5. Ion Channels Flashcards
What does it mean to say that the ion channels of nerve cells are heterogeneous?
In different parts of the nervous system different types of channels carry out specific signalling tasks.
What often results from the malfunctioning of ion channels?
Because of their key roles in electrical signalling, malfunctioning of ion channels can cause a wide variety of neurological diseases.
Why do ion channels play a crucial role in both the normal physiology and pathophysiology of the nervous system?
Ion channels are often the site of action of drugs, poisons, or toxins. Thus ion channels have crucial roles in both the normal physiology and pathophysiology of the nervous system.
In addition to ion channels, nerve cells contain a second important class of proteins with a similar function. What are these?
In addition to ion channels, nerve cells contain a second important class of proteins specialised for moving ions across cell membranes, the ion trans- porters or pumps.
What role do these ion pumps play in neuronal signalling?
These proteins do not participate in rapid neuronal signalling but rather are important for establishing and maintaining the concentration gradients of physiologically important ions between the inside and outside of the cell.
What three important properties do ion channels have?
(1) They recognise and select specific ions
(2) They open and close in response to specific electrical, mechanical, or chemical signals
(3) They conduct ions across the membrane.
What causes the large flow if electric charge during an action potential?
The channels in nerve and muscle conduct ions across the cell membrane at extremely rapid rates, thereby providing a large flow of electric charge. Up to 100 million ions can pass through a single channel each second. This current causes the rapid changes in membrane potential required for signalling
What is a surprising feature of these ion channels given the fast turnover rate?
In light of such an extraordinary rate of ion flow, channels are surprisingly selective for the ions they allow to permeate. Each type of channel allows only one or a few types of ions to pass.
How do ion channels ‘know’ when to open?
Many channels open and close in response to a specific event: Voltage-gated channels are regulated by changes in membrane potential, ligand-gated channels by chemical transmitters, and mechanically gated channels by pressure or stretch. However, some channels are normally open in the cell at rest. The ion flux through these “resting” channels contributes significantly to the resting potential.
How are ion channels limited in a manner in which ion pumps are not?
Ion channels are limited to catalyzing the passive movement of ions down their thermodynamic concentration and electrical gradients. For example, Na+ ions enter a cell through voltage-gated Na+ channels during an action potential because the external Na+ concentration is much greater than the internal concentration; the open channels allow Na+ to diffuse into the cell down its concentration gradient. With such passive ion movement the Na+ concentration gradient would eventually dissipate were it not for
ion pumps.
Name four ions in which ion pumps manage
Different types of ion pumps maintain the concentration gradients for Na+ , K+ , Ca 2+ , Cl- , and other ions.
These pumps differ from ion channels in two important details. Describe these
First, whereas open ion channels have a continuous water-filled pathway through which ions flow unimpeded from one side of the membrane to the other, each time a pump moves an ion, or a group of a few ions, across the membrane, it must undergo a series of conformational changes. As a result, the rate of ion flow through pumps is 100 to 100,000 times slower than through channels.
Second, pumps that maintain ion gradients use energy, often in the form of adenosine triphosphate (ATP), to transport ions against their electrical and chemical gradients. Such ion movements are termed active transport.
Describe the plasma membrane of nerve cells
The plasma membrane of all cells, including nerve cells, is approximately 6 to 8 nm thick and consists of a mosaic of lipids and proteins. The core of the membrane is formed by a double layer of phospholipids. Embedded within this continuous lipid sheet are integral membrane proteins, including ion channels.
How does the lipid membrane differ to that of the ions within and outside in terms of what they attract?
The lipids of the membrane do not mix with water—they are hydrophobic. In contrast, the ions within the cell and those outside strongly attract water molecules—they are hydrophilic.
Why do the ions attract water?
Ions attract water because water molecules are dipolar: Although the net charge on a water molecule is zero, charge is separated within the molecule. The oxygen atom in a water molecule tends to attract electrons and so bears a small net negative charge, whereas the hydrogen atoms tend to lose electrons and therefore carry a small net positive charge. As a result of this unequal distribution of charge, positively charged ions (cations) are strongly attracted electrostatically to the oxygen atom of water, and negatively charged ions (anions) are attracted to the hydrogen atoms. Similarly, ions attract water; in fact they become surrounded by electrostatically bound waters of hydration
What is meant by ‘electrostatic’? **
relating to stationary electric charges or fields as opposed to electric currents.
Can an ion move away from water into the noncharged hydrocarbon tails of the lipid bilayer in the membrane? Why not or how?
