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Chapter2 - ION CHANNELS AND Chapter 2 FIGURE 2.1 Cell...

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Unformatted text preview: ION CHANNELS AND SIGNALING 25 26 Chapter 2 FIGURE 2.1 Cell Membrane and Ion Channel. (A) The cell mem- brane is composed of a lipid bilayer embedded with proteins. Some of the proteins traverse the lipid layer, and some ofthese membrane-span- ning proteins form membrane chan- nels. (B) This schematic represen- tation shows a membrane channel in cross section, with a central water- filled pore and channel “gate” (G). The gate opens and closes irregu- larly; the probability of opening may be regulated by the membrane po- tential, by the binding of a ligand to the channel, or by other biophysical or biochemical conditions. A sodium ion, surrounded by a single shell of water molecules, is shown to scale in the pore for size comparison. In Chapter 1 we discussed how the transfer of information in the nervous system is me- diated by two types of electrical signals in nerve cells: graded potentials that are localized to specific regions of the nerve cell membrane, and action potentials that are propagated along the entire length of a neuronal process. These signals are superimposed on a steady electrical potential across the cell membrane called the resting membrane potential. De- pending on cell type, nerve cells at rest have steady membrane potentials ranging from about —30 mV to almost —100 mV, the negative sign meaning that the inside of the mem— brane is negative with respect to the outside. Signaling in the nervous system is mediated by changes in the membrane potential: In sensory receptors, an appropriate stimulus, such as touch, sound, or light, causes local de- polarization (making the membrane potential less negative) or hyperpolarization (mem- brane potential more negative). Similarly, neurotransmitters at synapses act by de- polarizing or hyperpolarizing the postsynaptic cell. Action potentials, which are large, brief pulses of depolarization, propagate along axons to carry information from one place to the next in the nervous system. All such changes in membrane potential are produced by the movement of ions across the nerve cell membrane. For example, inward movement of positively charged sodium ions reduces the net negative charge on the inner surface of the membrane or, in other words, causes depolarization. Conversely, outward movement of positively charged potas- sium ions results in an increase in net negative charge, causing hyperpolarization, as does inward movement of negatively charged chloride ions. How do ions move across the cell membrane, and how is their movement regulated? The major pathway for rapid movement of ions into and out of the cell is through ion channels, which are protein molecules that span the membrane and form pores through which ions can pass. Ion currents are regulated by controlling the opening and closing of such channels. Knowledge of the functional behavior of ion channels has provided an es— sential advance in our understanding of how electrical signals are generated. PROPERTIES OF ION CHANNELS The Nerve Cell Membrane Cell membranes consist of a fluid mosaic of lipid and protein molecules. As shown in Figure 2.1A, the lipid molecules are arranged in a bilayer about 6 nm thick, with their polar, hydrophilic heads facing outward and their hydrophobic tails extending to the middle of the layer. The lipid is sparingly permeable to water, and virtually impermeable to ions. Embedded in the lipid bilayer are protein molecules—some on the extracellular (A) (B) side, some facing the cytoplasm, and some spanning the membrane. Many of the mem- brane—spanning proteins form ion channels. Ions such as potassium, sodium, calcium, or chloride move through such channels passively, driven by concentration gradients and by the electrical potential across the membrane. Other membrane—spanning proteins function as transport molecules (pumps and transporters) that move substances across the membrane against their electrochemical gradients. Transport molecules maintain the ionic composition of the cytoplasm by pumping back across the cell membrane ion species that have moved down their electro— chemical gradients into or out of the cell. They also perform the important function of carrying metabolic substances such as glucose and amino acids across cell membranes. Properties of transport molecules are discussed in Chapter 4. What Does an Ion Channel Look Like? The molecular composition of ion channels and their configuration in the cell membrane are discussed in detail in Chapter 3, but it is useful at this point to have some idea of the general physical features of an ion channel protein. These are illustrated by Figure 2.113. The protein spans the membrane and has a central water—filled pore open to both the in- tracellular and the extracellular spaces. On each side the pore widens to form a vestibule (or mouth), and the restricted region within the plane of the