David A. Rubenstein, ... Mary D.

Frame, in Biofluid Mechanics

(Second Edition), 2015

10.4 Aquaporins
Aquaporins are transmembrane proteins that regulate the flow of water into and out of
cells. For many years, it was believed that water movement into and out of the cell was
(1) not regulated in any manner and (2) could be accounted for by simple diffusion
across the cell membrane. However, the rapid movement of water in aqueous
humor formation could not be described by simple diffusion. In the early 1990s,
aquaporins were discovered, and it was found that they can selectively control water
movement into and out of cells. One of the critical functions of aquaporins is that while
they allow the passage of water, they prevent the passage of ions. If aquaporins allowed
ions through their channels, all ion concentration gradients across the cell membrane
would approach zero (i.e., all ions would be in equilibrium) and the cell would not be
able to perform many of its critical functions that depend on the concentration gradient
of various molecules (e.g., many transport processes that rely on an electrochemical
gradient would fail). Also, if ions were allowed to pass through aquaporins, the amount
of energy that cells would expend on maintaining the necessary ion concentration
gradient, if the cell could maintain a concentration gradient under these conditions,
would exceed the amount of energy produced during cellular respiration.
Aquaporins are found in a high concentration in the epithelial cells that produce
aqueous humor (as well as other epithelial cells that allow water to move readily across
their membrane, e.g., epithelial cells in the kidney). These pores allow water molecules
through in a single file. To understand how aquaporins regulate water movement into a
single file and prevent the movement of other ions, it is important to understand the
three-dimensional structure of the aquaporin protein. An aquaporin channel is composed
of six transmembrane α-helices, with both the amino and carboxyl terminal on the
cytoplasmic side of the membrane. Two of the five loops that connect the six
transmembrane helices are extremely hydrophobic. One of these loops is on the
intracellular side of the membrane and the other is on the extracellular side of the
membrane. The two hydrophobic loops contain a three amino acid sequence, termed the
NPA (Asparagine–Proline–Alanine) motif. The NPA motif folds back into the channel
aquaporin created by the six transmembrane helices. In three-dimensional space, the
folding back of these two domains resembles an hourglass shape (or a bottle neck for
flow). This hourglass constriction restricts water molecules to a single file as they are
passing through the channel. Also, the hydrophobic portion “coats” one side of the
channel (the reason for this and the effect that his has on ions will be discussed later).
The restriction of water most likely occurs due to an electric field created by the charges
on the protein structure, inducing the majority of the channel’s core to be hydrophobic.
This electric field also dictates the direction of the water molecules as the flow through
the channel. As water molecules enter the channel, they typically are oriented with the
oxygen atom facing the entrance of the channel. As the molecules enter the NPA motif,
the water molecules flip, so that the oxygen atom is facing toward the channel’s exit. It
is believed that the orientation of oxygen changes due to a hydrogen-bonding event with
the two asparagine molecules within the NPA motif. Therefore, because each water
molecule must be reoriented to pass through the aquaporin channel and it can only be
reoriented by interacting with the two asparagine molecules within the NPA motif, only
one water molecule can flow through the channel at a time. Through these two
restrictions it has been observed that the permeability of aquaporin channels toward
water molecules is on the order of 6E−14 cm3/s, which allows approximately 109 water
molecules through each pore per second.
There is a second constriction of the aquaporin channel, usually toward the extracellular
side of the cell membrane, which acts to restrict the movement of other molecules
through the channel. This selectivity filter is termed the aromatic/arginine selectivity
filter in aquaporin channels. The selectivity filter is a grouping of amino acids that
interact with only water molecules and helps them through the narrowing created by this
filter. Other molecules that do not interact with the selectivity filter cannot pass through
this narrowing. The aromatic ring weakens the hydrogen bonds between water
molecules and then the partial negative charge on the oxygen atom interacts with the
positive charge on the arginine. The interaction between the oxygen and the arginine
allows water through the channels and prevents the passage of other molecules,
especially protons.
All of the restrictions are physically smaller than hydrated ions; remember that all ions
that are in the body are hydrated (for instance a sodium ion, which has a positive charge,
will typically traverse through the biological substrates, with four water
molecules associated with it; the partial negative charge on the water’s oxygen weakly
hydrogen bonds to the sodium). However, ions in solution can dehydrate and this is how
they typically pass-through ions channels. To dehydrate an ion, a significant amount of
energy would be needed and typically this is counterbalanced by other binding events.
In the case of the aquaporin channel, the location where the ions would need to become
dehydrated to pass through the pore restriction is associated with hydrophobic amino
acid regions. A hydrophobic surface cannot provide a temporary bonding event for
a hydrophilic ion. Thus, an ion would need an energy source to break the water
hydrogen bonding events and not create new hydrogen bonding events. This significant
amount of energy is not readily available, which effectively prevents ions from moving
through aquaporin channels.
Four aquaporin channels associate with each other in the membrane, so that in one
location there are four possible passageways for water to move through the cell
membrane. Each aquaporin channel can have a slightly different protein structure. There
are at least four different aquaporins in mammals and upward of 10 aquaporin channels
found within plants. The different structures between the aquaporin molecules may
allow for the movement of a small quantity of ions or other solutes (such
as glycerol and small sugars) through the channels, although most aquaporin channels
restrict ion/solute movement. Also, there are some aquaporin channels that respond to
stimuli from external hormones or other paracrine molecules. Upon stimulation, the rate
of water movement (and possibly the direction of movement) can be altered.
It is important to remember that aquaporins do not actively transport water across the
cell membrane, instead they facilitate the diffusion of water across the cell membrane.
Due to the slow diffusion of water across the lipid bilayer, aquaporins effectively
increase the overall rate of water movement across the cell membrane.