An ion cannot move away from water into the noncharged hydrocarbon tails of the lipid bilayer in the membrane unless a large amount of energy is expended to overcome the attraction between the ion and the surrounding water molecules. For this reason it is extremely unlikely that an ion will move from solution into the lipid bilayer, and therefore the bilayer itself is almost completely impermeable to ions.
Why can ion channel selectivity not be based solely on the diameter of the ion?
Selectivity cannot be based solely on the diameter of the ion because K , with a crystal radius of 0.133 nm, is larger than Na+ (crystal radius of 0.095 nm).
How can a water-filled channel conduct ions at high rates and yet be selective? How, for instance, does a channel allow K+ ions to pass while excluding Na+ ions?
Because ions in solution are surrounded by waters of hydration, the ease with which an ion moves in solution (its mobility or diffusion constant) depends on the size of the ion together with the shell of water surrounding it. The smaller an ion, the more highly localised is its charge and the stronger its electric field. As a result, smaller ions attract water more strongly. Thus, as Na+ moves through solution its stronger electrostatic attraction for water causes it to have a larger water shell, which tends to slow it down relative to K .
Because of its larger water shell, Na+ behaves as if it is larger than K+ . In fact, there is a precise relationship between the size of an ion and its mobility in solution: the smaller the ion, the lower its mobility. We therefore can construct a model of a channel that selects K+ rather than Na+ simply on the basis of the interaction of the two ions with water in a water-filled channel
Although this model explains how a channel can select K+ and exclude Na+ , where does it fall short? How has this developed since?
It does not explain how a channel could select Na+ and exclude K+ . Moreover, the model cannot account quantitatively for the very high ionic selectivity exhibited by biological K+ channels.
Even though we now know that ions can cross membranes by means of a variety of transport macro-molecules, the Na -K pump being a well-characterised example, many properties of membrane ion conductances do not fit the carrier model.
What is the most important characteristic of ion channels which is not accounted for in this model? How do we know of this characteristic?
Most important is the rapid rate of ion transfer across membranes. This transfer rate was first examined in the early 1970s by measuring the transmembrane current initiated when the neurotransmitter acetylcholine (ACh) binds its receptor in the cell membrane of skeletal muscle fibers at the synapse between nerve and muscle. Using measurements of membrane current noise, small statistical fluctuations in the mean ionic current induced by ACh, Bernard Katz and Ricardo Miledi concluded that the current conducted by a single ACh receptor is 10 million ions per second. In contrast, the Na+ -K+ pump transports at most 100 ions per second.
What is this rate of transport evidence against?
If the ACh receptor acted as a carrier, it would
have to shuttle an ion across the membrane in 0.1 μs
(one ten-millionth of a second), an implausibly fast
rate. The 100,000-fold difference in rates strongly suggests that the ACh receptor (and other ligand-gates receptors) must conduct ions through a channel.
But we are still left with the problem of what makes a channel selective. How did Bertil Hille explain selectivity?
To explain selectivity, Bertil Hille extended the pore theory by proposing that channels have narrow regions that act as molecular sieves. At this selectivity filter an ion must shed most of its waters of hydration to traverse the channel; in their place weak chemical bonds (electrostatic interactions) form with polar (charged) amino acid residues that line the walls of the channel. Because shedding its waters of hydration is energetically unfavourable, the ion will traverse a channel only if its energy of interaction with the selectivity filter compensates for the loss of the energy of interaction with its waters of hydration. Ions traversing the channel are normally bound to the selectivity filter for only a short time (less than 1 μs), after which electrostatic and diffusional forces propel the ion through the channel. In channels where the pore diameter is large enough to accommodate several water molecules, an ion need not be stripped completely of its water shell.
How is this chemical recognition and specificity of ion channels established? Describe one theory
According to one theory was developed in the early 1960s by George Eisenman, a binding site with a high negative field strength—for example, one formed by negatively charged carboxylic acid groups of glutamate or aspartate — will bind Na+ more tightly than K+. This selectivity results because the electrostatic interaction between two charged groups, as governed by Coulomb’s law, depends inversely on the distance between the two groups.
Because Na has a smaller crystal radius than K , it will approach a negative site more closely than K+ and thus will derive a more favourable free-energy change on binding. This compensates for the requirement that Na+ lose some of its waters of hydration to traverse the narrow selectivity filter. In contrast, a binding site with a low negative strength—one that is composed, for example, of polar carbonyl or hydroxyl oxygen atoms—would select K over Na .
At such a site the binding of Na+ would not provide a sufficient free-energy change to compensate for the loss of the ion’s waters of hydration, which Na+ holds strongly. However, such a site would be able to compensate for the loss of a K+ ion’s associated water molecules as the larger K+ ions interact more weakly with water.
