Slide 1 Membrane Transport Mechanisms II and the Nerve Action Potential Slide 2 Apical Basolateral Epithelia Microvilli Tight junction Basal Lamina Lie on a sheet of connective tissue (basal lamina) Tight Junctional Complexes: Structural Allow paracellular transport Apical membrane; brush border (microvilli) increases surface area Apical (mucosal, brush border, lumenal) and basolateral (serosal, peritubular) membranes have different transport functions Capable of vectorial transport An epithelium is an uninterrupted sheet of cells joined together by a continuous hoop, called the tight junction. Epithelial sheets line the inner and outer surfaces of the body. For example, the GI tract is lined in its entirety by epithelial cells. There are a variety of types of epithelial cell, but they all have a common feature in that they lie on a sheet of connective tissue, termed the basal lamina shown here in pink. The tight junctional complexes we talked about in the first lecture serves two purposes. First, they are attachment points and maintains the integrity of the cell sheet and also will allow the movement of water, solutes and even cells from one body compartment to another. The apical (free) surface of almost all epithelial cells have at least a few short finger-like extensions, called microvilli ("tiny hairs"). Epithelia that are specialized for absorption, like that of the small intestine is, have a brush border, which consists of a large number of these microvilli on each cell. The apical surface of other epithelial cells, like some of the ones lining the trachea, have cilia ("eyelashes"), which are extensions that look a little like microvilli, but are longer and have a different structure and function. Within the cytoplasm at the core of each cilium is a bundle of microtubules.
These can slide over each other when motor proteins (dynein) attached to them are activated by ATP. When they slide, the whole cilium bends and pushes against the fluid at the cell's surface. There are specialized epithelial cells that secrete for example mucous secreting cells. This is one type of secretion, which involves exocytosis. Slide 3 This slide demonstrates the movement of solute and water across the epithelial layer. Water can traverse the lipid bilayer. There are also water channels expressed in the plasma membrane that enhances water flux. Crystallographic structure of the aquaporin 1 (AQP1) channel Slide 4 Models of Ion Transport in Mammalian Cells e.g. Cl - secretory cell Transepithelial potential difference NEGATIVE POSITIVE APICAL/ MUCOSAL SIDE Cl - K + K + Cl - K+ BASOLATERAL/ SEROSAL/ BLOOD SIDE H 2 O Paracellular Transcellular So here is a typical cell model. Lets consider some of the key points to remember. First, the cell is polarized and can be separated into an apical, also called the brush border or mucosal side and basolateral, or serosal side. The apical membrane faces toward the lumen. So, for example, if you imagine your gut is a tube the center of the tube is the lumen and the outside of the tube would face the blood. So here in this cell, the apical membrane is lumenal and the basolateral side faces the blood. Solutes and water can move either between the cells, this is termed paracellular; or through the cells, termed transcellular. The tight junctions play yet another role in polarizing the epithelium and prevent the lateral
movement of ions from the apical side to the basolateral side or vice versa. In addition to membrane potentials, there can also be transepithelial potentials. That is the lumenal side of the cell can have a different potential with respect to the blood side. This potential can also play a role in the movement of charged molecules. For example in the case where the lumen is negative, a positively charged ion will move down its electrical gradient. Lets use the example shown here to introduce how membrane transporters and channels functionally interact to enable NaCl secretion. 