Recently, a functional IP₃R ortholog (CO.IP₃R-A) capable of IP₃-induced Ca²⁺ release has been discovered in Capsaspora owczarzaki, a close unicellular relative to Metazoa. In contrast to mammalian IP₃Rs, CO.IP₃R-A is not modulated by Ca²⁺, ATP or PKA. Protein-sequence analysis revealed that CO.IP₃R-A contained a putative binding site for anti-apoptotic Bcl-2, although Bcl-2 was not detected in Capsaspora owczarzaki and only appeared in Metazoa. Here, we examined whether human Bcl-2 could form a complex with CO.IP₃R-A channels and modulate their Ca²⁺-flux properties using ectopic expression approaches in a HEK293 cell model in which all three IP₃R isoforms were knocked out. We demonstrate that human Bcl-2 via its BH4 domain could functionally interact with CO.IP₃R-A, thereby suppressing Ca²⁺ flux through CO.IP₃R-A channels. The BH4 domain of Bcl-2 was sufficient for interaction with CO.IP₃R-A channels. Moreover, mutating the Lys17 of Bcl-2's BH4 domain, the residue critical for Bcl-2-dependent modulation of mammalian IP₃Rs, abrogated Bcl-2's ability to bind and inhibit CO.IP₃R-A channels. Hence, this raises the possibility that a unicellular ancestor of animals already had an IP₃R that harbored a Bcl-2-binding site. Bcl-2 proteins may have evolved as controllers of IP₃R function by exploiting this pre-existing site, thereby counteracting Ca²⁺-dependent apoptosis.

最近，在Capsaspora owczarzaki 中发现了一种能够 IP ₃诱导 Ca ²⁺释放的功能性 IP ₃ R 直向同源物（CO.IP ₃ R-A），这是一种与后生动物相关的紧密单细胞。与哺乳动物 IP ₃ Rs 相比，CO.IP ₃ R-A 不受 Ca ²⁺、ATP 或 PKA 的调节。蛋白质序列分析表明，CO.IP ₃ R-A 含有一个推定的抗凋亡 Bcl-2 结合位点，尽管 Bcl-2 在Capsaspora owczarzaki 中未检测到，仅出现在后生动物中。在这里，我们检查了人类 Bcl-2 是否可以与 CO.IP ₃ R-A 通道形成复合物并调节它们的 Ca在 HEK293 细胞模型中使用异位表达方法的²⁺ -flux 特性，其中所有三个 IP ₃ R 同种型都被敲除。我们证明人类 Bcl-2 通过其 BH4 域可以在功能上与 CO.IP ₃ R-A相互作用，从而抑制通过 CO.IP ₃ R-A 通道的Ca ²⁺通量。Bcl-2 的 BH4 域足以与 CO.IP ₃ R-A 通道相互作用。此外，突变 Bcl-2 的 BH4 结构域的 Lys17，这是 Bcl-2 依赖性调节哺乳动物 IP ₃ Rs 的关键残基，废除了 Bcl-2 结合和抑制 CO.IP ₃ R-A 通道的能力。因此，这增加了动物的单细胞祖先已经拥有 IP 的可能性₃ R 含有 Bcl-2 结合位点。Bcl-2 蛋白可能已经通过利用这个预先存在的位点进化为 IP ₃ R 功能的控制器，从而抵消 Ca ²⁺依赖性细胞凋亡。