What is Coloumb’s law? **
According to Coulomb, the electric force for charges at rest has the following properties: Like charges repel each other; unlike charges attract.
He figured out that the force between two charges is equal to some constant times the first charge times the second charge divided by the distance between them squared.
What is charge? **
It’s a property of objects and particles like mass. We know that like repels like and opposites attract but eve the names positive and negative are arbitrary. o one fundamentally knows what it is.
Is the current thinking that ion channels are selective because of specific chemical interactions or because of molecular sieving based on pore diameter?
It is currently thought that ion channels are selective both because of specific chemical interactions and because of molecular sieving based on pore diameter.
X-ray crystallographic and other structural analy- ses have been informative in the study of enzymes and other soluble proteins. Why have they only recently bee applied to membrane proteins?
They have only recently been applied to integral membrane proteins, such as ion channels, because their transmembrane hydrophobic regions make them difficult to crystallise.
What alternative method has been informative regarding the structure of these channels?
For the past 35 years single-channel recording has provided important functional information that has yielded significant insights into channel structure.
Before it became possible to resolve the small amount of current that flows through a single ion channel in biological membranes, how was channel function studied?
Before it became possible to resolve the small amount of current that flows through a single ion channel in biological membranes, channel function was studied in artificial lipid bilayers.
Describe how Paul Mueller and Donald Rudin developed a technique for studying lipid bilayers in the early 60’s
In the early 1960s Paul Mueller and Donald Rudin developed a technique for forming functional lipid bilayers by painting a thin drop of phospholipid over a hole in a nonconducting barrier that separates two salt solutions.
How did Paul Mueller and Donald Rudin study the permeability of this artificial lipid bilayer? What did they find?
Although lipid membranes are highly impermeable to ions, ionic permeability of the membrane increases dramatically when certain peptide antibiotics are added to the salt solution. Early studies with gramicidin A, a 15-amino acid cyclic peptide, were especially informative. Application of low concentrations of gramicidin A brings about small step- like changes in current across the membrane. These brief pulses of current reflect the all-or-none opening and closing of the single ion channel formed by the peptide.
What is the relationship between the current through a single gramicidin channel and the membrane potential?
The current through a single gramicidin channel varies with membrane potential in a linear manner; that is, the channel behaves as a simple resistor.
How can the amplitude of the single-channel current be obtained?
The amplitude of the single-channel current can thus be obtained from Ohm’s law, i = V/R, where i is the current through the single channel, V is the voltage across the membrane, and R is the resistance of the open channel.
What is the approximate resistance of a single open channel?
The resistance of a single open channel is approximately 8 × 10^10 ohms
We don’t usually speak of resistance when it comes to channels, what do we speak of instead and why?
In dealing with ion channels it is more useful to speak of the reciprocal of resistance or conductance (γ = 1/R), as this provides an electrical measure related to ion permeability.
How can conductance be used in Ohm’s law?
Thus, Ohm’s law can be expressed as
i = γ × V
What is the approximate conductance of the gramicidin A channel?
The conductance of the gramicidin A channel is approximately 12 × 10^−12 siemens (S), or 12 picosiemens (pS), where 1 S equals 1/ohm.
Have basic channel properties obtained from artificial membranes been confirmed? What is preventing this, or how has it been confirmed?
The insights into basic channel properties obtained from artificial membranes were later confirmed in biological membranes by the patch-clamp technique, developed by Erwin Neher and Bert Sakmann in 1976
Describe how the patch clamp technique was first applied to obtain evidence for these channel properties
A glass micropipette containing ACh—the neurotransmitter that activates the ACh receptors in the membrane of skeletal muscle—was pressed tightly against a frog muscle membrane. Small unitary current pulses representing the opening and closing of a single ACh receptor channel were recorded from the area of the membrane under the pipette tip. As with gramicidin A channels, the relation between current and voltage in the ACh receptor channel is linear, with a single-channel conductance of approximately 25 pS. This generates a unitary current of 2 pA (picoamperes) at a membrane potential of −80 mV (millivolts), which corresponds to a flux of around 12.5 million ions per second.
Christopher Miller independently developed a method for incorporating channels from biological membranes into artificial lipid bilayers. Describe this method
Biological membranes are first homogenised in a laboratory blender; centrifugation (a technique used for the separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed) of the homogenate then separates out a portion composed only of membrane vesicles. Under appropriate ionic conditions these membrane vesicles will fuse with a planar lipid membrane, incorporating any ion channel in the vesicle into the planar membrane.
This technique of incorporating channels from biological membranes into artificial lipid bilayers has two experimental advantages. Describe them
First, it allows recording from ion channels in regions of cells that are inaccessible to patch clamp; for example, Miller has successfully studied a K+ channel isolated from the internal membrane of skeletal muscle (the sarcoplasmic reticulum).
