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TPC2 polymorphisms associated with a hair pigmentation phenotype in humans result in gain of channel function by independent mechanisms

Significance Polymorphisms in the endolysosomal cation channel TPC2 have been suggested to lead to a shift in human hair color from brown to blond. In two further studies a role for TPC2 in melanosomal pH regulation was postulated. Electrophysiological data on how these polymorphisms affect channel gating and activity are, however, missing. We show here that both polymorphisms lead to a gain of channel function by different mechanisms. In M484L sensitivity to its endogenous ligand PI(3,5)P2 is strongly increased while in G734E channel inactivation by ATP is reduced. These findings are corroborated by molecular dynamics and ion substitution experiments. Furthermore, >100 blond- and brown/black-haired human individuals were genotyped and fibroblasts isolated from selected donors to confirm key in vitro findings. Two-pore channels (TPCs) are endolysosomal cation channels. Two members exist in humans, TPC1 and TPC2. Functional roles associated with the ubiquitously expressed TPCs include VEGF-induced neoangiogenesis, LDL-cholesterol trafficking and degradation, physical endurance under fasting conditions, autophagy regulation, the acrosome reaction in sperm, cancer cell migration, and intracellular trafficking of pathogens such as Ebola virus or bacterial toxins (e.g., cholera toxin). In a genome-wide association study for variants associated with human pigmentation characteristics two coding variants of TPC2, rs35264875 (encoding M484L) and rs3829241 (encoding G734E), have been found to be associated with a shift from brown to blond hair color. In two recent follow-up studies a role for TPC2 in pigmentation has been further confirmed. However, these human polymorphic variants have not been functionally characterized until now. The development of endolysosomal patch-clamp techniques has made it possible to investigate directly ion channel activities and characteristics in isolated endolysosomal organelles. We applied this technique here to scrutinize channel characteristics of the polymorphic TPC2 variants in direct comparison with WT. We found that both polymorphisms lead to a gain of channel function by independent mechanisms. We next conducted a clinical study with more than 100 blond- and brown/black-haired individuals. We performed a genotype/phenotype analysis and subsequently isolated fibroblasts from WT and polymorphic variant carriers for endolysosomal patch-clamp experimentation to confirm key in vitro findings.