Evidence for the Presence of Aquaporin-3 in Human Red Blood Cells

Abstract

A facilitated diffusion for glycerol is present in human erythrocytes. Glycerol transporters identified to date belong to the Major Intrinsic Protein (MIP) family of integral membrane proteins, and one of them, aquaporin-3 (AQP3), has been characterized in mammals. Using an antibody raised against a peptide corresponding to the rat AQP3 carboxyl terminus, we examined the presence of AQP3 in normal and Colton-null (aquaporin-1 (AQP1)-deficient) human erythrocytes. Three immunoreactive bands were detected on immunoblots of both normal and Colton-null red cells, very similar to the bands revealed in rat kidney, a material in which AQP3 has been extensively studied. By immunofluorescence, anti-AQP3 antibodies stained the plasma membranes of both normal and Colton-null erythrocytes. Glycerol transport was measured on intact erythrocytes by stopped-flow light scattering and on one-step pink ghosts by a rapid filtration technique. Glycerol permeability values, similar in both cell types, suggest that AQP1 does not represent the major path for glycerol movement across red blood cell membranes. Furthermore, pharmacological studies showed that Colton-null red cells remain sensitive to water and glycerol flux inhibitors, supporting the idea that another proteinaceous path, probably AQP3, mediates most of the glycerol movements across red cell membranes and represents part of the residual water transport activity found in AQP1-deficient red cells.Human erythrocytes are highly permeable to water, urea, and glycerol (1-3). The existence of membrane proteins that facilitate water and solute movements in these cells has been postulated, but most of these proteins remain to be characterized. In 1992, Agre and co-workers identified a very abundant protein of the human red blood cell as the first water-selective channel that was named aquaporin-1 (AQP1) 1 (4). Colton-null erythrocytes, lacking AQP1, exhibit a reduced osmotic water permeability (5). However, these cells are found to be residually permeable to water, suggesting the presence of additional, non-AQP1, water channels.The existence of a protein-mediated glycerol transport in erythrocytes has relied mostly on pharmacological evidence. In particular, inhibition of glycerol transport by sulfhydryl reagents, phloretin, and copper ions has been reported (1,3,6). Yet, the identity of the erythrocyte glycerol carrier remains unknown. The glycerol facilitators identified to date all belong to the Major Intrinsic Protein (MIP) family (7). They have been characterized in several organisms: GlpF, a bacterial glycerol permease facilitator (8); Fps1p, a yeast glycerol exporter (9); aquaporin-3 (AQP3), initially characterized in mammalian kidney (10 -12); and AQP7, recently identified in rat testis (13). Compared with other mammalian aquaporins, which are selective mostly for water, AQP3 is moderately permeable to water, but highly permeable to glycerol and possibly to urea (11,12,14). AQP3 expression has been reported in several mammalian tissues, including kidney, intestine, stomach...

Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation

“…The P gly obtained for hRBC using the stopped-flow were of the same order of magnitude as those reported for dihydroxy alcohols using this same methodology [29], but were one order larger than the described using rapid filtration with radioactive labeled glycerol [13].…”

“…Given that the AQP3 protein has been recognized as a main glycerol channel in human and rat erythrocytes [13] and that AQP3 gene was recently assigned to bovine 14 chromosome 8 [16], our first approach was to examine AQP3 expression in bRBC and correlate it with the channel function.…”

“…Compared with other mammalian orthodox aquaporins (namely with AQP1), AQP3 is moderately permeable to water, but highly permeable to glycerol and possibly to urea [1]. AQP3 expression has been reported in several mammalian tissues including kidney, epidermis, urinary, respiratory and digestive tracts [11] and in human erythrocytes [13].…”

“…Likewise, expression and function of the glycerol channel AQP3 has been detected in human and rat [13] but not in mouse erythrocytes [15]. As for bovines, despite the AQP3 gene has been identified in the bovine genome…”

An emerging consensus on aquaporin translocation as a regulatory mechanism

“…Chimeric proteins involving substitution of the AQP2 N-terminus for that of the AQP3 was able to re-direct localisation of AQP2 from its original site at the apical membrane to its new site at the basolateral membrane. Interestingly, despite this clear amino acid-sorting motif, membrane expression of AQP3 is not polarised in red blood cells (Roudier et al, 1998) or in epidermal keratinocytes and in the epidermis "basal cells"; AQP3 may be predominantly intracellular (Sougrat et al, 2002).…”