A cataract-causing mutation in the TRPM3 cation channel disrupts calcium dynamics in the lens

TRPM3 belongs to the melastatin sub-family of transient receptor potential (TRPM) cation channels and has been shown to function as a steroid-activated, heat-sensitive, calcium ion (Ca2+) channel. A missense substitution (p.I65M) in the TRPM3 gene of humans (TRPM3) and mice (Trpm3) has been shown to underlie an inherited form of early-onset, progressive cataract. Here we model the pathogenetic effects of this cataract-causing mutation using 'knock-in' mutant mice and human cell-lines. Trpm3 and its intron-hosted micro-RNA gene (Mir204) were strongly co-expressed in the lens epithelium and other non-pigmented and pigmented ocular epithelia. Homozygous Trpm3-mutant lenses displayed elevated cytosolic Ca2+ levels and an imbalance of sodium (Na+) and potassium (K+) ions coupled with increased water content. Homozygous TRPM3-mutant human lens epithelial (HLE-B3) cell-lines and Trpm3-mutant lenses exhibited increased levels of phosphorylated mitogen-activated protein kinase 1/extracellular signal-regulated kinase 2 (MAPK1/ERK2/p42) and MAPK3/ERK1/p44. Mutant TRPM3-M65 channels displayed an increased sensitivity to external Ca2+ concentration and an altered dose response to pregnenolone sulfate (PS) activation. Trpm3-mutant lenses shared downregulation of genes involved in insulin/peptide secretion and upregulation of genes involved in Ca2+ dynamics. By contrast, Trpm3-deficient lenses did not replicate the pathophysiological changes observed in Trpm3-mutant lenses. Collectively, our data suggest that a cataract-causing substitution in the TRPM3 cation channel elicits a deleterious gain-of-function, rather than a loss-of-function, mechanism in the lens. Mouse lens RNA samples (4 male and 4 female lenses per sample) were prepared in triplicate and cDNA libraries were synthesized, indexed, pooled, and sequenced. Samples represented WT, Trpm3-KO, Trpm3-mutant (I/M), and Trpm3-mutant (M/M).TRPM3属于瞬态受体电位黑素原亚家族（transient receptor potential melastatin, TRPM）阳离子通道，已被证实可作为类固醇激活、热敏感型钙离子（Ca²+）通道发挥功能。人类与小鼠的TRPM3基因中存在一处错义突变（p.I65M），该突变被证实是一种遗传性早发性进行性白内障的致病根源。本研究通过“敲入（knock-in）”突变小鼠及人类细胞系，模拟该致白内障突变的致病效应。Trpm3及其内含子编码的微小RNA（micro-RNA）基因Mir204在晶状体上皮及其他非色素化、色素化眼上皮组织中呈现显著共表达特征。纯合Trpm3突变小鼠的晶状体表现为胞质钙离子水平升高，钠（Na+）、钾（K+）离子失衡，并伴随含水量增加。纯合TRPM3突变的人类晶状体上皮（HLE-B3）细胞系及Trpm3突变小鼠晶状体中，磷酸化丝裂原活化蛋白激酶1/细胞外调节蛋白激酶2（MAPK1/ERK2/p42）与MAPK3/ERK1/p44的表达水平均升高。突变型TRPM3-M65通道对胞外钙离子浓度的敏感性增强，且对硫酸孕烯醇酮（PS）激活的剂量反应特征发生改变。Trpm3突变小鼠的晶状体中，胰岛素/肽类分泌相关基因表达下调，而钙离子动态平衡相关基因表达上调。与之相比，Trpm3基因敲除（Trpm3-KO）小鼠的晶状体并未重现Trpm3突变小鼠晶状体所表现出的病理生理变化。综上，本研究数据表明，TRPM3阳离子通道上的致白内障错义突变，通过有害的功能获得性（而非功能丧失性）机制引发晶状体病变。本研究设置3次生物学重复，每份样本取自4只雄性与4只雌性小鼠的晶状体，随后提取总RNA并合成互补DNA（cDNA）文库，经索引标记、混合后进行高通量测序。实验样本分为野生型（WT）、Trpm3基因敲除型（Trpm3-KO）、Trpm3杂合突变型（I/M）及Trpm3纯合突变型（M/M）四组。