Cell Membranes and Transport

Cell membranes

All cells are surrounded by a plasma membrane that forms the boundary between the living cyto­plasm and the extracellular space. The membrane retains the cytoplasmic contents and pre­vents intrusion by most molecules outside the cell. The mem­brane is flexible and semi-fluid, conforming dynam­ically to the changing forces of intracellular cytoskeletal proteins and the extra­cellular matrix and substrate. Red blood cells, for example, take their bicon­cave disk shape from their special­ized spring-like intra­cellular cyto­skeletal network. Nevertheless, they readily bend and squeeze unharmed in transient con­tor­tions as they rush through narrow capillaries. Inside nucle­ated cells, other mem­branes define organ­elles like the mitochondria, the nuc­leus, and the endo­plas­mic reticulum. The plasma membrane and the intra­cellular mem­branes share a common basic archi­tecture and a common origin. They are made of a molecular bilayer of lipids peppered with mem­brane proteins. We start by discussing the lipid component and then return to the proteins.

Membranes form a hydrophobic lipid bilayer

Typical membrane lipids are phospho­lipids and chole­sterol. These molecules are primarily hydro­pho­bic (“water fearing”) but bear a charged or polar group at one end that is hydrophilic (“water loving”) (Figure 1). We call this dual nature, amphipathic (“both pre­fer­ences”). Soaps and detergents are also amphi­pathic. This allows them to bring greasy substances into solu­tion in water (the cleaning action of soaps, for example, relies on breaking grease up into small droplets in water).

In the structure of phospho­lipids, we see two hydrophobic fatty acid chains attached to a glycerol scaffold at the glycerol 1- and 2-positions (Figure 1). The glycerol 3-position carries the hydrophilic phos­phate and head group. Common head groups include (Figure 2) serine, etha­nol­amine, choline, and inositol, each with its own special roles and forming phos­pho­lipids with names like phos­pha­tidyl serine, phospha­tidyl­inositol, and so forth. Amphipathic phos­pho­lipids spontan­eously form a lipid bilayer made of two lipid leaflets as a thin flexible sheet (Figure 3). This is a minimum energy state for phos­pho­lipids in water because it maxi­mizes the interactions of the hydrophobic fatty acid side chains with each other and away from water, and it maximizes the interaction of the hydro­philic head groups with water.

At physio­logical tem­per­atures, the lipid side chains are free to undergo rapid spon­taneous thermal motion, waving around in a fluid-like state while still remaining anchored to the gly­cerol, which carries the head group. In addition, each phos­pho­lipid molecule experiences spontaneous thermal rotation, stocha­stically spinning around its long axis, as well as lateral diffusion by Brown­ian motion in the plane of the membrane. All these motions make the lipid bilayer an ultrathin flex­ible, oily fluid. The thickness is just twice that of the two opposing fatty acid leaflets, about 3 nm, far thinner than the wave­length of light and therefore resolv­able only by electron microscopy.

Passive permeability of the bilayer

Some molecules easily cross the lipid bilayer membrane and some do not (Figure 4). Permeability is a measure of how easily molecules cross the mem­brane. Key permeability properties of cell mem­branes are well under­stood if we remember that the lipid bilayer is like a thin sheet of oil. Hydro­phobic molecules presented in the aqueous phase, being lipid soluble, pass easily into the oily lipid bilayer and cross the membrane barrier. Illustra­tive examples would include aspirin, steroid hormones, O₂, CO₂, and NO (Box A). We say the mem­brane is permeable to these hydrophobic mole­cules, which is good in these cases since their physio­logical actions require them to enter cells from the outside. Thus, the steroid hormone estro­gen is made in the ovaries but has to act on receptors in the nucleus of cells throughout the body. In all respiring cells, O₂ has to enter the cell con­tinuously, and CO₂ has to leave. Similarly, O₂ has to enter red blood cells in the lungs and be delivered to tissues in the capillaries during every circuit of the circu­lation, and the reverse for CO₂. Each of these steps requires the hydro­pho­bic steroid or gas molecules to dissolve in the plasma mem­brane and to pass across it and emerge on the other side, a process that takes only a small fraction of a second. For the same reasons, some degree of hydrophobicity is essential in the design of new drugs that are intended to be taken orally so that they can be absorbed across cells of the intestinal wall and cross into target tissues.