1. Active transport Na pump. Na gradient Na moves down its concentration grad via NaKCL. A basolateral K conductance sets the membrane potential of the basolateral membrane. Cl channels on the apical membrane open and allow movement of Cl form the cell to the lumen. Na moves paracellularly following the negatively charged Cl ion. Osmotic movement of water. Net result NaCl secretion. Slide 5 Absorptive Epithelia - e.g. Villus cell of the small intestine -driven glucose symport Lateral domain Carrier protein mediating passive transport of glucose Basal domain (Modified from: Alberts et al., Molecular Biology of the Cell, 4 th Ed. Garland Science, 2002) So now lets consider a cell that is capable of absorbing solute. Lets use the absorption of glucose in the GI tract as our example. Again we have the basolateral energy utilizing Na pump setting up a concentration gradient. The Na concentration gradient set up by the pump enables the influx of glucose via a secondary active transport mechanism the Na-glucose cotransporter/symporter. In this case the glucose concentration is high inside the cell and so glucose is moving against its concentration gradient from low to high. Now we have set up a
concentration gradient for glucose across the basolateral membrane and glucose now wants to move down its concentration gradient via a facilitated diffusion mechanism mediated by glucose transporters on the basolateral membrane. Note these transporters are different from the apical glucose transporters. Note the tight junctions are impermeable to glucose so once on the basolateral side it will stay there. It is worth noting that tight junctions serve many purposes and their integrity must be maintained. For example the tight junctional barrier protects against viral invasion, such as HIV transmission. One example of a situation where the breakdown in the barrier can be life threatening is when STDs affect the integrity of the barrier and allow viral entry. (Dezzutti CS. Mechanisms of HIV Transmission through Epithelial Cell Barriers. 12th World AIDS Conference. Geneva, June/July 1998). It is also worth noting that the paracellular pathway and the fibers of the tight junctions that make up the junctions are not simply a random mesh, rather they contain specialized proteins. One such example is paracellin 1, a renal tight junction protein that is important for renal Mg absorption.
Slide 6 Electrophysiological Technique: Patch Clamp Slide 7 Measuring Membrane Potentials Slide 8 Terminology and Electrophysiological Conventions Membrane potential (Vm) +100 mv Depolarize 0 mv -100 mv Hyperpolarize (Positive) OUTWARD I CURRENT -100 mv Reversal Potential (I=0) (Negative) V +100 mv INWARD CURRENT Here I introduce some of the terminology that you will hear. As the cell membrane potential becomes more positive it is said to depolarize. Conversely, as the cell membrane potential becomes more negative, the membrane potential is said to hyperpolarize. On the right hand side is a schematic of a current-voltage relationship. Much can be learned about the electrophysiological properties of either whole cells, portions of a cell membrane or single ion channels and transporters using the techniques we discussed in the previous slide. You may come across these IV relationships. Much information is obtained from such a graph. As the current goes from inward to outward the line traverses the X axis - the point at which this occurs is called the reversal potential i.e. the point at which the current is zero.
Slide 9 Diffusion of electrolytes through membrane channels The following are three important features of ion channels that influence flux : 1) Open probability (Po). Opening and closing of channels are random processes. The Po is the probability that the channel is in an open state. 2) Conductance. 1/R to the movement of ions. Where V=IR (Ohms law) from Byrne, J.H. & Shultz, S.G. An introduction to membrane transport and bioelectricity, 2 nd ed., 1994. 3) Selectivity. The channel pore allows only certain ions to pass through. The slope of the line is used to determine the conductance of an electrophysiological process. This can be for either macroscopic currents or single channel. The slope of the line when studying macroscopic currents in a cell is a combination of all individual conductances in the cell. The reversal potential would be determined using the GHK equation. For a single channel, the slope of the line is used to determine the conductance of an individual channel. The reversal potential in this case would be used to determine the selectivity of the channel being studied. Another term you may come across is open probability. This is the probability that a channel is in an open state. These three are characteristics that determine the overall flux - open probability, conductance and selectivity. In the next slide we will see how if single channel behavior changes, how this influences flux through a channel.
