Ion transport, Na + / K + ATPase

The plasma membrane of cells acts as a selectively permeable interface between the cell and its environment and between its cytosol and intracellular organelles. Permeability determines which substances can enter the cell, many of which may be necessary to maintain its vital processes and the synthesis of living substances. It also regulates the outflow of excretory material and water from the cell. The presence of a membrane establishes a net differences between the intracellular fluid in which the cell is bathed. This may be fresh or salt water in unicellular organisms grown in ponds or the sea, but in multicellular organisms the internal fluid, i.e., the blood, the lymph, and especially the interstitial fluid, is in contact with the outer surface of the cell membrane. One of the functions of the cell membrane is to maintain a balance between the osmotic pressure of the intracellular fluid and that of the interstitial fluid.

!ig"# $tructure of a typical cell membrane.

Motion in Membranes A functioning biological membrane is not a rigid, frozen structure. In fact, many of the lipid and protein components are in constant motion. This motion can be demonstrated in a direct way. If human and mouse cells, each carrying a distinctive fluorescent mar er in its plasma membrane, fused

together, the two inds of mar ers gradually become completely intermixed. This demonstrates that lateral diffusion %parallel to the membrane surface& can occur in the membrane. The rapidity with which such two'dimensional diffusion can occur depends on the membrane fluidity, which in turn depends on temperature and lipid composition. (nder physiological conditions, the average time re)uired for a phospholipid molecule to wander completely around a cell is on the order of seconds to minutes* membrane proteins also move, but more slowly.

!ig,# -xperimentally demonstration of membrane fluidity. .hen cells with surface membrane protein mar ed by flurescent tags are induced to fuse, the proteins gradually mix over the fused surface.

Transport Across Membranes A cell or an organelle cannot be either wholly open or wholly closed to its surroundings. Its interior must be protected from certain toxic compounds, and yet metabolites must be ta en in and waste products removed. +ecause the cell must contend with thousands of substance, it is not surprising that much of the complex structure of membranes is devoted to the regulation of transport. In all cells there is a difference in ionic concentration with the extracellular medium and an electrical potential across the membrane. These two properties are intimately related, since the electrical potential depends on an une)ual distribution of the ions on both sides of the membrane. The passage of molecules and ions across membrane may be result of two principal mechanisms. Permeability may be passive, if it occurs only because of physical laws such as diffusion or active if it re)uires energy for transport.

In the absence of an intervening membrane, when two solutions of different concentration are mixed a process of intermixing called diffusion occurs. !or example, if a contact with water, there will be a net movement %i.e., flux& of the solute from the region of higher concentration to that of a lower concentration. In this case the higher the difference in concentration between the two solutions %i.e., the concentration gradient&, the more rapid the rate of diffusion. The presence of a lipoprotein membrane, such as the plasma membrane, greatly modifies this diffusion or passive permeability. It can be considered that the passage of solute from one side of the membrane to the other occurs in three stages# %"& the solute leaves the a)ueous phase and enters the hydrophobic region of the membrane, %,& it traverses the lipid bilayer, and %/& it leaves the lipid phase to enter another a)ueous phase. !or most polar molecules stages" and / represent a considerable barrier. Overton demonstrated that substance that dissolve in lipids pass more easily into the cell, and 0ollander and +arlund, in their classic experiments with the cells of the plant 0hara, demonstrated that the rate at which substances penetrate depends on their solubility in lipids and the size of the molecule. The more soluble they are, the more rapidly they penetrate, and with e)ual solubility in lipids the smaller molecules penetrate at a faster rate. The permeability %P& of molecules across the membrane is# P 1 234t .ith 2, the partition coefficient* 3, the diffusion coefficient, which depends on the molecular weight* and t, the thic ness of the membrane. The partition coefficient in most cell membranes is similar to that of olive oil and water. The Thermodynamics of Transport It was shown that the free energy change, 5 6, for transporting " mole of a substance from a region in which its concentration is 0 " to a place where its concentration is 0, is given by 5 6 1 7T in 0,40" ''''' %"& According to this e)uation, if 0 , is less than 0", 5 6 is negative, and the process is thermodynamically favorable. As more and more substance is transferred %between two finite compartmens&, 0" decrease and 0, increases, until 0, 1 0". At this point 5 6 1 8, and the system is at e)uilibrium. (nless

