The exchange of functional domains among aquaporins with different transport characteristics

129

121

Excessive water uptake through Aquaporins (AQP) can be lifethreatening and reversible AQP inhibitors are needed. Here, we determined the specificity, potency, and binding site of tetraethylammonium (TEA) to block Aquaporin water permeability. Using oocytes, externally applied TEA blocked AQP1/AQP2/AQP4 with IC 50 values of 1.4, 6.2, and 9.8 M, respectively. Related tetraammonium compounds yielded some (propyl) or no (methyl, butyl, or pentyl) inhibition. TEA inhibition was lost upon a Tyr to Phe amino acid switch in the external water pore of AQP1/AQP2/AQP4, whereas the water permeability of AQP3 and AQP5, which lack a corresponding Tyr, was not blocked by TEA. Consistent with experimental data, multi-nanosecond molecular dynamics simulations showed one stable binding site for TEA, but not tetramethyl (TMA), in AQP1, resulting in a nearly 50% water permeability inhibition, which was reduced in AQP1-Y186F due to effects on the TEA inhibitory binding region. Moreover, in the simulation TEA interacted with charged residues in the C (Asp 128 ) and E (Asp 185 ) loop, and the A(Tyr 37 -Asn 42 -Thr 44 ) loop of the neighboring monomer, but not directly with Tyr 186 . The loss of TEA inhibition in oocytes expressing properly folded AQP1-N42A or -T44A is in line with the computationally predicted binding mode. Our data reveal that the molecular interaction of TEA with AQP1 differs and is about 1000-fold more effective on AQPs than on potassium channels. Moreover, the observed experimental and simulated similarities open the way for rational design and virtual screening for AQP-specific inhibitors, with quaternary ammonium compounds in general, and TEA in particular as a lead compound.

Section: Quaternary Ammonium Compounds Tested On Human Aqp1-brooks Etmentioning

confidence: 99%

Quaternary Ammonium Compounds as Water Channel Blockers

Detmers

Groot

Müller

et al. 2006

129

121

“…Substitution of tyrosine 222 and tryptophan 223 in the upper part of the sixth TM of AQPcic by the two corresponding amino acids of GlpF (a proline and a leucine, respectively) induced a switch from a water to a glycerol channel and a switch from a tetrameric to a monomeric state in nondenaturing detergent (20). Although the general relevance of this observation is unclear (2), functional analyses of AQP0-AQP2 chimeras have demonstrated that stability of loop E is crucial for MIP channels (21). When TM 5 was replaced in AQP0 by that of AQP2, it was concluded that the distal part of TM 5 is necessary for maximum water channel activity and contributes to formation of the aqueous pore and determination of the flux rate (22).…”

mentioning

confidence: 99%

Role of C-terminal Domain and Transmembrane Helices 5 and 6 in Function and Quaternary Structure of Major Intrinsic Proteins

Duchesne

Pellerin

Delamarche

et al. 2002

We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the Cterminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability.

“…AQP0, AQP1, and AQP4 share ϳ40% sequence identity with each other. Attempts to increase AQP0 water permeability by exchanging parts of AQP0 for corresponding parts of AQP1 have been ineffective (19).…”

mentioning

confidence: 99%

pH and Calcium Regulate the Water Permeability of Aquaporin 0

Németh-Cahalan

Hall

2000

241

236

Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca 2؉ on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 ؎ 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 ؎ 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca 2؉ increased water permeability 4.1 ؎ 0. acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca 2؉ may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.The major intrinsic protein (MIP, now designated aquaporin 0 and abbreviated AQP0) 1 of the optical lens was the first sequenced member of the aquaporins, an ancient family of proteins found in bacteria, plants, and animals (1-4). Preston et al. (5) discovered that CHIP 28 (now called aquaporin 1 (AQP1)), a protein abundant in red blood cells, facilitates the diffusion of water across the plasma membrane when expressed in Xenopus oocytes. AQP4 (previously called MIWC for mercurial-insensitive water channel) is expressed strongly in the brain and kidney collecting duct (6, 7). Work in several laboratories subsequently demonstrated that many members of the aquaporin family facilitate the diffusion of water and other nonelectrolytes (3, 8 -12). Among the aquaporins, AQP0 forms a water channel with a relatively low water permeability (13,14); the water permeability per molecule is 40 times higher for AQP1 (13,15). The structural basis of this large difference in water permeability is unknown. AQP0 and AQP1 form tetrameric arrays in their native membranes and when reconstituted in lipid vesicles (6 -18). AQP0, AQP1, and AQP4 share ϳ40% sequence identity with each other. Attempts to increase AQP0 water permeability by exchanging parts of AQP0 for corresponding parts of AQP1 have been ineffective (19).Although low in water permeability per molecule, AQP0 comprises more than 60% of the membrane protein in the normal vertebrate lens and therefore provides the major permeability pathway for water movement across the membranes of lens fiber cells. If it is defective or missing from an otherwise normal lens, a cataract results (20, 21). In a chimeric mouse model, cataract can be prevented by the presence of 20% normal cells, which presumably supply the requisite AQP0 (22). The role of AQP0 in maintaining normal lens conditions is uncertain, but it likely facilitates the intrinsic circulation of fluid in the lens that maintains lens transparency and homeostasis in the absence of blood vessels (23). pH and Ca 2ϩ are likely ca...