Second, it allows researchers to study how the composition of the membrane lipids influences channel function.
To what extent are all cells capable of signalling?
Most cells are capable of local signalling, but only nerve and muscle cells are specialised for rapid signalling over long distances.
How is the direction of the flux of ions through a channel determined?
The direction and eventual equilibrium for this flux are determined not by the channel itself, but rather by the electrostatic and diffusional driving forces across the membrane.
Ion channels allow particular types of ions to cross the membrane, selecting either cations or anions to permeate.
What are cations and anions?
Cation: a positively charged ion, i.e. one that would be attracted to the cathode in electrolysis.
Anion: Anions are ions that are negatively charged. Ions are charged atoms or molecules. If a balanced atom loses one or more electrons, it will become a positively charged cation.
What ions can pass through cation-selective channels? (4)
Some types of cation-selective channels
allow the cations that are usually present in extracellular fluid—Na+ , K+ , Ca2+ , and Mg2+ —to pass almost indiscriminately. However, many other cation- selective channels are permeable primarily to a single type of ion, whether it is Na+, K+ ,or Ca 2+.
What are most anion selective channels selective for?
Most types of anion-selective channels are also highly discriminating; they conduct only one physiological ion, chloride (Cl−).
How are the kinetic properties of ion permeation best described?
The kinetic properties of ion permeation are best described by the channel’s conductance, which is deter- mined by measuring the current (ion flux) through the open channel in response to an electrochemical driving force.
How is the net electrochemical driving force determined?
The net electrochemical driving force is determined by two factors: the electrical potential difference across the membrane and the concentration gradients of the permeant ions across the membrane. Changing either one can change the net driving force
Describe the relationship between the current and the driving force in ion channels in regards to their function characteristics
As we have seen, in some open channels the cur- rent varies linearly with driving force—that is, the channels behave as simple resistors. In others the cur- rent is a nonlinear function of driving force. This type of channel behaves as a rectifier—it conducts ions more readily in one direction than in the other because of asymmetry in the channel’s structure or environment. Whereas the conductance of a resistor-like channel is constant—it is the same at all voltages—the conductance of a rectifying channel is variable and must be determined by plotting current versus voltage over the entire physiological range of membrane potential (In figures)
How does the net electrochemical driving force depend on concentration gradients of the permeant ions across the membrane?
The rate of net ion flux (current) through a channel depends on the concentration of the permeant ions in the surrounding solution. At low concentrations the current increases almost linearly with concentration. At higher concentrations the current tends to reach a point at which it no longer increases. At this point the current is said to saturate.
This saturation effect is consistent with what idea involving transmembrane current?
This saturation effect is consistent with the idea that ion flux across the cell membrane is not strictly a function of laws of electrochemical diffusion in free solution but also involves the binding of ions to specific polar sites within the pore of the channel. A simple electrodiffusion model would predict that the ionic current should continue to increase as long as the ionic concentration also increases; that is, the more charge carriers in solution, the greater the current.
The relation between current and ionic concentration for a wide range of ion channels is well described by what equation? What could this suggest?
The relation between current and ionic concentration for a wide range of ion channels is well described by a simple chemical binding equation, identical to the Michaelis-Menten equation for enzymes, suggesting that a single ion binds within the channel during permeation. The ionic concentration at which current reaches half its maximum defines the dissociation constant, the concentration at which half of the channels will be occupied by a bound ion.
How strong is the binding between the ion and the channel and how do we know?
The dissociation constant in plots of current versus concentration is typically quite high, approximately 100 mM, indicating weak binding. (In typical interactions between enzymes and substrates the dissociation constant is below 1 μM.)
What does this weak binding of the ion and ion channel suggest?
This weak interaction indicates that the bonds between the ion and the channel are rapidly formed and broken. In fact, an ion typically stays bound in the channel for less than 1 μs. The rapid rate at which an ion unbinds is necessary for the channel to achieve the very high conduction rates responsible for the rapid changes in membrane potential during signalling.
What is meant by the term occlusion and how is it relevant to ion channels?
Some ion channels are susceptible to occlusion (the blockage or closing of a blood vessel or hollow organ) by certain free ions or molecules in the cytoplasm or extracellular fluid. Passage through the channel can be blocked by particles that bind either to the mouth of the aqueous pore or somewhere within the pore. If the blocker is an ionised molecule that binds to a site within the pore, it will be influenced by the membrane electric field as it enters the channel
Give an example of how free ion blocking a channel can be influenced by the membrane electric field as it enters the channel
If a positively charged blocker enters the channel from outside the membrane, then making the cytoplasmic side of the membrane more negative—which, according to convention, corresponds to a more negative membrane potential—will drive the blocker into the channel through an electrostatic attraction, increasing the block.
