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University of Toronto St. George
Cell and Systems Biology
Francis Bambico

Chapter 4 – Ion Channels and Signaling  The transfer of information in the nervous system is mediated by 2 types of electrical signals in nerve cells: o graded potentials, which are localized to specific regions of the nerve cellmembrane, o action potentials, which 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 restingmembrane potential  potentials ranging from -30mV to almost -100 mV  inside of the membrane is negative  signalling in the nervous system is mediated by changes in the membrane potential  stimulus causes local depolarization (membrane is less negative/more positive) or hyperpolarization (membrane potential more negative)  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 o depol: inward movement of Na+ reduces the net negative charge on the inner surface of the membrane o hyper: outward movement of K+ -> increase in net negativecharge/ inward movement of Cl-  the pathway for rapid movement of ions into and out of thecell is through ion channels -> proteinmolecules that span the membrane and form pores through which ions can pass  ion currents are regulated by controlling the rate at which these channels open and close Properties of Ion Channels The Nerve Cell Membrane  cell membranes consist of a fluid mosaic of lipid and protein molecules  the lipid molecules are arranged in a bilayer with their polar, hydrophilic heads facing outward and their hydrophobic tails extending to themiddle of the layer  the lipid is sparingly permeable to water and virtually impermeable to ions  proteinmolecules are embedded, some on the extracellularside, some on intracellular, some spanning  ions are driven by conc gradients and by electrical potentialacross themembrane  another set of membrane-spanning proteins function as transport molecules (pumps and transporters) thatmove substances across the membrane against their electrochemical gradients  they maintain the ionic composition of the cytoplasm by pumping back across the cell membrane ion species that have leaked through channels into or out of the cell  they also perform the imp function of carrying substances (glucose etc) across cell membranes What Does an Ion Channel Look Like?  The protein spans themembrane, with a central water-filled pore open to both the intracellular and extracellular spaces  On each side of the membrane, the pore widens to form a vestibule  Within the plane of the membrane, a segment of the pore is constricted and lined with a ring of negative charges to form a selectivity filter for cations and vice versa for anions  The channel contains a gate that opens and closes to control ion movement through the channel  The size varies considerably from 1 channel type to the next and some have additional structural features Channel Selectivity  Some are permeable to cations, some to anions  Some are selective for a single ion species  Others are relatively nonspecific, allowing the passage of even small organic cations  Anions channels involved in signaling tend to have low specificity but are referred to as Cl- channels, because Cl- is the major permeant anion in biological solutions Open and Closed States  Protein molecules are dynamic because of their thermal energies  At room temp, chemical bonds stretch and relax, and twist and wave around their eq positions  Numerous rapidmotions of the atoms occasionally allow groups to slide by one another in spite of mutual repulsive interactions that would otherwise keep them in place  In ion channel proteins, molecular transitions occur between open and close states virtually instantaneous  Open times vary randomly  Each channel has its own characteristicmean open time around which individual open times fluctuate  Some channels in the resting cell membrane open frequently; thus, the prob of finding such channels in the open state is relatively high o Most of these are K+ and Cl- channels o May be deactivated by a stimulus  Others are predominantly in the closed state, and the probof an individual channel opening is low o When such channels are activated by an appropriate stimulus, the prob of opening increase sharply  Certain channels can enter a conformational state in which activation nolonger occurs, even though the activating stimulus is still present o In channels that respond to depolarization, this condition is called inactivation o In channels that respond to chemical stimuli, the condition is known as desensitization  Open channel block: a largemolecule can bind to a channeland physically occlude the pore Modes of Activation  Some channels respond specifically to physical changes in the nerve cellmembrane o Voltage-sensitive Na channel is responsible for the regenerative depolarization that underlies the rising pahse of the action potential o Stretch-activated channels respond to mechanical distortion of the cell membrane  Others are activated when chemical agonists attach to binding sites on the channel protein o Ligand-activated channels are further divided into 2 subgroups depending on whether the binding sites are extracellular orintracellular o