Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee.
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee.
Am J Physiol Cell Physiol. 2021 Mar 1;320(3):C293-C303. doi: 10.1152/ajpcell.00454.2020. Epub 2020 Dec 23.
Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 µM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ∼90% by 10 µM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ dependent. At normal intracellular µ, LRRC8A + LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels.
肿胀激活的体积调节阴离子通道（VRAC）是由 LRRC8A 和至少一种其他 LRRC8 同源异构体组成的异源多聚体通道。已经描述了非天然 LRRC8A 和 LRRC8D 同六聚体的冷冻电镜（cryo-EM）结构。我们在这里证明，LRRC8A 同六聚体不能很好地再现 VRAC 的功能特性。与 VRAC 不同，异源表达于 HCT116 细胞中的 LRRC8A 通道对内源性离子强度（µ）降低的反应较差，并且对正常 µ 的细胞肿胀不敏感。将低 µ 与肿胀结合使用，可适度激活 LRRC8A，从而可以对其孔道特性进行表征。10µM 4-[(2-丁基-6,7-二氯-2-环戊基-2,3-二氢-1-氧代-1-茚满-5-基)氧基]丁酸（DCPIB）以电压非依赖性方式强烈抑制 VRAC。相比之下，DCPIB 对 LRRC8A 的抑制作用较弱且对电压敏感。cryo-EM 结构表明，DCPIB 抑制作用依赖于精氨酸 103。LRRC8A R103F 突变体对此一致，对 DCPIB 不敏感。然而，在同源位置用亮氨酸替换 R103 的 LRRC8 嵌合通道以电压非依赖性方式被 10µM DCPIB 抑制约 90%。LRRC8A 和 LRRC8C 的共表达产生了对 DCPIB 敏感的通道，其敏感性强烈依赖于 µ。在正常的细胞内 µ 下，LRRC8A+LRRC8C 异源二聚体表现出强烈的、电压非依赖性的 DCPIB 抑制作用，对 R103F 不敏感。在低 µ 条件下，DCPIB 抑制作用大大降低，并表现出电压依赖性。R103F 突变对最大 DCPIB 抑制作用没有影响，但消除了低 µ 条件下的电压依赖性。我们的研究结果表明，LRRC8A 的 cryo-EM 结构和异源表达的 LRRC8 异源多聚体通道的使用对基于 VRAC 突变的结构-功能分析构成了重大限制。天然 VRAC 功能最接近嵌合 LRRC8 同聚体通道模拟。
