PIP aquaporin pH-sensing is regulated by the length and charge of the C-terminal region.

Plant PIP aquaporins play a central role in controlling plant water status. The current structural model for PIP pH-gating states that the main pH sensor is located in loopD and that all the mobile cytosolic elements participate in a complex interaction network that ensures the closed structure. However, the precise participation of the last part of the C-terminal domain (CT) in PIP pH gating remains unknown. This last part has not been resolved in PIP crystal structures and is a key difference between PIP1 and PIP2 paralogues. Here, by a combined experimental and computational approach, we provide data about the role of CT in pH gating of Beta vulgaris PIP. We demonstrate that the length of CT and the positive charge located among its last residues modulate the pH at which the open/closed transition occurs. We also postulate a molecular-based mechanism for the differential pH sensing in PIP homo- or heterotetramers by performing atomistic molecular dynamics simulations (MDS) on complete models of PIP tetramers. Our findings show that the last part of CT can affect the environment of loopD pH sensors in the closed state. Results presented herein contribute to the understanding of how the characteristics of CT in PIP channels play a crucial role in determining the pH at which water transport through these channels is blocked, highlighting the relevance of the differentially conserved very last residues in PIP1 and PIP2 paralogues.

Gating in plant plasma membrane aquaporins: the involvement of leucine in the formation of a pore constriction in the closed state.

The control of water permeability in plant PIP2 aquaporins has become a paradigmatic case study of the capping mechanism for pore closure in water channels. From structural data, it has been postulated that the gating process in PIP2 involves a conformational rearrangement in cytosolic loopD that generates an obstruction to the transport of water molecules inside the aquaporin pore. BvPIP2;2 is a PIP2 aquaporin from Beta vulgaris whose pH response has been thoroughly characterized. In this work, we study the participation of Leu206 in BvPIP2;2 gating triggered by cytosolic acidification and show that this residue acts as a plug that blocks water transport. Based on data obtained from in silico and in vitro studies, we demonstrate that Leu206, one of the residues lining the pore, is responsible for ~ 60% of water blockage. Cell osmotic swelling experiments and atomistic molecular dynamics simulations indicate that the replacement of Leu206 by an Ala residue maintains high water permeability under conditions where the pore is expected to be closed. The present work demonstrates that Leu206, located at the cytoplasmic entry of the channel, constitutes a crucial pH-sensitive steric gate regulating water transport in PIP aquaporins.

Cooperativity in proton sensing by PIP aquaporins.

One of the most intriguing properties of plasma membrane intrinsic protein (PIP) aquaporins (AQPs) is their ability to modulate water transport by sensing different levels of intracellular pH through the assembly of homo- and heterotetrameric molecular species in the plasma membrane. In this work, using a phenomenological modeling approach, we demonstrate that cooperativity in PIP biological response cannot be directly attributed to a cooperative proton binding, as it is usually considered, since it could also be the consequence of a cooperative conformation transition between open and closed states of the channel. Moreover, our results show that, when mixed populations of homo- and heterotetrameric PIP channels are coexpressed in the plasma membrane of the same cell, the observed decrease in the degree of positive cooperativity would result from the simultaneous presence of molecular species with different levels of proton sensing. Indeed, the random mixing between different PIP paralogues as subunits in a single tetramer, plus the possibility of mixed populations of homo- and heterotetrameric PIP channels widen the spectrum of cooperative responses of a cell membrane. Our approach offers a deep understanding of cooperative transport of AQP channels, as members of a multiprotein family where the relevant proton binding sites of each member have not been clearly elucidated yet.

Tonoplast (BvTIP1;2) and plasma membrane (BvPIP2;1) aquaporins show different mechanosensitive properties.

Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied-out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2.