Department of Molecular Cell Physiology, Kyoto Prefectural University of Medicine, 465 Kajiicho Kamigyo-ward, Kyoto, 602-8566, Japan.
Department of Physiology, Osaka Medical College, 2-7 Daigakumachi, Takatsuki, 569-8686, Japan.
J Physiol. 2017 Sep 15;595(18):6121-6145. doi: 10.1113/JP274164. Epub 2017 Aug 14.
Calcium homeostasis modulator 1 (CALHM1), a new voltage-gated ATP- and Ca -permeable channel, plays important physiological roles in taste perception and memory formation. Regulatory mechanisms of CALHM1 remain unexplored, although the biophysical disparity between CALHM1 gating in vivo and in vitro suggests that there are undiscovered regulatory mechanisms. Here we report that CALHM1 gating and association with lipid microdomains are post-translationally regulated through the process of protein S-palmitoylation, a reversible attachment of palmitate to cysteine residues. Our data also establish cysteine residues and enzymes responsible for CALHM1 palmitoylation. CALHM1 regulation by palmitoylation provides new mechanistic insights into fine-tuning of CALHM1 gating in vivo and suggests a potential layer of regulation in taste and memory.
Emerging roles of CALHM1, a recently discovered voltage-gated ion channel, include purinergic neurotransmission of tastes in taste buds and memory formation in the brain, highlighting its physiological importance. However, the regulatory mechanisms of the CALHM1 channel remain entirely unexplored, hindering full understanding of its contribution in vivo. The different gating properties of CALHM1 in vivo and in vitro suggest undiscovered regulatory mechanisms. Here, in searching for post-translational regulatory mechanisms, we discovered the regulation of CALHM1 gating and association with lipid microdomains via protein S-palmitoylation, the only reversible lipid modification of proteins on cysteine residues. CALHM1 is palmitoylated at two intracellular cysteines located in the juxtamembrane regions of the third and fourth transmembrane domains. Enzymes that catalyse CALHM1 palmitoylation were identified by screening 23 members of the DHHC protein acyltransferase family. Epitope tagging of endogenous CALHM1 proteins in mice revealed that CALHM1 is basally palmitoylated in taste buds in vivo. Functionally, palmitoylation downregulates CALHM1 without effects on its synthesis, degradation and cell surface expression. Mutation of the palmitoylation sites has a profound impact on CALHM1 gating, shifting the conductance-voltage relationship to more negative voltages and accelerating the activation kinetics. The same mutation also reduces CALHM1 association with detergent-resistant membranes. Our results comprehensively uncover a post-translational regulation of the voltage-dependent gating of CALHM1 by palmitoylation.
钙稳态调节剂 1（CALHM1）是一种新的电压门控 ATP 和 Ca 通透通道，在味觉感知和记忆形成中发挥重要的生理作用。尽管 CALHM1 的门控在体内和体外存在明显差异，表明存在尚未发现的调节机制，但 CALHM1 的调节机制仍未得到探索。在这里，我们报告 CALHM1 的门控和与脂质微区的关联是通过蛋白质 S-棕榈酰化的翻译后过程来调节的，这是一种将棕榈酸可逆地连接到半胱氨酸残基上的过程。我们的数据还确定了负责 CALHM1 棕榈酰化的半胱氨酸残基和酶。CALHM1 的棕榈酰化调节为 CALHM1 门控的精细调控提供了新的机制见解，并提示了味觉和记忆中潜在的调控层。
最近发现的电压门控离子通道 CALHM1 的作用不断涌现，包括在味蕾中的嘌呤能神经递质传递和在大脑中的记忆形成，这凸显了其生理重要性。然而，CALHM1 通道的调节机制仍完全未知，阻碍了对其在体内的贡献的全面理解。CALHM1 在体内和体外的不同门控特性表明存在未被发现的调节机制。在这里，在寻找翻译后调节机制时，我们通过蛋白质 S-棕榈酰化发现了 CALHM1 门控和与脂质微区的关联的调节，这是半胱氨酸残基上唯一的蛋白质可逆脂质修饰。CALHM1 在位于第三和第四跨膜区域的近膜区的两个细胞内半胱氨酸残基上被棕榈酰化。通过筛选 23 种 DHHC 蛋白酰基转移酶家族成员，鉴定出催化 CALHM1 棕榈酰化的酶。用内源性 CALHM1 蛋白的表位标记在体内的小鼠味蕾中发现，CALHM1 被基础棕榈酰化。在功能上，棕榈酰化下调 CALHM1，而不影响其合成、降解和细胞表面表达。棕榈酰化位点的突变对 CALHM1 的门控有深远的影响，使电导-电压关系向更负的电压偏移，并加速激活动力学。相同的突变也减少了 CALHM1 与去污剂抗性膜的关联。我们的结果全面揭示了 CALHM1 的电压依赖性门控通过棕榈酰化的翻译后调节。
