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Molecules to Medicine- Cell Physiology 5. Membrane Transporters 20 Nov 9AM Bill Betz http://www.cuphys.net/cellphys [email protected] Reading: Alberts et al, 4th ed: pp 615-631. Lehninger, 4th ed: pp 389-411. Key words: primary active transport, secondary active transport, cotransporter, exchanger, electrogenic 5. Membrane Transporters Earlier we talked about the famous sodium-potassium pump, the ubiquitous transporter that renders cells ‘functionally impermeable’ to sodium ions, and keeps the cells from swelling uncontrollably. Today we will consider other types of carrier mechanisms. The first thing to note is that none of them, including the Na/K pump, actually ‘carries’ solute cargo across the membrane. There is an antibiotic (valinomycin) that actually diffuses back and forth across the membrane (shuttling potassium ions), but no biological molecule is known to act this way, so transporter is a better word than carrier. How do protein transporters work? For most, we don’t really know, but the general model of the Na/K pump (a tube with a gate on each end and dynamic (changeable) binding affinity) is probably a good model for the others as well (see Handout #4, page 5). Conceptually, matters are pretty simple. Some transporters act like ion channels, shuttling a single solute species in either direction. This is called facilitated diffusion (a historic term applied to molecules that shouldn’t be able to diffuse across lipid membranes because of their large size or charge, but do get across). The best known example of this is the glucose transporter (cartoon at right). The glucose transporter will transport glucose in either direction, and burns no energy in the process. Thus, it is not a pump. You might then wonder how cells accumulate glucose. The answer is that as soon as a glucose molecule gets into the cell, it is phosphorylated to Glucose-6-Phosphate, which doesn't fit on the transporter, and so is "trapped" inside. Glucose uptake by cells is regulated by insulin, a hormone secreted by specialized cells in the pancreas when plasma glucose levels rise. How does insulin turn on the transporter? It turns out that in the absence of glucose, the transporter is not even present in the plasma membrane; it is sequestered inside the cell, in the membrane of intracellular vesicles. Insulin triggers a biochemical cascade that causes the vesicle membranes to fuse with the surface membrane (exocytosis), exposing the glucose transporter to the ECF. The transporter then gets busy and ‘carries’ glucose inside. When insulin subsides, the transporter molecules are reinternalized (endocytosis). Pumps. Now we turn to transport proteins that can cause a substance to be accumulated or expelled against its electrochemical gradient. Dozens have been identified, pumping nearly every substance of importance in the body in one cell or another. The only evident exceptions are water and urea (the end product of nitrogen metabolism in mammals). Isn’t it odd that no water pumps exist anywhere in nature, given its importance? Water always moves down its concentration (osmotic) gradient. By definition, pumps move solute against an energy gradient, and so require an energy source. We subdivide pumps into two categories, depending on the source of that energy. Primary active transporters, like the Na/K pump, derive their energy directly from the splitting of ATP. There are no other ubiquitous primary active transporters in the plasma 1 membrane of cells. Some specialized cells have them, and you will study them later. For example, the cells in the stomach that secrete acid, and certain cells in the kidney geared for excreting protons from the body possess proton pumps in their plasma membranes that rely directly on ATP for their energy source. Inside cells (as opposed to the surface membrane) there are other primary active transporters. One pumps protons into intracellular membrane-bound organelles (endosomes, vesicles, lysosomes). Another pumps calcium ions into membrane-bound compartments. All proton and calcium pumps are oriented to pump these ions out of the cytoplasm. Inside mitochondria is a very special proton pump (the F1-ATPase, or ATP synthase) that, when running backwards, lets protons leak across a membrane and synthesizes, rather than hydrolyzes ATP. Secondary active transport is the mechanism by which most substances are pumped. In this case, the energy to do the direct work of pumping comes not from metabolism (ATP), but from a secondary source. Usually this energy source is the 'downhill leak' of Na+ into the cell. For example, cells can accumulate amino acids against their energy gradients. This active uptake is dependent on external Na+; if external Na+ is removed, amino acid uptake is abolished. Conversely, removing the amino acid reduces the entry of Na+. The carrier ingeniously captures the energy released by the inward leak of Na+ and instead of letting it escape as heat, uses it to pump the amino acid into the cell. There are two basic types of secondary active transporters, those that move different solute species in the same direction (cotransport), and those that move solute in opposite directions (antiport, or exchange). The cartoons below illustrate these two types (arrows pointing up mean the substance is pumped; down means leak). Secondary active transporters do not necessarily always run in the same direction. They will always tap the bigger leak to drive the smaller pump. Consequently, they can reverse direction sometimes. One of the most important examples of this is the sodium-calcium exchanger, which reverses direction in heart muscle cells every time the heart beats (more on this later). All secondary transport mechanisms depend ultimately on the Na+/K+ pump (and therefore on ATP). For example, if the Na+/K+ pump is blocked, cells fill up with Na+, and thus the Na+ electrochemical