Slide 10 Potassium Channel Slide 11 Common Gating Modes of Ion Channels Voltage-gating is only one way in which channels are opened. Other methods include ligand gating and mechanical gating. (Source: Alberts et al., Molecular Biology of the Cell, 4 th Ed. Garland Science, 2002) Slide 12
Slide 13 How the behavior of an ion channels can be modified to permit an increased ion flux: Control/ Wild-type: Closed state Open state An increase in conductance (more current flows/opening) but the open probability stays the same: Closed state Open state An increase in open probability (the channel spends more time in the open state, or less time in the closed state) but the conductance stays the same: Closed state Open state Here pink is used to represent the closed state of the channel, downward deflections are channel openings. Thus blue represents the open state. Modification of the channel protein, for example by phosphorylation can alter the channel conductance. Thus the deflections would become larger. Another way that ionic flux could be increased is to increase the channel open probability as shown on the lower figure. Here the channel spends less time in the closed state and more time in the open state, thus for a given period of time, more ions would be permitted to flow. Slide 14 Ionic currents through a single channel sum to make macroscopic currents VOLTAGE- dependent closure TIME- dependent closure Channel K + Channel VOLTAGE-GATED GATED CHANNELS Shown here are two examples of voltagegated channels at the macroscopic and single channel level. On the left is a recording of voltage-gated Na channel activity. A stepwise change in the potential induces these voltage-gated channels to open and a sudden increase in macroscopic current is observed. If one records these channels in the cell, many of these channels open together and a macroscopic current is elicited in response to this voltage pulse. The lower panel shows the activity of a single channel, where downward deflections represent inward current through the channel. Notice in this case, that the channels spontaneously begin to close in a time-dependent manner, illustrated clearly in the macroscopic current trace. Later we will be discussing the generation of the action potential and the importance of this channel behavior will become apparent. On the right hand side is an example of a voltage-gated K channel. Again a stepwise change in
potential elicits a channel opening, but notice here the channel only closes down when the potential is stepped back to its original value. Here openings are upward, representing outward K movement. Thus closure of this channel is voltage, not time dependent. Slide 15 The resting membrane potential (V m ) describes a steady state condition with no flow of electrical current across the membrane. V m depends at any time depends upon the distribution of permeant ions and the permeability of the membrane to these ions relative to the Nernst equilibrium potential for each. Goldman- -Hodgkin-Katz Equation We are now going to discuss the action potential of a nerve. Before we do so, let s remember a couple of key points. One: The resting membrane potential is when the cell is in a steady state condition and there is now net charge flow. This potential depends upon the relative permeability of the cell in question to any permeant ion. The GHK equation is used to determine the membrane potential relative to each ion s permeability. Slide 16 Membrane Potential (mv) -80 Resting potential The Nerve Action Potential 20 0-20 -40-60 Depolarizing phase Recorded from a rat hippocampal neuron: CMB Overshoot Threshold Repolarizing Phase -5 0 5 10 15 20 Time (ms) After-hyperpolarization Certain types of cells are called excitable, that is if the membrane potential is depolarized beyond a certain level, a large potential change ensues and an action potential can be elicited. This is an action potential recorded from a neuron illustrating several features. First, the resting potential of the cell is negative. A slow depolarization raises the membrane potential to a threshold level at which time an action potential is elicited in an all-ornone manner. The membrane potential rapidly depolarizes and becomes more and more positive reaching a crest known as the overshoot. The cell potential then repolarizes, progressively becoming more negative. For a period of time the cell is even more negative than at rest, this phase of the action potential is known as the after-hyperpolarization.
Slide 17 Slide 18 Changes in the underlying conductance of and K + underlie the nerve action potential from:boron, W.F. & Boulpaep, E.L., eds., Medical Physiology, 2003. Two types of conductance underly the nerve action potential. We will discuss these in more detail shortly. First a Na conductance which is responsible for the upstroke of the action potential and second a K conductance which is responsible for repolarization of the cell. We will consider each of these in due course but first let s consider the underlying forces on sodium and potassium. Slide 19 Chemical and electrical gradients prior to initiation of an action potential K + + At rest, the cell membrane potential (V m-rest ) is generated by ion gradients established by the Na- pump. The K + conductance (permeability) is high, conductance is extremely low, hence V m-rest is strongly negative. We learned how the Na pump generates an inwardly directed Na gradient and an outwardly directed K gradient. It is important to remember that the Na pump only generates the gradients, it is the presence of ion channels that leads to the generation of membrane potentials. So at rest, the neuron has a negative potential - closer to the reversal potential of K, which is approximately -90mV, than Na which has a reversal potential of approximately +70mV. There is also a Cl conductance which contributes to the resting potential.
