Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42°C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37°C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.References

In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico-chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport.

The MIP (major intrinsic protein) proteins constitute a channel family of currently 150 members that have been identified in cell membranes of organisms ranging from bacteria to man. Among these proteins, two functionally distinct subgroups are characterized: aquaporins that allow specific water transfer and glycerol channels that are involved in glycerol and small neutral solutes transport. Since the flow of small molecules across cell membranes is vital for every living organism, the study of such proteins is of particular interest. For instance, aquaporins located in kidney cell membranes are responsible for reabsorption of 150 liters of water/ day in adult human. To understand the molecular mechanisms of solute transport specificity, we analyzed mutant aquaporins in which highly conserved residues have been substituted by amino acids located at the same positions in glycerol channels. Here, we show that substitution of a tyrosine and a tryptophan by a proline and a leucine, respectively, in the sixth transmembrane helix of an aquaporin leads to a switch in the selectivity of the channel, from water to glycerol.Based on amino acid sequence, members of the MIP 1 family are predicted to share a common topology consisting in 6 transmembrane domains connected by 5 loops (A-E). From biochemical and biophysical data, a model representing these proteins as "hourglasses" has been proposed (1) (Fig. 1A). In this model, the channel pore is constituted by the junction of loops B and E that overlap midway between the leaflets of the membrane. Recently, the three-dimensional structure of the first identified aquaporin, AQP1 (2), has been obtained and has defined that the protein complex is constituted by four monomers (3-5). Each monomer is formed by six tilted ␣ helices spanning the membrane bilayer and surrounding a central density zone. This zone represents likely the narrowest segment of the water pore and may be constituted by loops B and E according to the hourglass model. As opposed to the increasing amount of data aiming to determine aquaporins structure, no study concerning glycerol channels has been reported, but considering their high level of identity, it was assumed that they had the same structural organization. Using a biochemical approach, we showed recently that an insect aquaporin, AQPcic (6), is tetrameric in cell membrane, like AQP1, whereas the glycerol channel of Escherichia coli (GlpF) is a monomer (7). These results suggest that oligomerization of MIP proteins could be involved in transport selectivity. In order to elucidate molecular mechanisms that are accountable of the channel selectivity, we have developed a strategy consisting in a systematic comparison of the physico-chemical properties of amino acids at each position in multiple sequence alignments (8). We have identified five positions (P1-P5) corresponding to amino acid residues conserved in aquaporins and glycerol channels but with highly different physico-chemical properties in the two subgroups. Interestingly, four positions (P2, P3, P...

These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens.

The major intrinsic protein (MIP) family includes water channels aquaporins (AQPs) and facilitators for small solutes such as glycerol (GlpFs). Velocity sedimentation on sucrose gradients demonstrates that heterologous AQPcic expressed in yeast or Xenopus oocytes behaves as an homotetramer when extracted by n-octyl ␤-D-glucopyranoside (OG) and as a monomer when extracted by SDS. We performed an analysis of GlpF solubilized from membranes of Escherichia coli or of mRNA-injected Xenopus oocytes. The GlpF protein extracted either by SDS or by nondenaturing detergents, OG and Triton X-100, exhibits sedimentation coefficients only compatible with a monomeric form of the protein in micelles. We then substituted in loop E of AQPcic two amino acids predicted to play a role in the functional/structural properties of the MIPs. In two expression systems, yeast and oocytes, the mutant AQPcic-S205D is monomeric in OG and in SDS. The A209K mutation does not modify the tetrameric form of the heterologous protein in OG. This study shows that the serine residue at position 205 is essential for AQPcic tetramerization. Because the serine in this position is highly conserved among aquaporins and systematically replaced by an acid aspartic in GlpFs, we postulate that glycerol facilitators are monomers whereas aquaporins are organized in tetramers. Our data suggest that the role of loop E in MIP properties partly occurs through its ability to allow oligomerization of the proteins.

In Xenopus oocytes, the water permeability of AQP0 (P f ) increases with removal of external calcium, an effect that is mediated by cytoplasmic calmodulin (CaM) bound to the C terminus of AQP0. To investigate the effects of serine phosphorylation on CaM-mediated Ca 2؉ regulation of P f , we tested the effects of kinase activation, CaM inhibition, and a series of mutations in the C terminus CaM binding site. Calcium regulation of AQP0 P f manifests four distinct phenotypes: Group 1, with high P f upon removal of external Ca 2؉ (wild-type, S229N, R233A, S235A, S235K, K238A, and R241E); Group 2, with high P f in elevated (5 mM) external Ca 2؉ (S235D and R241A); Group 3, with high P f and no Ca 2؉ regulation (S229D, S231N, S231D, S235N, and S235N/I236S); and Group 4, with low P f and no Ca 2؉ regulation (protein kinase A and protein kinase C activators, S229D/S235D and S235N/I236S). Within each group, we tested whether CaM binding mediates the phenotype, as shown previously for wild-type AQP0. In the presence of calmidazolium, a CaM inhibitor, S235D showed high P f and no Ca 2؉ regulation, suggesting that S235D still binds CaM. Contrarily, S229D showed a decrease in recruitment of CaM, suggesting that S229D is unable to bind CaM. Taken together, our results suggest a model in which CaM acts as an inhibitor of AQP0 P f . CaM binding is associated with a low P f state, and a lack of CaM binding is associated with a high P f state. Pathological conditions of inappropriate phosphorylation or calcium/CaM regulation could induce P f changes contributing to the development of a cataract.In the equatorial region of the lens, metabolically active epithelial cells differentiate into less active fiber cells and form the cortical region of the lens. In the core of the lens, mature fiber cells lack nuclei and organelles (1-3). Mature lens fiber cells are characterized by slow protein turnover and increasing posttranslational modifications such as proteolytic cleavage, glycation, and phosphorylation. In the cortex of the lens, there is an elevated level of phosphorylated proteins, demonstrating the significance of phosphorylation in lens differentiation (3-5). Gap junctions, water channels, Na ϩ /K ϩ pumps, and Ca 2ϩ , K ϩ , Na ϩ , and Cl Ϫ channels act in concert to maintain cellular homeostasis, possibly by generating an intrinsic lens circulation (6 -9). Such a proposed intrinsic circulation would move nutrients from the aqueous humor to core fiber cells and waste products away from the central part of the lens toward the surface.AQP0, 4 the water channel of lens fiber cells, is essential for normal lens structure development and for maintaining lens transparency and focusing power. Lenses of AQP0 knock-out mice (AQP0 Ϫ/Ϫ ) present congenital cataracts, fail to develop normal structure, and lose 80% of their water permeability (10 -13). When AQP0 and AQP0-LTR (a naturally occurring C-terminal mutant of AQP0) are expressed together in transgenic mice, the lenses show incomplete fiber cell development. In vitro expression of wil...

We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777–6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573–580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.

The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium. The MIPs can be classified into three functional subgroups : aquaporins, glycerol facilitators and aquaglyceroporins. Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria. However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology. Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis. This MIP is shown to be permeable to glycerol, like E. coli GlpF, and to water, like E. coli AqpZ. This is the first characterization of a microbial MIP that has a mixed function. This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.