On the other hand, molecules that are polar (sugars) or charged (ions) are hydrophilic and do not enter into oil. They are reluc­tant to leave water and do not enter or cross the lipid bilayer easily. Examples include Na⁺, ATP, and glucose (Box A). Cell membranes are relatively imper­meable to them, which allows the cell to develop ion gradients across mem­branes and to retain its high-energy molecules, meta­bolites, and genetic material inside. Many important physiological responses involve changes of membrane permeability to charged and polar molecules. In electrophysiology we will see that the ion-imper­meable lipid bilayer can be thought of as an electri­cal insulator. It allows a small charge imbalance across the mem­brane to generate an electrical potential difference across the membrane – the mem­brane potential. The electrical responses of cells involve changes of permeability to ions and ion movements through the carefully orchestrated opening and closing of ion channels in the plasma membrane. In this way, as we shall see later, the plasma membrane is the seat of all electrical signaling in nerve and muscle.

Proteins add functions to cell membranes

A more complete view of biological mem­branes would show a thin lipid sea with many proteins inserted and floating in it (Figure 5). Some proteins, the integral mem­brane proteins, have one or more hydro­phobic trans­membrane segments and never leave the mem­brane (Box B). Others, the peripheral proteins, bind to other membrane proteins and lipid head groups but do not have any trans­mem­brane segments. They may associate and disso­ciate dyna­mically from the mem­brane compart­ment depending on the state of the cell.

Almost all integral membrane proteins are initially inserted into membranes of the endo­plasmic reticulum (ER) during protein synthesis as the nascent peptide chain is length­ened by protein translation. The ER membrane then buds off a membrane vesicle that passes to the Golgi with its new membrane proteins. Eventually, more budding from the Golgi sends a vesicle further to, e.g., the plasma mem­brane.

Details of mem­brane origin and traffic will be discussed in a separate session empha­sizing cell biology of mem­brane budding and endo- and exo­cytosis. They explain how all membranes have a similar archi­tecture since they derive from the same source. Mem­brane proteins contri­bute many biological functions to the mem­brane including transport and enzy­matic cata­lysis (Box B). Although the basic architecture of the membrane is conserved, the different mem­branes of a cell are special­ized by having different sets of functional proteins to allow them to perform different functions, so both at the level of gene expres­sion and in the events of mem­brane traffic, decisions are made as to which protein belongs where.

Now we will emphasize certain transmembrane proteins that facilitate movement of ions across the membrane, sometimes coup­ling with transport of other small mole­cules. One can consider them catalysts that enable regu­lated trans­mem­brane move­ments of a small select number of molecules. They include ion channels, coupled trans­porters, and ion ATPase pumps (Figure 6). We introduce them superficially first.

(A) The ion channels are the sim­plest. When they are open, they act like a pore that allows ions to flow. They have ion selec­tivity – a prefer­ence for certain ions. The direction of ion flow is set entirely by two thermo­dy­namic forces on the ions: (i) the ion concen­tration gradient across the membrane, and (ii) an electrical force if there is a difference in electrical poten­tial across the membrane. We can say that the direction of ion flux is thermo­dyna­mically downhill, and, more tech­nically, we say it is down the electro­chemical gradient. The ion channels in Figure 6 are selec­tive for K⁺, Na⁺, or Cl^– and would be called K⁺ chan­nels, Na⁺ channels, or Cl^– channels. Some ion channels are less selective, passing a range of small cations or a range of small anions. Ion channels have an addi­tional feature: most of them open and close their pore in response to stimuli such as a neuro­trans­mitter, a voltage change, heat, or an intracellular second mes­senger. We say they are gated. This gating allows them to react dynamically to other stimuli.  The figure also shows a water channel, a pore that allows osmotic flow of water across the membrane, e.g. in the kid­ney. As a peek forward to the next few weeks, Table 1 lists the proper­ties of ion channels you will be encoun­tering soon.

(B) The next category in Figure 6, the coupled trans­porters, couples the movement of one molecule (here an organic solute or bicarbonate or Ca²⁺) to the downhill flow of another (Na⁺ or Cl^–). The action is like that of an enzyme that in this case has two substrates. Nothing happens until both substrates are present. They bind and induce con­for­mational changes of the protein that exposes each of them to the opposite side of the mem­brane. Such transporters are sometimes called “carriers.” The first example shown is a Na⁺-coupled solute trans­porter, of which there are many types (for sugars, amino acids, neuro­transmitters, etc.). Here Na⁺ and the coupled solute move in the same direc­tion into the cell, so it is called a co-transporter. These cotransporters use the free ener­gy of Na⁺ allowed to flow down­hill (down its electro­chemi­cal gradient) into the cell, to drive the cotransport of the other sub­strate. They can drive uphill trans­port of solutes, cre­ating a concen­tration gradient. Such a process is called secon­dary active transport, an active trans­port that does not use ATP as its direct source of energy. It is analo­gous to using the cascading of water over a waterfall (Na⁺ entry) to do some work (solute transport).