membrane contains a gate that can open and close to regulate the passage of ions. The size of the protein varies considerably from one channel type to the next, and some have additional structural features. Figure 2.18 represents a channel of medium di— mensions. A sodium ion, hydrated by a single shell of water molecules, is shown in the pore for size comparison. Channel Selectivity Membrane channels vary considerably in their selectivity: Some are permeable to cations, some to anions. Some cation channels are selective for a single ion species. For example, some allow permeation of sodium almost exclusively, others of potassium, still others of calcium. Others are relatively nonspecific, allowing the passage of even small organic cations. Anion channels involved in signaling tend to have low specificity, but they are re— ferred to as “chloride channels” because chloride is the major permeant anion in biologi— cal solutions. In addition, some channels (called connexons) connect adjacent cells and allow the passage of most inorganic ions and many small organic molecules. Connexons are discussed in Chapter 7. Open and Closed States Although for simplicity we must represent protein molecules as static structures, they are never still. Because of their thermal energy, all large molecules are inherently dy- namic. At room temperature, chemical bonds stretch and relax, and twist and wave around their equilibrium positions. Although individual movements are of the order of only 10‘'2 m in magnitude, with frequencies approaching 10[3 Hz, such atomic trem— bling can underlie much larger and slower changes in conformation of the molecule. The reason is that numerous rapid motions of the atoms occasionally allow groups to slide by one another in spite of mutual repulsive interactions that would otherwise keep them in place. Such a transition, once achieved, can last for many milliseconds, or even seconds. Hemoglobin provides an example: The binding sites for oxygen to the heme groups are buried inside the molecule, and not immediately accessible. Oxygen binding, and its subsequent escape, can be accomplished only by the dynamic formation of tran— sient access pathways to the heme pocket; thus, the molecule “breathes” in order to per— form its function.1 In ion channel proteins, molecular transitions occur between open and closed states, with transitions between states being virtually instantaneous. If we examine the behavior of any given channel, we find that open times vary randomly: Sometimes the channel opens for only a millisecond or less, sometimes for very much longer (see Figure 2.4). Ion Channels and Signaling 1Karplus, M., and Petsko, G. A. 1990. Nature 347: 631-639. 27 28 Chapter 2 FIGURE 2.2 Modes of Channel Activation. The probability of chan- nel opening is influenced by a variety of stimuli. (A) Some channels re- spond to changes in the physical state ofthe membrane, specifically changes in membrane potential (voltage-activated) and mechanical distortion (stretch-activated). (B) Ligand-activated channels respond to chemical agonists, which attach to binding sites on the channel protein. Neurotransmitters, such as glycine and acetylcholine, act on extracellular binding sites. Included among a wide variety of intracellular ligands are calcium ions, subunits of G proteins, and cyclic nucleotides. However, each channel has its own characteristic mean open time (I) around which these durations of opening fluctuate. Some channels in the resting cell membrane open frequently; the probability of find- ing such channels in the open state is relatively high. Most of these are potassium and chloride channels associated with the resting membrane potential. The remainder are predominantly in the closed state, and the probability that an individual channel will open is low. When such channels are activated by an appropriate stimulus, the probabil- ity of openings increases sharply. On the other hand, channels that open frequently at rest may be deactivated by a stimulus; that is, their frequency of opening is decreased. An im— portant point to remember is that activation or deactivation of a channel means an in- crease or decrease in the probability ofchannel opening, not an increase or decrease in mean open time of the channel. In addition to activation and deactivation, two other factors regulate current flow through ion channels. One is that certain channels can enter a conformational state in which activation no longer occurs, even though the activating stimulus is still present. In channels that respond to depolarization of the cell membrane, this condition is called inactivation; in channels that respond to chemical stimuli, the condition is known as desensitization. The second mechanism is open channel block.