1.A.8 The Major Intrinsic Protein (MIP) Family

The Major Intrinsic Protein (MIP) of the human lens of the eye (Aqp0), after which the MIP family was named, represents about 60% of the protein in the lens cell. In the native form, it is an aquaporin, but during lens development, it becomes proteolytically truncated. The channel, which normally houses 6-9 water molecules, becomes constricted so only three remain, and these are trapped in a closed conformation (Gonen et al., 2004a,b). These truncated tetramers form intercellular adhesive junctions (head to head), yielding a crystalline array that mediates lens formation with cells tightly packed as required to form a clear lens (Gonen and Walz, 2006). Lipids crystallize with the protein (Gonen et al., 2005).

The MIP family is large and diverse, possessing over 100 members that all form transmembrane channels. These channel proteins function in water, small carbohydrate (e.g., glycerol), urea, NH₃, CO₂, H₂O₂ and ion transport by energy-independent mechanisms. For example, the glycerol channel, Fpslp of Saccharomyces cerevisiae mediates uptake of arsenite and antimonite (Wysocki et al., 2001). Ion permeability appears to occur through a pathway different than that used for water/glycerol transport and may involve a channel at the 4 subunit interface rather than the channels through the subunits (Saparov et al., 2001). MIP family members are found ubiquitously in bacteria, archaea and eukaryotes. Phylogenetic clustering of the proteins is largely according to phylum of the organisms of origin, but one to three clusters are observed for each phylogenetic kingdom (plants, animals, yeast, bacteria and archaea) (Park and Saier, 1996). One of the plant clusters includes only tonoplast (TIP) proteins, with another includes plasma membrane (PIP) proteins (see below).

The known aquaporins cluster loosely together as do the known glycerol facilitators. MIP family proteins are believed to form aqueous pores that selectively allow passive transport of their solute(s) across the membrane with minimal apparent recognition. Aquaporins selectively transport water (but not glycerol) while glycerol facilitators selectively transport glycerol but not water. Some aquaporins can transport NH₃ and CO₂. Glycerol facilitators function as solute nonspecific channels, and may transport glycerol, dihydroxyacetone, propanediol, urea and other small neutral molecules in physiologically important processes. Some members of the family, including the yeast Fps1 protein (TC #1.A.8.5.1) and tobacco NtTIPa (TC #1.A.8.10.2) may transport both water and small solutes.

Zardoya and Villalba (2001) have conducted phylogenetic analyses of the MIP family, analyzing 153 homologues. They divided the proteins into six major 'paralogous' groups: (1) GLPs, or glycerol-transporting channel proteins, which include mammalian AQP3, AQP7, and AQP9, several nematode paralogues, a yeast paralogue, and Escherichia coli GLP; (2) AQPs, or aquaporins, which include metazoan AQP0, AQP1, AQP2, AQP4, AQP5, and AQP6; (3) PIPs, or plasma membrane intrinsic proteins of plants, which include PIP1 and PIP2; (4) TIPs, or tonoplast intrinsic proteins of plants, which include αTIP, γTIP, and δTIP; (5) NODs, or nodulins of plants; and (6) AQP8s, or metazoan aquaporin 8 proteins. Of these groups, AQPs, PIPs, and TIPs cluster together as noted above.

In agreement with their divergent sequences, human AQP1-9 have very different physiological functions. They are involved in (1) nephrogenic diabetes insipidus, (2) brain water balance and hearing and (3) salivary secretion (Li and Verkman, 2001).

Several reports of MIP family proteins transporting ions may or may not be physiologically significant. For example, the influx of arsenite and antimonite via the Fps1 protein into yeast cells is well documented (Wysocki et al., 2001). Similarly, these compounds are taken up via aquaporins in Leishmania (Gourbal et al., 2004). Moreover, AQP6 of renal epithelia have been reported to transport anions at low pH (Yasui et al., 1999). Demonstration of the involvement of the cyanobacterial channel protein (TC #1.A.8.4.1) in copper homeostasis suggests that it may transport Cu²⁺. Finally, Yang et al. (2005) showed that arsenite exists the Mesorhizobium meliloti cell by downhill movement through AqpS (1.A.8.15.1). The physiological functions of many MIP family proteins are unknown.