Where do they ion channel blockers typically originate?
Although blockers are often toxins or drugs that originate outside the body, some are common ions that are normally present in the cell or its environment
Name four examples of physiological blockers of certain classes of channels.
Physiological blockers of certain classes of channels include Mg 2+, Ca 2+, Na +, and polyamines such as spermine.
What has been observed in every ion channel so far studied concerning its states?
In all ion channels so far studied the channel protein has two or more conformational states that are relatively stable. Each of these stable conformations represents a different functional state. For example, each ion channel has at least one open state and one or two closed states.
What is the transition of a channel between these different states
The transition of a channel between these different states is called gating.
Assess how much we know about the molecular mechanisms of gating
Relatively little is known about the molecular mechanisms of gating. Although the picture of a gate swinging open and shut is a convenient image, it probably is accurate only for certain cases (for example the inactivation of Na+ and K+ channels)
Instead of the concept of the gate swinging open and shut, what is a more common gating mechanism?
More commonly, channel gating involves widespread changes in the channel’s conformation. For example, evidence from high-resolution electron microscopy and image analysis suggests that the opening and closing of gap junction channels involve a concerted twisting and tilting of the six subunits that make up the channel.
What has similar evidence unveiled about the gating of ACh and K+ channels? (2)
Similar evidence indicates that ACh receptor channels open through a coordinated twisting and bending of the α-helixes of each of the five subunits that form the channel pore.
In K+ channels movement of a ring of α-helixes that forms the internal mouth of the pore is thought to act as a gate.
In what two ways is it proposed that the molecular rearrangements of ion channels enhance ion conduction?
The molecular rearrangements that occur during the transition from closed to open states appear to enhance ion conduction through the channel not only by creating a wider lumen, but also by positioning relatively more polar amino acid constituents at the surface that lines the aqueous pore.
What is the primary function of ion channels?
The primary function of ion channels in neurons is
to generate transient electrical signals
What three major gating mechanisms have evolved to control channel opening?
Ligand gating: Certain channels are opened by the binding of chemical ligands, known as agonists.
Voltage gating: Other ion channels are regulated by changes in membrane potential.
Stretch or pressure gating: Some channels are regulated by mechanical stretch of the membrane
Where do ligands bind in order to activate gating? (3)
Some ligands bind directly to the channel either at an extracellular or intracellular site; transmitters bind at extracellular sites whereas certain cytoplasmic constituents, such as Ca 2+, cyclic nucleotides and GTP-binding proteins, bind at intracellular sites.
Other ligands activate cellular signalling cascades, which can covalently modify a channel through protein phosphorylation.
(Diagram in docs)
Give an example of of voltage-gated channels besides those activated by an action potential or PSP
Some voltage-gated channels act as temperature sensors; changes in temperature shift their voltage gating to higher or lower membrane potentials, giving rise to heat- or cold-sensitive channels.
The rapid gating actions necessary for moment-to- moment signalling may also be influenced by certain long-term changes in the metabolic state of the cell. Give an example of how this may be the case
The gating of some K+ channels is sensitive to intracellular levels of ATP. Some channel proteins contain a subunit with an integral oxidoreductase catalytic domain that is thought to alter channel gating in response to the redox state of the cell.
The previous answer asserted the following:
The gating of some K+ channels is sensitive to intracellular levels of ATP. Some channel proteins contain a subunit with an integral oxidoreductase catalytic domain that is thought to alter channel gating in response to the redox state of the cell.
What is meant by:
1) an integral oxidoreductase catalytic domain
2) the redox state of the cell **
1) An oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. A catalytic domain is the region of an enzyme that interacts with its substrate to cause the enzymatic reaction.
2) The redox state can be defined as an equilibrium state between oxidants (substances that take an electron from other molecules such as free radicals) and anti oxidants (Any substance that can inhibit the oxidation of other compounds).
Why is the concept of a redox state in a cell relevant? **
Oxidants, including free radicals and other reactive species, are continuously produced in the cell. As it is impossible to completely prevent oxidant production, several antioxidant systems have evolved in the cell. In order to maintain a healthy status, oxidants and antioxidants should be in equilibrium. However, this equilibrium is very difficult to maintain in the cell.
What happens when this equilibrium state between oxidants and antioxidants is disrupted in a cell? **
When this equilibrium between oxidant and antioxidant is disrupted, tilting the equilibrium toward an oxidised state, oxidative stress is produced. Oxidative stress is involved in the physiopathology of several diseases, including cardiovascular disease, cancer, diabetes, and many others.
