Zhao Pan, Moss Fraser J, Occhipinti Rossana, Geyer R Ryan, Huffman Dale E, Meyerson Howard J, Boron Walter F
Department of Physiology & Biophysics, Case Western Reserve University School of Medicine Cleveland, OH 44106.
Department of Pathology, Case Western Reserve University School of Medicine Cleveland, OH 44106.
bioRxiv. 2025 May 18:2025.03.05.639948. doi: 10.1101/2025.03.05.639948.
Many have believed that oxygen crosses red blood cell (RBC) membranes by dissolving in lipids that offer a finite resistance to diffusion or, alternatively, no resistance at all. In this first in a series of three interrelated papers, we examine these idea in murine RBCs. In this first paper, analyses of hemoglobin (Hb) absorbance spectra during off-loading from mouse RBCs indicate that RBC membranes do indeed offer resistance to diffusion, and that the resistance would be far higher if not for the presence of -permeable channels. Two agents-both excluded from the RBC interior-markedly reduce the rate constant for off-loading : p-chloromercuribenzenesulfonate (pCMBS) reduces membrane permeability ( by ~82% (computed from in paper #3), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) by ~56%. Because neither likely produces these effects via membrane lipids, we examined RBCs from mice genetically deficient in aquaporin-1 (AQP1), the Rh complex ( = Rhesus blood group-associated A glycoprotein, RhAG + Rhesus blood group D antigen, RhD), or both. The double knockout (dKO) reduces by ~55%, and pCMBS+dKO, by ~91%. Proteomic analyses of RBC membranes, flow cytometry, hematology (see paper #2), and mathematical simulations (paper #3) rule out meaningful contributions from other membrane proteins, RBC geometry, or extracellular unconvected fluid (EUF). Our work represents a paradigm shift for physiology by identifying the first two animal channels, and points to the existence of at least a third, all of which could be subject to physiological regulation and pharmacological intervention.
许多人认为，氧气通过溶解在对扩散有一定阻力或根本没有阻力的脂质中来穿过红细胞（RBC）膜。在这三篇相互关联的系列论文的第一篇中，我们在小鼠红细胞中研究了这些观点。在这第一篇论文中，对从小鼠红细胞卸载过程中血红蛋白（Hb）吸收光谱的分析表明，红细胞膜确实对扩散有阻力，而且如果没有水通道，阻力会高得多。两种均被排除在红细胞内部的试剂显著降低了卸载的速率常数：对氯汞苯磺酸盐（pCMBS）使膜通透性降低约82%（根据论文#3中的计算），4,4'-二异硫氰酸根合芪-2,2'-二磺酸盐（DIDS）使膜通透性降低约56%。由于两者都不太可能通过膜脂质产生这些影响，我们研究了来自水通道蛋白-1（AQP1）基因缺陷、Rh复合物（Rh = 恒河猴血型相关A糖蛋白，RhAG + 恒河猴血型D抗原，RhD）基因缺陷或两者均缺陷的小鼠的红细胞。双敲除（dKO）使膜通透性降低约55%，pCMBS + dKO使膜通透性降低约91%。对红细胞膜的蛋白质组学分析、流式细胞术、血液学（见论文#2）和数学模拟（论文#3）排除了其他膜蛋白、红细胞几何形状或细胞外非对流流体（EUF）的重要贡献。我们的工作通过鉴定出首批两个动物水通道，代表了对生理学的一种范式转变，并指出至少还存在第三个水通道，所有这些水通道都可能受到生理调节和药物干预。