Extracellular  Cation channels in the postsynaptic membranes of skeletal muscle that are activated by Ach o Intracellular  May be sensitive to local changes in conc of a specific ion  1 type of K+ channel is activated by an increase in intracellular [Ca] in the adjacent cytoplasm, usually to repolarize the membrane  Other ligands include the cyclic nucleotides Measurement of Single-Channel Currents Intracellular Recording with Microelectrodes  The first experiments designed to examine the properties of membrane channels were done using glass microelectrodes to record membrane potentials or membrane currents from whole cells  In 1949, Ling andGerard adapted the glass microelectrode for intracellular recording from single cells  The technique provided amethod for accurate measurements of resting membrane potentials, action potentials, and responses to synaptic activation in muscle fibers and neurons  A sharp glass micropipette, with a tip diameter of less than0.5 µm and filled with a conc salt solution, serves as an electrode and is connected to an amplifier to record the potential as the tip of the pipette  Penetration into the cytoplasm is signaled by the sudden appearance of the resting potential  If the penetration is successful, themembrane seals around the outer surface of the pipette, so that the cytoplasm remains isolated from the ECF Channel Noise  In the early 1970s, Katz and Miledi did pioneering experiments on frogmuscle fibers to examine the characteristics of the noise produced by Ach at the neuromuscular junction  They noticed that during the depolarization, fluctuations in the electrical recording were larger than the normal baseline fluctuations at rest  This increase in noise was due to the random opening and closing of the Ach-activated channels  By applying noise analysis techniques, they were able to obtain info about the behavior of the individual channels activated by Ach  If the single channel currents are relatively large, then the noise will be large as well  Channels that open for a relatively long time will produce only low-freq noise Patch Clamp Recording  Patch clamp recording methods provide direct answers to questions of obvious physiological interest about channels  Neher, Sakmann and their colleagues developed patch clamp: involve sealing the tip of a small glass pipette to the membrane of a cell  Havingmade a seal to form a cell-attached patch, we can then pull the patch from the cell to form an inside-out patch, with the cytoplasmic face of the patchmembrane facing the bathing solution  Alternatively, after forming a cell-attached patch, we can apply slight additional suction to rupture the membrane inside the patch and thereby provide access to the cell cytoplasm -> currents 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  Each of these configurations has an advantage, depending upon the type of channels studied and the kind of information one wishes to obtain  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 pulse of current, reflecting the opening and closing of single channels  One feature of whole-cell recordingis that substances canmove between the cytoplasm and the pipette – this exchange canbe useful in providing a method for changing the preexisting intracellular ion conc to those in the pipette  On the other hand, particularly in a small cell, important cytoplasmic components can be lost rapidly into the pipette solution -> can be avoided via perforated patch: the patch pipette is loaded with a pore-forming substance and a seal is formed to the cell  After a delay, pores are formed in the patch that allow whole-cell currents to be recorded without loss of intracellular macromolecules Single-Channel Currents  In their simplest form, single-channel current pulses appearirregularly, with nearly fixed amplitudes and variable durations  However, in some cases, current records are more complex o Channels exhibit open states withmore than one current level where the open channels often close to smaller “substrate” levels o In addition, channels may display complicated kinetics (channel openings in bursts)  Patch clamp techniques offer advantages for studying the behavior of channels 1. The isolation of a small patch of membrane allows us to observe the activity of only a few channels 2. They very high resistance of the seal enables us to record extremely small currents Channel Conductance  The kinetic behavior of a channel (the durations of its closed and open states) provide info about the steps involved in channel activation and the rate constants associated with these steps  The channel current is a directmeasure of how rapidly ions move through a channel  The current depends on both the channel properties and transmembrane potential  Outside-out membrane patch which contains a single, spontaneously active channel that is permeable to K+  The soln in both the pipette and bath contain 150 mM K+  because the conc are equal, there is no net movement  When a voltage of +20mV is applied, each channel opening results in a pulse of outward current, because positively charged K+ ions are driven outward through the channel by the electrical gradient between thepipette soln and the