MIP family channels consist of homotetramers (e.g., GlpF of E. coli; TC #1.A.8.1.1, AqpZ of E. coli; TC #1.A.8.3.1, and MIP or Aqp0 of Bos taurus; TC #1.A.8.8.1). Each subunit spans the membrane six times as putative α-helices and arose from a 3-spanner-encoding genetic element by a tandem, intragenic duplication event. The two halves of the proteins are therefore of opposite orientation in the membrane. However, a well-conserved region between TMSs 2 and 3 and TMSs 5 and 6 dip into the membrane, each loop forming a half TMS.

Several MIPs within all domains of life have been shown to facilitate the diffusion of reduced and non-charged species of the metalloids silicon, boron, arsenic and antimony (Bienert et al., 2008). Metalloids encompass a group of biologically important elements ranging from the essential to the highly toxic. Consequently, all organisms require efficient membrane transport systems to control the exchange of metalloids with the environment. Recent genetic evidence has demonstrated a crucial role for specific MIPs in metalloid homeostasis (Bienert et al., 2008).

The crystal structure of the glycerol facilitator of E. coli was solved at 2.2 Å resolution (Fu et al., 2000). Glycerol molecules line up in single file within the amphipathic channel. In the narrow selectivity filter of the channel, the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor and donor atoms. The two conserved D-P-A motifs in the loops between TMSs 2 and 3 and TMSs 5 and 6 form the interface between the two duplicated halves of each subunit. Thus each half of the protein forms 3.5 TMSs surrounding the channel. The structure explains why GlpF is selectively permeable to straight chain carbohydrates, and why water and ions are excluded. Aquaporin-1 (AQP1) and the bacterial glycerol facilitator, GlpF can transport O₂, CO₂, NH₃, glycerol, urea, and water to varying degrees. For small solutes permeating through AQP1, a remarkable anticorrelation between permeability and solute hydrophobicity was observed whereas the opposite trend was observed for permeation through the membrane (Hub and Groot, 2008). AQP1 is thus a selective filter for small polar solutes, whereas GlpF is highly permeable to small solutes and less permeable to larger solutes. 

Aquaporin-1 (Aqp1) from the human red blood cell has been solved by x-ray crystallography to 3.8 Å resolution (Murata et al., 2000). The aqueous pathway is lined with conserved hydrophobic residues that permit rapid water transport. Water selectivity is due to a constriction of the inner pore diameter to about 3 Å over the span of a single residue, superficially similar to that in the glycerol facilitator of E. coli.

AqpZ, a homotetramer (tAqpZ) of four water-conducting channels that facilitate rapid water movements across the plasma membrane of E. coli, has been solved to 3.2 Å resolution. All channel-lining residues in the four monomeric channels are found orientated in nearly identical positions with one marked exception at the narrowest channel constriction, where the side chain of a conserved Arg-189 adopts two distinct conformational orientations. In one of the four monomers, the guanidino group of Arg-189 points toward the periplasmic vestibule, opening up the constriction to accommodate the binding of a water molecule through a tridentate H-bond. In the other three monomers, the Arg-189 guanidino group bends over to form an H-bond with carbonyl oxygen of Thr-183 occluding the channel. Therefore, the tAqpZ structure reveals two distinct Arg-189 conformations associated with water permeation through the channel constrictions. Alternating between the two Arg-189 conformations disrupts continuous flow of water, thus regulating the open probability of the water pore. Further, the difference in Arg-189 displacements is correlated with a strong electron density found between the first transmembrane helices of two open channels, suggesting that the observed Arg-189 conformations are stabilized by asymmetrical subunit interactions in tAqpZ (Jiang et al., 2006).

The 3-D structures of the open and closed forms of plant aquaporins, PIP1 and PIP2, have been solved (Törnroth-Horsefield et al., 2006). In the closed conformation, loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation, loop D is displaced up to 16 Å, and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins. In plants it regulates water intake/export in response to water availability and cytoplasmic pH during anoxia (Törnroth-Horsefield et al., 2006).

The transport reaction for channel proteins of the MIP family is:

H₂O (out) H₂O (in) (e.g., aquaporins) or

solute (out) solute (in) (e.g., glycerol facilitators).

References:

Bienert, G.P., M.D. Schüssler, and T.P. Jahn. (2008). Metalloids: essential, beneficial or toxic? Major intrinsic proteins sort it out. Trends Biochem. Sci. 33: 20-26.