBEST1 is highly expressed, to explore their physiological relevance. Additionally, we aim to resolve the complex structure of hBEST1 and hPYCR3 to elucidate the mechanisms underlying their structural modulation. Ongoing studies aim to elucidate the structural and functional mechanisms underlying hPYCR3-dependent modulation of hBEST1, offering new insights into the interplay between proline metabolism and ion channel regulation. * Keywords: BEST1, PYCR3, whole-cell patch-clamp, Co-Immunoprecipitation