gradient is reduced. Because this is the energy source for secondary active transport, all of these transport mechanisms suffer. Some secondary active transporters are electrogenic, in that one cycle produces a net charge transfer across the membrane. For example, Na/amino acid transporters are electrogenic, because one cycle transfers a net positive charge (Na+) into the cell. Other secondary active transporters are not electrogenic; an example is the Na/K/2Cl cotransporter, which each cycle moves one sodium ion, one potassium ion, and two chloride ions into the cell. The main feature of electrogenic secondary active transporters is that their activity is governed by the membrane potential (as described in the Appendix). Electrically silent transporters could not care less about 2 membrane potential. Here are some important examples of secondary active transporters driven by the inward sodium leak: Calcium transport. There is a huge electrochemical gradient for calcium ions across cell membranes. In fact, no other ion is further from equilibrium than calcium. The extracellular (ionized) calcium concentration (about 1 mM) is nearly 10,000 times greater than the intracellular concentration (about 0.0002 mM, or 200 nM); thus its concentration gradient is inward. And the electrical gradient is also inward, of course, because the ICF is electrically negative and calcium is positively charged. From the Nernst equation, ECa is calculated to be about +111 mV (ECa = (60/2)*log(1/0.0002) = +111 mV). Thus, given the opportunity (i.e., an open calcium channel), Ca++ ions will always leak into cells, so there must be a pump to extrude them. The Na/Ca exchanger’s main job is to pump calcium ions out of the cell. The inward leak of sodium ions provides the energy source. The Na/Ca exchange pump takes on a special significance in the heart, where it actually switches direction during each heartbeat. (You will study muscle contraction shortly, and will learn that calcium ions are necessary for contraction.) At rest (while the ventricles are refilling with blood during diastole), the exchanger runs forward, pumping Ca++ out (keeping the muscle relaxed) as Na+ leaks in. When the ventricles contract (systole), the exchanger switches direction, letting Ca++ leak into the cell, where it strengthens the force of contraction. The direction of Ca++ movement is controlled by the value of the membrane potential; when Vm is more negative than about -60 mV, the sodium leak rules, and drives the outward pumping of calcium; when Vm is more positive than -60 (during the action potential), the pump reverses direction, and calcium leaks in (pumping sodium out). Digitalis. For centuries it has been known that an extract of the beautiful purple foxglove can help a weak heart beat stronger. Digitalis (and related drugs) exert their action by acting directly on, not the Na/Ca exchanger, but the Na/K pump! In fact, digitalis blocks the Na/K pump. How does this lead to an increase in the strength of contraction of heart muscle? Blocking the Na/K pump of course allows intracellular sodium ion concentration to increase. In turn, this reduces the energy available to all sodium-driven secondary active transporters, including the Na/Ca exchanger. Thus, digitalis indirectly inhibits the Na/Ca exchanger, allowing intracellular calcium ion concentration to rise, which increases cardiac contractility. Hydrogen ions (protons) are also pumped out of most cells by a Na+/H+ exchange carrier, which operates under the same principles as the Na/Ca exchanger. Protons are harder to study than UFO's, because they capriciously vanish and reappear as they bind to and unbind from various buffers. Typically, only about 1 in a million protons is free, as H+; the rest are hiding, bound to buffers. The free concentration of protons in the ICF is about 100 nM (pH=7.0); in the ECF it’s even lower, about 40 nM (pH=7.4). From the Nernst equation, EH = -24 mV. That means in cells with membrane potentials more negative than -24 mV (most cells), H+ must be pumped out of the cell. The mechanism is a secondary active transport system, in which the inward leak of Na+ drives the outward pumping of H+. Chloride ions are pumped into some cells by a secondary active transport process (Na/K/2Cl cotransporter). As a result, ECl moves in a positive direction, away from the resting membrane potential. The (non-existent) ‘H+/K+ exchanger’. There are several clinical situations that suggest the presence of a system that will exchange K+ for H+, and vice versa. For example, infusing K+ causes 3 acidemia (the K+ is taken up by cells ‘in exchange’ for H+), and infusing acid causes hyperkalemia (elevated (hyper-) potassium (-kal- for Latin kalium) in the blood (-emia)). While it is conceptually simple (and useful) to think in terms of an H/K exchanger, the reality is that such a transporter probably does not exist. Rather, the process evidently involves different transporters, perhaps working in pairs in parallel, the upshot being hydrogen/potassium exchange. For example, hyperkalemia will cause extra K+ uptake via the Na/K pump. Hyperkalemia also will depolarize cells (by shifting EK in a positive direction), and the change in membrane potential can affect the rate of activity of electrogenic transporters. One such transporter, which transports 3 bicarbonate ions and one sodium ion from the ICF to the ECF, is inhibited by depolarization. Thus, the reduction in activity will reduce bicarbonate extrusion. Because bicarbonate is a base, its slower extrusion will cause acidemia. So, which is easier to remember, the idea of a (non-existent) H/K exchanger, or trying to keep track of several different cotransporters and exchangers that might be playing a role? Below are 10 cartoons of membrane transporters. One of them has been mislabeled. Which one? answer: the last one (it is a sodium-bicarbonate cotransporter, not an exchanger) 4