Institute of Biophysics, Johannes Kepler University of Linz, 4020 Linz, Austria.
Department of Chemistry and Biochemistry, University of Berne, 3012 Bern, Switzerland.
Biomolecules. 2022 Jun 8;12(6):804. doi: 10.3390/biom12060804.
The highly calcium-selective transient receptor potential vanilloid-type channel TRPV6 is important for epithelial Ca transport. Proper regulation of the inherently constitutively active TRPV6 channels is intricate in preserving Ca homeostasis, whereby structural and functional data suggest that lipids hold an essential role. Altered expression levels or specific TRPV6 mutations may lead to diseases, hence, TRPV6 represents an interesting target for pharmacological modulation. Recent cryo-EM data identified that the specific TRPV6 blocker cis-22a binds, apart from the pore, to a site within the tetrameric channel that largely matches a lipid binding pocket, LBS-2. Therein, cis-22a may replace a lipid such as cholesterol that is bound in the open state. Based on site-directed mutagenesis and functional recordings, we identified and characterized a series of residues within LBS-2 that are essential for TRPV6 inhibition by cis-22a. Additionally, we investigated the modulatory potential of diverse cholesterol depletion efforts on TRPV6 activity. While LBS-2 mutants exhibited altered maximum currents, slow Ca-dependent inactivation (SCDI) as well as less inhibition by cis-22a, TRPV6 activity was resistant to cholesterol depletion. Hence, lipids other than cholesterol may predominate TRPV6 regulation when the channel is expressed in HEK293 cells.
高度钙选择性瞬时受体电位香草素型通道 TRPV6 对上皮细胞 Ca 转运很重要。固有组成性激活的 TRPV6 通道的适当调节对于维持 Ca 平衡至关重要，结构和功能数据表明脂质起着重要作用。表达水平的改变或 TRPV6 的特定突变可能导致疾病，因此 TRPV6 是药理学调节的一个有趣靶点。最近的冷冻电镜数据表明，特定的 TRPV6 阻断剂 cis-22a 结合除了孔以外，还结合在四聚体通道内的一个位点，该位点在很大程度上与脂质结合口袋 LBS-2 匹配。在该位点，cis-22a 可能取代在开放状态下结合的脂质，如胆固醇。基于定点突变和功能记录，我们鉴定并表征了 LBS-2 内一系列对 cis-22a 抑制 TRPV6 至关重要的残基。此外，我们还研究了不同胆固醇耗竭方法对 TRPV6 活性的调节潜力。虽然 LBS-2 突变体表现出改变的最大电流、缓慢的 Ca 依赖性失活（SCDI）以及对 cis-22a 的抑制作用降低，但 TRPV6 活性对胆固醇耗竭具有抗性。因此，当通道在 HEK293 细胞中表达时，除了胆固醇之外的脂质可能在 TRPV6 调节中占主导地位。
