Tóth Balázs, Iordanov Iordan, Csanády László
Department of Medical Biochemistry and MTA-SE Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary Department of Medical Biochemistry and MTA-SE Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary.
Department of Medical Biochemistry and MTA-SE Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary Department of Medical Biochemistry and MTA-SE Ion Channel Research Group, Semmelweis University, Budapest H-1094, Hungary
J Gen Physiol. 2015 May;145(5):419-30. doi: 10.1085/jgp.201511377.
Transient receptor potential melastatin 2 (TRPM2), a Ca(2+)-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid-adenine-dinucleotide (NAAD), and NAAD-2'-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified-specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2'-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest.
瞬时受体电位褪黑素2（TRPM2）是一种Ca(2+)通透阳离子通道，与缺血后神经元细胞死亡、白细胞活化和胰岛素分泌有关，可被细胞内ADP核糖（ADPR）激活。此外，当透析入细胞时，吡啶二核苷酸烟酰胺腺嘌呤二核苷酸（NAD）、烟酸腺嘌呤二核苷酸（NAAD）和NAAD - 2'-磷酸（NAADP）已被证明可激活TRPM2，或增强其被ADPR的激活作用。然而，直接作用于TRPM2蛋白的精确核苷酸子集尚不清楚。在这里，我们使用异源表达、亲和纯化的特异性ADPR水解酶从ADPR或ADPR - 2'-磷酸（ADPRP）的大量污染中纯化吡啶二核苷酸的商业制剂。将纯化的NAD、NAAD或NAADP直接应用于内翻膜片中TRPM2通道的胞质面，结果表明它们均不刺激门控，也不影响ADPR对通道的激活，这表明这些二核苷酸均不直接与TRPM2结合。相反，我们的实验首次确定ADPRP是一种具有潜在生物学意义的真正直接TRPM2激动剂。
