The flow of water across cell membranes is fundamental to the physiology of all organisms. It is now clear that osmosis is not sufficient for this purpose; rather aquaporin (AQP)4
channels are required to ensure appropriate membrane permeability to water molecules (1
). Over the past two decades, the molecular basis of selective water passage through the AQP pore (3
), as well as the structural biology of the family (5
), have been established. However, there is a gap in our current understanding of how AQPs regulate the flow of water in and out of cells to meet the constant and rapid changes in local water availability that challenge them.
Membrane permeability to water is a function of the properties of the AQP pore as well as the abundance of AQP molecules in the cell membrane. The AQP pore is acknowledged widely to be constitutively open and highly specific. Some AQPs are permeable only to water, whereas others (e.g.
the aquaglyceroporins) are permeable to both water and small non-ionic molecules such as glycerol, urea, and ammonia (3
). Regulation via gating mechanisms, which allow open and closed states, has been reported for some plant and microbial AQPs (7
). However, this is not a widely accepted regulatory mechanism for mammalian AQPs (8
Regulation of AQP abundance, the number of pores per unit plasma membrane, is possible via
several mechanisms. Direct regulation by AQP gene expression and/or AQP protein degradation can be achieved over a time scale from hours to days (9
). Indirect, receptor-mediated mechanisms (11
) also have been described that account for more rapid regulation of AQP abundance on a time scale of minutes (13
). The best studied example of this is the regulation of AQP2 translocation in human kidney cells, which is dependent on vasopressin-mediated activation of protein kinase A by the G protein-coupled receptor, vasopressin V2
). Of the 13 known AQPs in the human body, AQP1 (16
), AQP3 (17
), and AQP5 (18
) also have been shown to undergo translocation to the plasma membrane in response to hormonal activation of specific G protein-coupled receptors.
Neither gene expression nor indirect, receptor-mediated translocation can explain the direct regulation of AQPs that may be necessary to respond to the rapidly changing extracellular environment on a time scale of seconds. We recently demonstrated that increased translocation of AQP1 is triggered on this rapid timescale by hypotonic stimulus in a specific protein kinase C (PKC)- and microtubule-dependent manner (19
). Furthermore, returning the extracellular environment to its original tonicity reversed this dynamic subcellular localization. In contrast, a hypotonic stimulus had little effect on AQP2 localization in the absence of the vasopressin V2
receptor required for AQP2 translocation (19
The change in cell volume that results from the transport of water across biological membranes is thought to be dependent on PKC and calcium, as well as the presence of transient receptor potential (TRP) channels and AQPs (20
). The data presented here provide evidence of a mechanistic link between these elements. In this study, we combined laser scanning confocal microscopy of chimeras of AQP1 with green fluorescent protein (AQP1-GFP), calcium imaging, and mutagenesis to determine that AQP1 translocation underpins the regulation of cellular water flow, as measured by changes in cell volume. Our data show that manipulating rapid AQP1 translocation, which can be observed in primary astrocytes as well as model cell lines, modulates changes in cell volume and that this rapid subcellular localization of AQP1 requires extracellular calcium influx, TRP channels, calmodulin, and specific phosphorylation at two known PKC sites, Thr-157 and Thr-239. We therefore suggest that the regulation of AQPs provides the rapid homeostatic control required by cells in a constantly changing osmotic environment.