other factors are involved, this e)uilibrium is the ultimate state approached by transport across any membrane. If the molecules are wandering into the membrane at random, the number entering from each side will be proportional to the concentration on that side. .hen the concentrations become e)ual, the rates of transport in the two directions will be the same, and no net transport will occur. There are three circumstances under which this e)ualization can be circumvented and each is important in the behavior of real membranes# ". A substance may be preferentially bound by macromolecules confined to one side of the membrane or may be chemically modified once it crosses. It may be found that compound A is more concentrated inside a cell %in terms of total moles of A per unit volume& than outside. +ut much of A may be bound to some cellular macromolecules or may have been modified* that portion does not really count in e)uation %"&, which simply states that the concentration of free A on the two sides must be e)ual at e)uilibrium. An appropriate example is oxygen in erythrocytes. ,. A membrane electrical potential may be maintained across a membrane that influences the distribution of ions. This tendency can be expressed )uantitatively in the following way. !or an ion of charge 9, the free energy change for transport across a cell or organelle membrane now involves two contributions# the normal concentration term, as given in e)uation %"&, plus a second term describing the energy change %or wor involved& in moving a mole of ions across the potential difference. 56 1 7T In 0in 4 0out : 9! 5; ''''%,& <ere ! is the !araday constant %=>.? @ mol '"A'"&, and 5; is the membrane potential in volts. If 5; is negative and 9 is positive, the 9! 5; term in e)uation %,& ma es a negative contribution to 56. That is, the transport of cations into the cell is favored. !or anions, of course, the opposite is true, they will be driven out. If the potential difference is somehow maintained, the e)uilibrium state %56 1 8& will correspond to the same concentration of ions on the two side of the membrane. <owever, energy must be expended continually to eep up the potential difference* otherwise migration of ions would neutralize it. 0onversely, e)uation %,& may be interpreted to mean that if a difference in ionic concentration is maintained, a potential will be produced across the membranes.

/. If some thermodynamically favored process is coupled to the transport, then the 56 for this process must be included in the free energy e)uation. This is the general case of active transport, for which we can write 56 1 7T In 0in 4 0out : 56B ''''''%/& The )uantity 56B could correspond to some thermodynamically favored reaction %li e ATP hydrolysis& that was somehow coupled to the process of transport. This e)uation is a generalization of e)uation %,&, now allowing a variety of process C not Dust those that maintain an electrical potential difference C to participate in the transport. Active Transport: Transport Against A Concentration Gradient !acilitated diffusion is useful in many biochemical processes, but in some situations it is imperative that cells or cellular compartments be able to transport substances against concentration gradients, even very unfavorable ones. To ta e an extreme example, under some circumstances a calcium ion ratio of /8,888 must be established across membranes of the sarcoplamic reticulum in muscle fibres. According to the e)uation %"&, this ratio corresponds to 56 1 : ,>.> @4mol, a formidable barrier. Eevertheless, this ratio is built up and maintained in living cells. $uch transport against a gradient re)uires a free energy source of some ind. Altogether, it is estimated that most cells spend about ,?F of their ATP Dust on active transport. <owever, the hydrolysis of ATP can be coupled to transport in a number of different ways, some of them rather indirect. Ion Pumps: Direct Coupling of ATP Hydrolysis to Transport The best' nown physiological example of active transport is the maintenance of sodium and potassium gradients across the plasma membranes of cells. The fluid surrounding cells in most animals is about "G8 mH in Ea: concentration of about "8 mH and a 2: concentration of about "88 mH in their cytosol.

!ig/# !low of ions across membrane.

-ven though Ea: and 2: pass very slowly through membranes by passive diffusion, such ine)ualities would ultimately vanish unless something were done to eep 2: moving in and Ea: out. This movement is accomplished by the action of the sodium'potassium pump. This molecular machine consists of large chains %I& of ""/ ilodaltons each and smaller %J& subunits of ?? ilodaltons each. The I subunit is directly involved in the transport process and is an enzyme that hydrolyzes ATP. The free energy change in that reaction is used to drive the transport. The I subunit traverses the membrane "8 times, forming a multihelix channel. The site for ATP binding and phosphorylation lies on the cytoplasmic side. !acing the outside are multiple sites for binding of cardiotonic steroids, including ouabain and digitoxin %digitalis&. The J subunit has a single membrane'traversing helix and carries a large %,8 3a& polysaccharide on the outer surface. The role of the polysaccharide or the J'protein is un nown. Although the single IJ pair will function in reconstituted systems, the actual )uarternary structure in cell membranes remains uncertain. Hany researcher believes that %IJ& , dimers predominate. The sodium'potassium pump is only one member of a large class of ATPase that function in active transport.
!ig Ga# A model for I subunit of the Ea:, 2:'ATPase. The molecule is shown in the open'to'cytoplasm conformation, with two 2:%green& bound in the channel. The phosphorylation site is indicated, as well as the multiple sites for ouabain binding %blac dots&. Kocation of the transmembrane helices and overall conformation are hypothetical. The J subunit is not shown %fig"a&, but passes through the membrane adDacent to the I subunit %!igGb&.

!igGa.