The second group of coupled transporters exchanges ions from opposite sides of the membrane. Shown are examples of Cl^–/­bicarbonate exchangers and Na⁺/Ca²⁺ exchangers. All these coupled trans­porters are fully reversible, operating in whichever direction thermodynamics dictates, and are capable of secondary active transport. For completeness, we also mention a type of transporter that has only one substrate and no coupled ion flow (Figure 6). This transport process, called facili­tated diffusion, is by necessity thermodynamically downhill for the single substrate. A notable exam­ple is the Glut4 glucose transporter inserted into the plasma membrane by the action of insulin to allow passage of glucose in either direction.

(C) The final example in Figure 6 is an ion pump that couples transport of Na⁺ and K⁺ to the free energy of hydrolysis of ATP, the metabolic dollar bill. Now both ions are moved uphill (in opposite directions) at the direct expense of metabolic energy. Such pumps that consume ATP directly are said to perform primary active trans­port. They generate the ion gradients across the membrane that are important for ion movements in coupled transporters and in ion channels. Before considering how the pumps work, we will discuss the ion imbalances that these pumps set up.

Four ion gradients

Gradients of ions across the plas­ma membrane are key for physio­logy and medi­cine–indeed for life. The word gradient means a differ­ence in concentration on the two sides of the mem­brane. Recall that salts like NaCl disso­ciate in water into the free cations and anions – Na⁺ (“sodium ion”) and Cl^– (“chloride ion”) in the case of NaCl.  This makes an electro­lyte solu­tion. With respect to ions, our plas­ma (the extra­cellular medi­um for cells) is like a dilute ver­sion of seawater, reflect­ing the origins of animals in early oceans. As in seawater, the con­cen­trations of Na⁺ and Cl^– are high and K⁺ is low (Table 2). In contrast, inside the cell, the Na⁺ is low, Cl^– is often low, and K⁺ is high. Resting cellular free Ca²⁺ is extra­ordi­narily low, making Ca²⁺ a good ion for intra­cellu­lar signaling as we will see later. Note that the table has a footnote explaining two conven­tions for the units of extra­cellular con­cen­tration that will be important for you as a clinician. Here we will use inter­national scientific units, but in the clinic you will use the other. Shortly we shall see that ion gradients allow cells to make electrical signals. Later curriculum blocks show how ion gradients help to regulate cellular and body volume and to transport essential meta­bo­lites into cells by Na⁺-coupled trans­porters. Right now, it would be important to com­mit to memory the direction of these four important cellular ion gradi­ents, remem­bering which ions are high outside, and which are high inside. These gradients are an essential concept for under­standing electrical excitability and cell physiology.

Ion pumps make ion gradients

The existence of ion gradients tells us that the cell is not at equilibrium and that some work has been done to make such an imbalance. As is always the case, doing work requires expenditure of energy; this is the job of the ATPase ion pumps. In cell membranes, there are several pumps in this protein/gene family specialized for active trans­port of Na⁺, K⁺, Ca²⁺, and protons. We start with the Na⁺/K⁺-ATPase, some­times just known as the Na⁺-pump for short.

Pumps are integral membrane proteins; more specifically, they are enzymes. Like other enzymes, they have a reac­tion velocity that increases with substrate concentration and that saturates when the sub­strates are abundant. Also, their reaction is cyclic with substrates binding and products unbinding in a fixed and cyclic stoichiometry. The unusual feature com­pared with most enzymes is that the substrate is picked up in one compartment and through a conformational change the product is released in a different compart­ment. They operate across membranes. The reaction for the Na⁺/K⁺-ATPase in the plasma mem­brane, already diagrammed in Figure 6, can be written as a chemical formula:

E + 3 Na⁺_i + 2 K⁺_o + ATP    ->    E + 3 Na⁺_o + 2 K⁺_i + ADP + P

where E stands for the enzyme, the numbers indicate the stoichio­metry of the reaction, subscript “o” means outside the cell, and subscript “i” means inside the cell, except that P_i is the conven­tional abbreviation for “inorganic phosphate.” Let’s turn this into words: 3 Na⁺ ions are moved from the inside to the outside, 2 K⁺ ions are moved from the outside to the inside, and 1 ATP is broken down to ADP plus inorganic phos­phate in the cyto­plasm. Some known substeps of the pump cycle can be repre­sented by a cyclic cartoon (Figure 7). Because this pump operates all the time in all our cells, the cytoplasmic Na⁺ is removed and replaced by K⁺, creating and maintaining the Na⁺/K⁺ gradients of Table 2. Some cells, like kidney cells and most excitable cells, must move ions rapidly to accomplish their transport and excitability functions. They have many copies of the Na⁺-pump in their plasma membrane. Others, like red blood cells, have very few ion channels and can get by with a low density of Na⁺-pumps. Red blood cells therefore do not need to consume glucose and produce ATP as rapidly as a kidney cell would.

This pump can be blocked by cardiac glyco­sides like digitalis (related to Digoxin used clinically) and ouabain. When this is done experi­mentally, the ion gradients of the cell gradually run down over hours. Such experiments also show that as much as 25-40% of the energy meta­bolism of a kidney cell is devoted to providing the ATP for the Na⁺/K⁺-ATPase. Clinically, digitalis analogs are used at very low concentration to reduce the Na⁺ gradient just a little in the heart. As you will learn later in the Cardio­vascular Block, a decreased Na⁺ gradient reduces the activity of the Na⁺/Ca²⁺ exchan­ger (see Figure 6), which is powered by the Na⁺ gradient. Therefore, some additional cyto­plasmic Ca²⁺ accu­mulates inside the cardiac cells, boosting muscle contraction and helping to ameliorate congestive heart failure.

The movement of ions through channels is the foundation of electrical signaling.  The properties of membranes and proteins contained within them described thus far lay much of the foundation for studying electrical signaling. We focus on ion channels. We introduce some of the essential components of electrical signaling here, and return to this in the chapters on membrane potentials and action potentials.

Diffusion and voltage

The movement of ions through an open ion channel is dictated by two forces.  The first is the chemical or diffusive force created by differences in the concentration of the ions on the two sides of the membrane.  In the example of Figure 7, K+ and Cl- are initially at higher concentration on the left side of the beaker.  If the membrane dividing the beaker permits both K⁺ and Cl^– to cross, both will move from left to right, down their concentration gradient.  Equilibrium in this case is achieved when the concentrations of K⁺ and Cl^– are the same on both sides of the membrane,

The second force influencing the flow of ions through a channel is the electrical force created by voltage differences.  In Figure 8, as soon as the battery is turned on, the negative electrode (on the right) will attract positively-charged cations (Na+) and the positive electrode will attract negatively-charged anions.

Membrane conductance  (Figure 9A)

A beaker with electrolyte solutions is divided into two com­part­ments called IN and OUT by a mem­brane contain­ing only K⁺ channels (in Figure 7, the membrane was permeable to both K⁺ and Cl^–). The IN and OUT com­part­ments contain equal concentrations of dissol­ved KCl and each has an immersed electrode (e.g. a wire, green) connected to a voltage-pulse gen­er­ator. The pulse generator chan­ges the potential on the wire for brief periods and can make the IN side briefly negative, pos­itive, or unchanged. Figure 2B plots the results as blue triangles. These triangles show the measured current I_K carried by K⁺ ions versus the applied voltage. (i) When the trans­mem­brane voltage is kept at 0 mV, no net K⁺ current flows (sym­bols at the origin). There is no force on the K⁺ ions. (ii) When the IN voltage is made nega­tive, there is an inward K⁺ current, the negative electrical potential pulls K⁺ ions towards the IN com­part­ment. (iii) when the IN voltage is made positive, there is out­ward K⁺ current.

The blue triangles in Figure 9B describe a straight line through the origin. The conductance is the slope of this line. This illustrates Ohm’s law

I_K = G_K E

which says that K⁺ current I_K is linearly pro­por­tional to G_K (the conductance to K⁺) and the electrical driving force E. Remember that the conductance is the inverse of the resistance (G = 1/R), which connects the equation above to the more familiar form of Ohm’s law (E = I R).  Ohm’s law tells us that more force (more E) means more current.

The blue line and blue symbols in Figure 9B des­cribe the current when all the K⁺ channels are open. If we close half of them, we get the red line. As you might expect, the flow of K⁺ is reduced to half, the slope is only half, and the con­duc­tance of the mem­brane is half. Electri­cal conduc­tance is easy for electro­physi­ologists to mea­sure and con­ven­iently tells us how many ion channels are open. This con­cept is used repea­tedly when we start with electro­physio­logy.