bath  When the inside is made negative by 20 mV, current flows in the other direction through the open channel into the pipette  Figure: The effect of voltage on the size of the current  The relationship is linear; the current (I) through the channel is proportional to the voltage applied to it: V=RI  V: voltage; R: resistance (slope); g – channel conductance  I = gV = g(V-Vo)  Vo is the voltage across the membrane at which the currentis 0  V-V0 is the driving force for current through the channel  Channel conductance is a measure of the ability of the channel to pass current  For a particular voltage, a high conductance channel will carry a lot of current, while a low conductance channel will carry only a little Conductance and Permeability  The conductance of a channel depends upon 2 factors: 1. The ease with which ions can pass through the open channel; this is an intrinsic property of the channel: Channel permeability 2. The conc of ions in theregion of the channel – no ions, no flow  If only a few K+ are present, then for a given permeability and a given potential, the channel current will be smaller than when K are present in abundance  Open channel -> permeability  Permeability + ions -> conductance Equilibrium Potential  Outside-out patch with [K]+ of 3mM in the bath and 90 mM in the electrode  Under those conditions, K+ will move through the channels to the bath down their conc gradient, even when no potential is applied to the pipette  If the pipette is made + with respect to the bath, the potential gradient across the membrane will accelerate the outward K movement and the channel current will increase  If the pipette is made negative, outward movement of K will be retarded and the channel current will decrease  With sufficiently large negativity, K will flow inward across the membrane against their conc gradient  K current through the channel depends on both the electrical potential across the membrane and on the [K] gradient -> electrochemical gradient  The potential that just balances the [K] gradient is called the potassium equilibrium potential, Ek  When themembrane potential is atEk, the driving force forK current is 0; at any other potential, the driving force is V-Ek  The equilibrium potential depends only on the ion conc on either side of themembrane and not on the properties of the channel or the mechanism of ion permeation through the channel Nernst Equation  The potential required to balance a given K conc differenceacross themembrane depends on the different between the log of the conc:  R: thermodynamic gas constant; T: the absolute temperature; z: the valence of the ion; F: Faraday  RT/zF has the dimensions of volts; RT/zF=58  It is sometimes more convenient to use the log of the conc ratio, rather than ln  The rate of diffusion of an ion down a conc gradient is not strictly related to its conc o Ions are subject to interactions with one another (electrostatic attraction or repulsion) o The result is that the effective conc of the ion is reduced o The effective conc of an ion in soln is called its activity Nonlinear Current-Voltage Relations  Another feature of the current-voltage relation of the figure above is that it’s not linear  As we move away from the equilibrium potential in the depolarizing direction, the outward current increases more and morerapidly as the potential approaches 0  When themembrane is mademore negative, the inward current increases more slowly with hyperpolarization becauseof the dependence of conductance on conc  The farther wemove away from the eq potential, the moreprominent the effect becomes, so that the current-voltage relation has a marked upward curvature  This relation also occur in some channels because the channels themselves rectify, allowing ions to move through the pores in one directionmuchmore readily than in the other Ion Permeation through Channels  Ions can pass through channels by diffusion through a water-filled pore  But channels themselves interact with the ions o Eg. Because they are charged, ions in soln are always accompanied by close apposed water molecules  If the pore is relatively narrow, then an ion must acquire a certain amount of energy in order to escape from its associated waters of hydration and squeeze through the neck of the channel  Once in the channel, the ion may be attracted to or repelled by electrostatic charges lining the channel wall, or it may be bound to sites from which it must escape  Channel models that deal with interactions that affect both ion selectivity and rate of ion flux are calledEyring rate theory models; more successful than simple diffusion models  The neuron makes use of standing electrochemical gradients to generate ion movements, and hence to generate electrical signal  Gradients aren’t dissipated because cells use metabolic energy to maintain the ionic composition of the cytoplasm Summary  Electrical signals in the nervous system are generated by the movement of ions across the nerve cell membrane. These ionic currents flow through the aqueous pores of membrane proteins known as ion channels  Channels vary in their selectivity: some cation channels allow only Na, K, or Ca to pass, while others