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...

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of α and β group connexins (Cx)and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed betweenα and β-type connexins; two are located in the N-terminal domain(P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an α-type connexin) with the corresponding residues of Cx32 (a β-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent,trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of severalβ-connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues(positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of α and β connexins.

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.