Thus there are both chemical and electrical forces that can influence ion movement. NOTE: the electrogenic nature of the Na-pump contributes only about 10% to the membrane potential. The remaining 90% depends on the pump indirectly. Slide 20 A stimulus raises the intracellular potential to a threshold level and voltage-gated channels open instantaneously Stimulus Na+ + + + + + + + + + 1. The membrane becomes permeable to and there is a rapid influx due to due to both electrical and chemical gradients. The cell membrane potential becomes progressively, but rapidly, more positive - i.e. it depolarizes As the cell is stimulated, the cell membrane potential is depolarized and reaches a threshold potential (approximately -50 - -20 mv ) which leads to the opening of voltage-gated Na channels. There is a large concentration gradient for Na to flow into the cell and also a strong electrical influence for positive charge movement into the cell given the negative resting membrane potential. Thus under the influence of both strong concentration and electrical gradients as soon as the Na channels open Na moves into the cell and the cell depolarizes. Slide 21 Membrane Potential (mv) 20 0-20 -40-60 -80 0 5 10 15 20 Time (ms) K + Cl - The rapid upstroke, or depolarizing phase, is due to an increase in conductance of the cell membrane due to activation of voltage-gated channels. An all-or-none response. The cell potential moves toward E Na due to chemical and electrical driving forces. It does not reach E Na. -100-50 0 +50 +100 +150 E ion Thus, the inward movement of Na results in the upstroke of the action potential. The cell membrane potential moves toward Ena because of the increase in permeability as Na channels open. The potential does not quite reach Ena however because the these channels exhibit time dependent closure, or inactivation, as we discussed earlier. A the same time the Na channels are beginning to inactivate, voltage-gated K channels activate. The net result is that the membrane potential
never reaches Ena. Slide 22 + + + + 2. channels + + + + begin to close: + + + K + + + + + + 4. Outward K + flux as voltagedependent K+ channels open hyperpolarization K + K + - - - - - - - - K + 3. Outward K + gradient K + 5. Cell repolarizes So here at stage 2 the Na channels are inactivating. The cell is now depolarized, so lets consider the forces on K. Remember the Na pump sets up a large concentration gradient for K. In addition as the membrane potential has become positive due to the efflux of Na, there is an additional electrical gradient that favors the movement of positive charge out of the cell. Thus as the voltage-gated K channels open, there are both concentration and electrical gradients for K efflux. These channels open in response to depolarization and the net result is repolarization of the cell. Slide 23 Membrane Potential (mv) 20 0-20 -40-60 -80 0 5 10 15 20 Time (ms) As the cell depolarizes, the channels inactivate and the permeability to is reduced. Voltage-gated K + channels open and the cell membrane potential becomes permeable to K + thereby driving V m toward E K. The continued opening of K + channel causes a brief afterhyperpolarization before the cell returns to its resting membrane potential. K + Cl - Ca 2+ -100-50 0 +50 +100 +150 E ion Thus the repolarization of the cell in response to increased K efflux and decreased Na influx is shown here in purple. Now the cell is highly permeable to K and the cell membrane potential moves toward EK. Unlike the Na channels that closed in a time dependent manner, the K channels close in response to membrane potential. As a result the cell moves very close to EK and becomes strongly hyperpolarized. For a brief time there is a hyperpolarization of the cell membrane
potential beyond the resting membrane potential.this is known as an afterhyperpolarization. Eventually, these voltage-gated K channels close and the cell membrane potential returns to its resting state. Slide 24 Gates Regulating Ion Flow Through Voltage-gated gated Channels DEPOLARIZING V m REST ACTIVATED (UPSTROKE) INACTIVATED out REPOLARIZATION HYPERPOLARIZATION in Activation gate Inactivation gate Slide 25 REFRACTORY PERIODS During RP the cell is incapable of eliciting a normal action potential Absolute RP (ARP): no matter how great the stimulus an AP cannot be elicited. channel inactivation gate is closed. Relative RP (RRP): Begins at the end of the absolute PR and overlaps with the afterhyperpolarization. An action potential can be elicited but a larger than normal stimulus is required to bring the cell to threshold.