are less selective. Anion channels are relatively nonselecive for smaller anions but pass mainly Cl because of the relative abundance of Cl in EC and ICF  Channels fluctuate between open and closed states. Each channel has a characteristic mean open time. When channelsare activated, their prob of opening increase. Deactivation reduces opening frequency. Channels may also be inactivated of blocked  Channels can be classified by their mode of activation: stretch-activated, voltage-activated, and ligand-activated  Ions move through channels passively in response to conc and electrical gradients across the membrane  The net flux of ions through a channel down a conc gradient can be reduced by an opposing electrical gradient. The electrical potential that reduces the net flux to exactly 0 is called the equilibrium potential for that ion species. The relation between eq potential and the conc gradient is given by the Nernst Eqn  The driving force formovement of an ion across the membrane is the diff between its eq potential and the actual membrane potential. The flow of ionic current through a channel depends on the driving force for the ion in question and on the conductance of the channel for that ion. The conductance depends, in turn, on theintrinsic ionic permeability of the channel and, in addition, on the inside and outside ion conc Chapter 6 – Ionic Basis of the Resting Potential  At rest, a neuron has a steady electrical potential across its plasma membrane, the inside being negative  Intracellularly: [K] is higher, [Na] and [Cl] are lower  The tendency for K to move out of the cell and Cl to move in is opposed by the membrane potential  The concentrations of the cell aremaintained by the Na-K exchange pump, which transports 3 Na out and 2 K in  The resting membrane potential depends on the K eq potential, the Na eq potential, the relative permeability of the cell membrane to the 2 ions, and the pump ratio  The Cl eq potential may be +/- relative to the resting membrane potential, depending on the Cl transport processes  Electrical signals are generated primarily by changes in permeability of the cell membrane to ions  Permeability increases are due to activation of ion channels A Model Cell  This cell contains K, Na, Cl, and a large anion species, and it is bathed in a soln of Na, KCl  It is permeable to K and Cl, but not to Na or to the internal anion  3 major requirements for a cell to remain in a stable cond: 1. The intracellular and extracellular soln must each be electrically neutral 2. The cellmust be in osmotic balance. Otherwise, water will enter/leave the cell. Osmotic balance is achieved when the total conc of solute particles inside the cell is equal to that on the outside 3. There must be no net movement of any particular ion into or out of the cell IonicEquilibrium  K ions do not diffuse out of the cell until the conc on either side of the membrane is equal because the process is self limiting: as the K ions diffuse outward, + charges accumulate on the outer surface of themembrane and excess negative charges are left on the inner surface  The electrical gradient slows the efflux of positively charged K ions and when the potential becomes sufficiently large, further net efflux of K is stopped  Both the tendency for K ions to leave the cell and for Cl to diffuse inward are opposed by the membrane potential  Because the conc ratios for the 2 ions are of exactly the same magnitude (1:30), their eq potentials are the same  The model cell can exist indefinitely without any net gain orloss of ions Electrical Neutrality  The charge separation across the membrane of our model cell means that there is an excess of anions inside the cell and of cations outside  K ions diffusing outward collect as excess cations against the outer membrane surface, leaving excess anions closely attracted to the inner surface  Both the K ions and the counter ions they leave behind are removed from the intracellular bulk solution  The outer layer of cations and inner layer of anions, of equal and opposite charges, are not in free solution but are held to the membrane surface bymutual attraction -> the membrane acts as a capacitor, separating and storing charge  The point is that although the identities of the ions in the layer are constantly changing, their total number remains constant, and the bulk solution stays neutral Membrane Potentials in Squid Axons  The first experiments measuringmembrane potential were done on giant axons that innervate the mantle of the squid  The axons are up to 1 mm in diameter, and their large size permits the insertion of recording electrodes into their cytoplasm tomeasure transmembrane potential directly  Hodgkin and Huxley initiated many experiments on squid axon  Experiments onisolated axons are usually done in seawater, with the ratio of intra to extra [K] being 40:1  In these cond, themembrane potential is -65 to -70  Bernstein’s hypothesis was tested by measuring the resting membrane potential and comparing it with the K eq potential at various extracellular K conc  As with ourmodel cell, these changes would not be expected to produce a sign
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