These regulators control the entry of a channel into one of what three functional states?
Closed and activatable (resting), open (active), or closed and nonactivatable (refractory).
What is required for this change in the functional state of a channel?
A change in the functional state of a channel requires energy.
How is this energy provided in voltage gated channels?
In voltage-gated channels the energy is provided by the movement of a charged region of the channel protein through the membrane’s electric field. This region, the voltage sensor, contains a net electric charge because of the presence of basic (positively charged) or acidic (negatively charged) amino acids. The movement of the charged voltage sensor through the electric field imparts a change in free energy to the channel that alters the equilibrium between the closed and open states of the channel. For most voltage-gated channels, channel opening is favoured by making the inside of the membrane more positive (depolarisation).
How is gating driven in transmitter-gated channels?
In transmitter-gated channels gating is driven by the change in chemical-free energy that results when the transmitter binds to a receptor site on the channel protein.
How is energy provided in mechanically activated channels?
For mechanically activated channels the energy associated with membrane stretch is thought to be transferred to the channel either through the cytoskeleton or more directly by changes in tension of the lipid bilayer.
The stimuli that gate the channel also control the rates of transition between the open and closed states of a channel. Describe how this is true for voltage-gated channels
For voltage-gated channels the rates are steeply dependent on membrane potential. Although the time scale can vary from several microseconds to a minute, the transition tends to require a few milliseconds on average. Thus, once a channel opens it stays open for a few milliseconds before closing, and after closing it stays closed for a few milliseconds before reopening. Once the transition between open and closed states begins, it proceeds virtually instantaneously (in less than 10 μs, the present limit of experimental measurements), thus giving rise to abrupt, all-or-none, step-like changes in current through the channel.
How do ligand-gated and voltage-gated channels enter refractory states through different processes than voltage-gated channels?
Ligand- gated channels can enter the refractory state when their exposure to the agonist is prolonged. This process is called desensitisation.
Many, but not all, voltage-gated channels enter a refractory state after opening, a process termed inactivation. In the inactive state the channel is closed and can no longer be opened by positive voltages. Rather, the membrane potential must be returned to its initial negative resting level before the channel can recover from inactivation so that it can again open in response to depolarisation.
Describe the mechanisms underlying desensitisation
The mechanisms underlying desensitisation of ion channels are not yet completely understood. In some channels desensitisation appears to be an intrinsic property of the interaction between ligand and channel, although in others it is a result of phosphorylation of the channel molecule by a protein kinase.
In voltage-gated Na+ and K+ channels, what is channel inactivation thought to result from? How is this different to Ca 2+ channels?
In voltage-gated Na+ and K+ channels inactivation is thought to result from a conformational change, controlled by a subunit or region of the channel separate from that which controls activation.
In contrast, the inactivation of certain voltage-gated Ca2+ channels is thought to require Ca2+ influx. An increase in internal Ca2+ concentration inactivates the Ca2+ channel by binding to the regulatory molecule calmodulin, which is permanently associated with the Ca2+ channel protein
Exogenous factors, such as drugs and toxins, can also affect the gating control sites of an ion channel. Do they typically open or close it?
Most of these agents tend to close the channel but a few open it.
How can the binding of exogenous agents to ion channels work?
Some bind to the same site at which the endogenous agonist normally binds and thereby interfere with normal gating.
Some exogenous substances act in a noncompetitive manner and affect the normal gating mechanism without directly interacting with the transmitter- binding site. For example, binding of the drug diazepam (Valium) to a regulatory site on Cl− channels that are gated by γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, prolongs the opening of the channels in response to GABA
How strong is the binding of exogenous agents to ion channels when binded to the same site?
The binding can be weak and reversible, as in the blockade of the nicotinic ACh- gated channel in skeletal muscle by curare, a South American arrow poison. Or it can be strong and irreversible, as in the blockade of the same channel by the snake venom α-bungarotoxin.
In what type of ion channel are these indirect effects of exogenous agents found?
This type of indirect, allosteric effect is found in voltage- or stretch-gated channels as well.
Biochemical and molecular biological studies have provided a basic understanding of channel structure and function.
Describe our basic understanding of their common structure
All ion channels are large integral-membrane proteins with a core transmembrane domain that contains a central aqueous pore spanning the entire width of the membrane. The channel protein often contains carbohydrate groups attached to its external surface. The pore-forming region of many channels is made up of two or more subunits, which may be identical or different. In addition, some channels have auxiliary subunits that modify their functional properties. These subunits may be cytoplasmic or embedded in the membrane
To what extent do we know the genes responsible for ion channels?
The genes for all the major classes of ion channels have been cloned and sequenced.