$unction of

a!"#!%ATPase or &odium%Potassium Pump:

In order to maintain the cell potential, cells must eep a low concentration of sodium ions and high levels of potassium ions within the cell %intracellular&. Outside cells %extracellular&, there are high concentrations of sodium and low concentrations of potassium, so diffusion occurs through ion channels in the plasma membrane. In order to eep the appropriate concentrations, the sodium'potassium pump pumps sodium out and potassium in through active transport.

!igGb. Alpha and beta units. The mechanism of a!"#! pump is:

The pump, with bound ATP, binds / intracellular Ea: ions. ATP is hydrolyzed, leading to phosphorylation of the pump at a highly conserved aspartate residue and subse)uent release of A3P. A conformational change in the pump exposes the Ea: ions to the outside. The phosphorylated form of the pump has a low affinity for Ea: ions, so they are released. The pump binds , extracellular 2: ions. This causes the dephosphorylation of the pump, reverting it to its previous conformational state, transporting the 2: ions into the cell. The unphosphorylated form of the pump has a higher affinity for Ea: ions than 2: ions, so the two bound 2: ions are released. ATP binds, and the process starts againL

Present estimates indicate that about two 2 : ions are pumped into the cell and three Ea: ions are pumped out for every ATP hydrolyzed. .hen an ion is transported against an electrochemical gradient, an extra consumption of oxygen is re)uired. It is calculated that "8 percent of the resting metabolism of a frog muscle is used for transport of sodium ions. This consumption may increase to ?8 per cent in some experimental conditions in which the muscle is stimulated. That the resting membrane potential is due to active transport may be demonstrated in plant and animal cells that have been metabolically bloc ed by anoxia or specific poisons. In this case lea age of 2 : occurs and the potential may decrease to zero. This is clearly observed when anoxia is combined with the poisoning of glycolysis. As suggested by 2rogh in "=G>,

the membrane potential is not really at e)uilibrium but in a Msteady stateN involving the constant expenditure of energy.

". ,. /. G. ?. >.

/ Ea: are ta en from inside. ATP phosphorylates I subunit, stimulating conformational change. Ea: expelled into surroundings. Pump open to outside, ready to start second half of cycle. Two 2: accepted from outside. 3ephosphorylation stimulates conformational change* , 2:expelled to inside and pump returns to initial state.

An interesting example of active transport has been provided by experiments with isolated frog s in. The epithelium is specialized to transport Ea : from the pond water to the interstitial fluids, and by this mechanism the frog can trap this essential ion for use in different tissues. The isolated s in can be ept alive for many hours and used as a wall between two chambers in which the ionic concentrations and other factors are changed experimentally. +y means of this s in has been demonstrated# the inside surface is positive with respect to the outside. The sodium ions are transported from the outer toward the inner surface, and the current produced is due to the flux of sodium. It was also observed that the antidiuretic hormone of the neurohypophysis stimulates the transport of sodium and water.

The active transport of ions is fundamental to maintenance of the osmotic e)uilibrium of the cell, the re)uired concentration of anions and cations and the special ions needed for the functioning of the cell. Together with the extrusion of Ea:, which is continuously pumped out by the cell, there is an exit of water molecules. In this way the cell eeps its osmotic pressure constant. Potassium ions, which are concentrated inside the cell, must pass against a concentration gradient. This can be achieved by a MpumpingN mechanism at the expense of energy. $odium ions are also transported by an active process, which is sometimes called the Msodium pumpN. 3espite the transport against of thermodynamic principles. The only re)uirement is that ATP hydrolysis and transport be coupled. This coupling is apparently accomplished in a multistep process. -xtensive study has led to a model for the mechanism of the Ea: ' 2: pump. It is proposed that the pump can exist in two inds of conformations, one open to cytosol, the other one open to the cellOs surroundings. Transition to the cytosol'open conformation, which allows 2: release and Ea: upta e, is triggered by binding of ATP and release of phosphate. Transition to the outside'open state, which permits Ea : release and 2: upta e, occurs upon phosphorylation of the I subunits and release of A3P. The outside'open state designated -'P has an especially high affinity for cardiotonic steroids, li e digitoxin and ouabain. These agents inhibit the Ea:'2: pump by loc ing it in this conformation. $uch inhibition has maDor effects on muscles, especially in the heart. The accumulation of Ea : in cells leads to measure to reduce it, including a 0a ,:' Ea: exchange process catalyzed by another pump. The resulting increase in 0a ,: in the sarcoplasmic reticulum of heart muscle cells leads to much stronger contractions. This is why substances li e digitoxin and ouabain are used as heart stimulants.

!igG# A schematic diagram of the functional cycle of the Ea:'2: pump. The I subunit is believed to have two states, one open to the outside %dar brown&, the other to the inside %blue&. A dot %P& between two symbols indicates noncovalent binding, a line %I& indicates covalent attachment %as in phosphorylation&