- This occurs when, for example, a large molecule (such as a toxin) binds to a channel and physically occludes the pore. Another example is the block of some cation channels by magnesium ions, which do not themselves permeate the channel, but bind in its inner mouth and prevent the permeation of other cations. Modes of Activation Figure 2.2 summarizes the modes of channel activation. Some channels respond specifi— cally to physical changes in the nerve cell membrane. Prominent in this group are the voltage-activated channels. An example is the voltage—sensitive sodium channel, which is responsible for the regenerative depolarization that underlies the rising phase of the action potential (Chapter 6). Also in this group are stretch—activated channels, which respond to mechanical distortion of the cell membrane. Stretch receptors are found, for example, in mechanoreceptors in the skin (Chapter 17). (A) Channels activated by physical changes in the cell membrane Voltage-activated Stretch-activated (B) Channels activated by ligands Extracellular activation Intracellular activation Other channels are activated when chemical agonists attach to binding sites on the channel protein. These ligand-activated channels are further divided into two subgroups, depending on whether the binding sites are extracellular or intracellular. Channels that respond to extracellular activation include, for example, cation channels in the postsyn- aptic membranes of skeletal muscle that are activated by the neurotransmitter acetyl- choline, released from presynaptic nerve terminals (Chapter 9). This activation allows sodium to enter the cell, causing muscle depolarization. Ligand-activated channels responding to intracellular stimuli include channels that are sensitive to local changes in the concentration of specific ions. For example, calcium— activated potassium channels are activated by local increases in intracellular calcium and, in many cells, play a role in repolariZing the membrane during termination of the action potential. Other intracellular ligands include the cyclic nucleotides: Cyclic GMP, for ex- ample, is responsible for the activation of sodium channels in retinal rods, thereby play- ing an important role in visual transduction (Chapter 19). These classifications are not rigid: For example, calcium—activated potassium channels are also voltage—sensitive, and some voltage—activated channels are sensitive to intracellu- lar ligands. MEASUREMENT OF SINGLE-CHANNEL CURRENTS Patch Clamp Recording Experimental tools have been devised to measure ion fluxes through individual channels, first indirectly by analysis of membrane noise,“ and later by direct observation, using patch clamp recording methods“)5 These methods provide direct answers to questions of obvious physiological interest about channels; for example, how much current does a sin— gle channel carry? how long does a channel stay open? how do its open and closed times depend on voltage? on the activating molecule? The development of the patch clamp by Erwin Neher, Bert Sakmann, and their col- leagues has contributed enormously to our knowledge of the functional behavior of mem- brane channels. In patch clamp recording, the tip of a small glass pipette (with an internal diameter of about 1 pm) is sealed to the membrane of a cell. Under ideal conditions, with slight suction on the pipette, a seal resistance of greater than 109 ohms (hence the term “giga- ohm seal”) forms around the rim of the pipette tip between the cell membrane and the glass (Figure 2.3A—B). When the pipette is connected to an appropriate amplifier, small cur- rents across the patch of membrane inside the pipette tip can be recorded (Figure 2.3F). The high-resistance seal ensures that such currents flow through the amplifier rather than escaping through the rim of the patch. The recorded events consist of rectangular pulses of current, reflecting the opening and closing of single channels. In other words, we can ob- serve in real time the activity of single protein molecules in the membrane. In their simplest form the current pulses appear irregularly, with nearly fixed ampli— tudes and variable durations (Figure 2.4A). In some cases, however, records of current are more complex: For example, channels may exhibit open states with more than one cur- rent level, as in Figure 2.4B, where the open channels often close to smaller “substate” lev- els. In addition, channels may display complicated kinetics. For example, channel openings may occur in bursts (Figure 2.4C). In summary, patch clamp techniques offer two advantages for studying the behavior of channels: First, the isolation ofa small patch of membrane allows us to observe the ac- tivity of only a few channels, rather than the thousands that may be active in an intact cell. Second, the very high resistance of the seal enables us to observe extremely small cur- rents. As a result, we are able to obtain accurate measures of the amplitudes of single- channel currents and to analyze the kinetic behavior of the channels. Recording Configurations with Patch Electrodes Patch clamp methods permit other recording configurations. Having made a seal to form a cell-attached patch, we can then pull the patch from the cell to form an inside-out patch Ion Channels and Signaling 29 Erwin Neher (left) and Bert Sakmann (right) 2Katz, 8., and Miledi, R. 1972.]