What does our ability of cloning and sequencing the genes of ion channels allow for?
The amino Ca2+ acid sequence of a channel, inferred from its DNA sequence, can be used to create a structural model of the channel protein. Regions of secondary structure— the arrangement of the amino acid residues into α-helixes and β-sheets—as well as regions that are likely to correspond to membrane-spanning domains of the channel are predicted based on the structures of related proteins that have been experimentally determined using electron and X-ray diffraction analysis.
What has this genetic analysis of ACh receptor channels identified?
This type of analysis identified the presence of four hydrophobic regions, each around 20 amino acids in length, in the amino acid sequence of a subunit of the ACh receptor channel. Each of these regions is thought to form an α-helix that spans the membrane
How have additional insights into channel structure and function been obtained by comparing the amino acid sequences of the same type of channel from different species?
Regions that show a high degree of similarity (that is, have been highly conserved through evolution) are likely to be important in maintaining the structure and function of the channel. Likewise, conserved regions in different but related channels are likely to serve a common biophysical function. For example, voltage-gated channels selective for different cations have a specific membrane-spanning segment that contains positively charged amino acids (lysine or arginine) spaced at every third position along an α-helix. This motif is observed in all voltage-gated cation channels, but not in transmitter-gated channels, suggesting that this charged region is important for voltage gating.
Once a structure is proposed, what is the next step?
Once a structure for a channel has been proposed, it can be tested in several ways.
Give an example of how the structure of proteins can be tested
Antibodies can be raised against synthetic peptides that correspond to different hydrophilic regions in the protein sequence. Immunocytochemistry can then be used to determine whether the antibody binds to the extracellular or cytoplasmic surface of the membrane, thus defining whether a particular region of the channel is extracellular or intracellular.
The functional consequences of changes in a channel’s primary amino acid sequence can be explored through a variety of techniques. Describe one particularly versatile technique
One particularly versatile approach is to use genetic engineering to construct channels with parts that are derived from the genes of different species—so-called chimeric channels. This technique takes advantage of the fact that the same type of channel can have somewhat differ- ent properties in different species (e.g differences in conductance). By comparing the properties of a chimeric channel to those of the two original channels, one can assess the functions of specific regions of the channel. This technique has been used to identify the membrane-spanning segment that forms the lining of the pore of the ACh receptor channel
Ion channels are integral membrane proteins composed of several subunits. What may these subunits be composed of relative to each other?
Ion channels can be constructed as hetero-oligomers from distinct subunits, as homo-oligomers from a single type of subunit, or from a single polypeptide chain organised into repeating motifs, where each motif functions as the equivalent of one subunit (Docs)
How can the roles of different amino acid residues or stretches of residues be tested?
The roles of different amino acid residues or stretches of residues can be tested using site-directed mutagenesis, a type of genetic engineering in which specific amino acid residues are substituted or deleted.
How can one explore the functions of amino acids and proteins in ion channels without the application of genetic manipulation?
Finally, one can exploit the naturally occurring mutations in channel genes. A number of inherited and spontaneous mutations in the genes that encode ion channels in nerve or muscle produce changes in channel function that can underlie certain neurological diseases. Many of these mutations are caused by localised changes in single amino acids within channel proteins, demonstrating the importance of that region for channel function.
What has underscored the great diversity of ion channels in multicellular organisms?
The great diversity of ion channels in a multicellular organism is underscored by the recent sequencing of the human genome. Our genome contains nine genes encoding variants of voltage-gated Na+ channels, 10 genes for different Ca+ channels, over 75 genes for K+ channels, 70 genes for ligand-gated channels, and more than a dozen genes for Cl− channels.
What does it mean to say that most of the ion channels that have been described in nerve and muscle cells fall into a few gene superfamilies?
Members of each gene superfamily have similar amino acid sequences and transmembrane topology and, importantly, related functions. Each superfamily is thought to have evolved from a common ancestral gene by gene duplication and divergence. Several superfamilies can be further classified into families of genes with more closely related structure and function.
Give three examples of the super gene families and what they encode
One superfamily encodes ligand-gated ion channels that are receptors for the neurotransmitters ACh, GABA, glycine, or serotonin.
Gap-junction channels, which bridge the cytoplasm of two cells at electrical synapses , are encoded by a separate gene superfamily.
The genes that encode the voltage-gated ion channels responsible for generating the action potential
belong to another family.
*A number of other families of channels have been identified, distinct from those considered above.
What do the receptors of the ligand-gated ion channel superfamily have in common?
All of these receptors are composed of five subunits, each of which has four transmembrane α-helixes. In addition, the extracellular domain that forms the receptor for the ligand contains a conserved loop of 13 amino acids flanked by pair of cysteine residues that form a disulfide bond.
