Institute of Physiology, Medical Faculty, RWTH Aachen University Hospital, D52057 Aachen, Germany.
Int J Mol Sci. 2021 Apr 28;22(9):4637. doi: 10.3390/ijms22094637.
The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P), commonly referred to as PIP, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP. We demonstrate a crucial role of PIP, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP that were dependent on the species variant became apparent. While depletion of PIP via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca in a species-specific manner.
人类凋亡通道 TRPM2 可被细胞内 ADR-核糖和钙刺激。最近的研究表明，该通道在不同物种中具有明显的特异性激活机制。我们的目的是分析磷脂酰肌醇 4,5-二磷酸（PI(4,5)P），通常称为 PIP，对不同 TRPM2 同源物的功能影响。此外，我们还希望确定 TRPM2 和 PIP 之间的相互作用位点。我们证明了 PIP 在人类、斑马鱼和海葵 TRPM2 同源物的激活中起着关键作用。利用转染了 TRPM2 的 HEK-293 细胞的内向外膜片钳记录，我们发现 PIP 的差异作用取决于物种变体。虽然通过多聚赖氨酸耗尽 PIP 会均匀地导致 TRPM2 的完全失活，但通过人工 PIP 恢复通道活性的效果差异很大。人类 TRPM2 是最不敏感的物种变体，因此最容易受到跨膜 PIP 含量变化的调节。此外，在膜界面腔中高度保守的带正电荷氨基酸残基的突变会不同程度地降低三种 TRPM2 同源物对 PIP 的敏感性。我们的结论是，膜界面腔作为 TRPM2 的统一 PIP 结合位点，在 ADPR 和 Ca 的存在下以物种特异性的方式促进通道激活。