. Phys- iol. 224: 665—699. 3Anderson, C. R., and Stevens, C. F. 1973.]. Physiol. 235: 665—691. 4Neher, E., Sakmann, B., and Stein— bach, J. H. 1978. PflL'igers Arch. 375: 219-228. 5Hamill, O. P., et al. 1981. PflL‘igers Arch. 391: 85—100. 30 Chapter 2 FIGURE 2.3 Patch Clamp Recording. (A—E) Patch (A) configurations, represented schematically. The electrode forms a seal on contact with the cell membrane (A), which is converted to a gigaohm seal by gentle suction (B). Records may then be made from the patch of mem- brane within the electrode tip (cell-attached patch). Pulling away from the cell results in the formation of a cell-free vesicle, whose outer membrane can then be ruptured to form an inside-out patch (C). Alternatively, (B) the membrane within the electrode tip may be ruptured by further suction to obtain a whole-cell recording (D) or, by pulling, to obtain an outside-out patch (E). (F) Record- ing arrangement. The patch electrode is connected to an amplifier that converts channel currents to voltage sig- nals. The signals are then displayed on an oscilloscope trace or computer screen so that amplitudes and dura- tions of single-channel currents can be measured. (A—E after Hamill et al., 1981.) (C) INSIDE-OUT PATCH (E) OUTSIDE—OUT PATCH Electrode l-ip Cell i Suction (Gigaohm seal) CELL ATTACHED / Suction\\ (D) WHOLE-CELL RECORDING Pull (low Ca2+) % Pull l (F) Current—to- voltage converter — T : Oscilloscope (see Figure 2.3C), with the cytoplasmic face of the patch membrane facing the bathing solution. Alternatively, with slight additional suction we can rupture the membrane in— side the patch to provide access to the cell cytoplasm (Figure 2.3D). In this condition cur— rents are recorded from the entire cell (whole—cell recording). Finally, we may first obtain a whole—cell recording and then pull the electrode away from the cell to form a thin neck of membrane that separates and seals to form an outside—out patch (see Figure 2.3E). Each of these configurations has an advantage, depending on the type of channel we are studying and the kind ofinformation we wish to obtain. For example, if we wish to apply a variety of chemical ligands to the outer membrane of the patch, then an outside—out patch is most convenient. One feature of whole—cell recording with a patch pipette is that substances can ex— change between the cell cytoplasm and the pipette. This exchange (sometimes referred to as dialysis) can be useful in providing a method for changing intracellular ion concentra— tions to those predetermined when the pipette is filled. On the other hand, particularly if Ion Channels and Signaling 31 (A) 20 M Closed(C) Open (0) ----- (B) 4pA C ( ) (0) --------------- 1 ‘- ---- - -------- 2pA (C) l——L_—L.___J—.__I—._1—_.__I—_.__I_ 0 200 400 600 800. _. - Time (ms) the cell is small, important cytoplasmic constituents can be lost rapidly into the pipette solution. Such loss can be avoided by using a perforated patch.6 The patch pipette is loaded with a pore-forming substance, such as the antibiotic nystatin, and a seal to the cell is formed. After a delay, pores form in the patch, allowing whole-cell currents to be recorded. Intracellular Recording with Microelectrodes Before patch clamp techniques were developed, properties of membrane channels were deduced from experiments in which glass microelectrodes were used to record mem- brane potential or membrane current from whole cells. The introduction of the glass mi- croelectrode for intracellular recording from single cells by Ling and Gerard7 in 1949 was at least as important as the introduction of patch clamp recording three decades later. The technique provided a method for accurate measurements of resting membrane po— tentials, action potentials, and responses to synaptic activation in muscle fibers and neu— rons (Chapter 1). The intracellular recording technique is illustrated in Figure 2.5A. A sharp glass mi- cropipette, with a tip diameter of less than 0.5 pm and filled with a concentrated salt so— lution (e.g., 3 M KCl), serves as an electrode and is connected to an amplifier to record the potential at its tip. When the pipette is pushed against the cell membrane, penetration into the cytoplasm is signaled by the sudden appearance of the resting potential. If the penetration is successful, the membrane seals around the outer surface of the pipette, so that the cytoplasm remains isolated from the extracellular fluid. Intracellular Recording of Channel Noise In the early 1970s, Katz and Miledi2 did pioneering experiments on frog muscle fibers, in which they used intracellular recording techniques to examine the characteristics of the “noise” produced by acetylcholine (ACh) at the neuromuscular junction. At this FIGURE 2.4 Examples of Patch Clamp Recordings. (A) Glutamate— activated channel currents recorded in a cell-attached patch from locust muscle occur irregularly, with a sin- gle amplitude and varied ope...
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