What is another name for the ligand-gated ion channels?
The extracellular domain that forms the receptor for the ligand contains a conserved loop of 13 amino acids flanked by pair of cysteine residues that form a disulfide bond. As a result, this receptor superfamily is referred to as the cys-loop receptors.
How can ligand-gated channels be classified?
Ligand-gated channels can be classified by their ion selectivity in addition to their agonist. The genes that encode glutamate receptor channels belong to a separate gene family.
Describe the structure of the gap junction channels encoded by the second gene superfamily
A gap- junction channel is composed of 12 identical subunits, each of which has four membrane-spanning segments. The gap-junction channel is formed from a pair of hemichannels, one each in the pre- and postsynaptic cell membranes that join in the space between two cells. Each hemichannel is made of six identical subunits, each containing four transmembrane α-helixes.
How conserved are the structures of the voltage-gate architectures? Describe a typical one
All voltage-gated channels have a similar architecture, with a core motif composed of six transmembrane segments termed S1–S6. The S5 and S6 segments are connected by an extended strand of amino acids, the P-region, which dips into and out of the external surface of the membrane to form the selectivity filter of the pore.
Describe voltage-gated K+, Na+ and Ca2+ channels
Voltage-gated Na+ and Ca2+ channels are composed of a large subunit that contains four repeats of this basic motif ( six transmembrane segments termed S1–S6, with S5 and S6 connected by P-region). Voltage-gated K+ channels are composed of four separate subunits, each containing one motif. This structure is shared by other, more distantly related channel families
The major gene family encoding the voltage-gated K+ channels is distantly related to two families of K+ channels, each with distinctive properties and structure. Describe these families
One family consists of the genes encoding inward-rectifying K+ channels, which are open at the resting potential and close rapidly during depolarisation. Each channel subunit has only two transmembrane segments connected by a pore-forming P-region. A second family is composed of subunits with two repeated pore-forming segments. These channels may also contribute to the resting K+ conductance.
The sequencing of the genomes of a variety of species has led to the identification of additional ion channel gene families, found in organisms from bacteria to humans. Describe two families that were found which also have a P-region
Channels with related P-regions have been identified that are only very distantly related to the family of voltage-gated channels. These channels include the glutamate-gated channels, in which the P-region is inverted: It enters and leaves the internal surface of the membrane
Finally, the transient receptor potential (TRP) family of nonselective cation channels (named after a mutant Drosophila strain in which light evokes an abnormal transient receptor potential in photoreceptors) comprises a very large group of channels that contain a P-region.
Describe the structure of ion channels in the TRP family
Like the voltage-gated K+ channels, TRP channels also contain six transmembrane segments but are usually gated by intracellular ligands.
What function do TRP family ion channels carry out?
TRP channels are important for Ca 2+ metabolism ( the process by which your body converts what you eat and drink into energy) in all cells, visual signalling in insects, and pain, heat, and cold sensation in the nervous system of higher animals. Recent evidence implicates TRP channels in mechano- sensation and hearing in insects and fish and in certain taste sensations in mammals.
A number of other families of channels have been identified, distinct from those considered previously. Name two of these
These include Cl− channels that help set the resting potential of certain nerve and skeletal muscle cells, and a class of ligand-gated cation channels activated by ATP, which functions as a neurotransmitter at certain synapses.
To what extent has the identification of all major ion channels been completed?
With the completion of the human genome project, it is likely that nearly all of the major classes of ion channel genes have now been identified.
However, the diversity of ion channels is even greater than the large number of ion channel genes. How is this the case? (2)
Because most channels in a given subfamily are composed of multiple subunits, each of which may be encoded by a family of closely related genes, combinatorial permutations of these subunits can generate a diverse array of heteromultimeric (a protein containing two or more different polypeptide chains) channels with different functional properties.
Further diversity can be produced by alternative splicing of precursor mRNA transcribed from a single gene. Finally, the sequence of a transcript can be altered by a process termed RNA editing. As with enzyme isoforms, variants of a channel with slightly different properties may be expressed at distinct stages of development, in different cell types throughout the brain, and even in different regions within a cell. These subtle variations in structure and function presumably allow channels to perform highly specific functions.
Describe research results which indicate that variants of a channel with slightly different properties may be expressed at distinct stages of development. Also describe the underlying biomechanics observed
The image in docs shows examples of currents through individual acetylcholine receptor channels which were recorded from frog skeletal muscle at three stages of development: early (1.1 days), intermediate (2.4 days), and late (48 days).
In immature muscle the single channels have a relatively small conductance and a relatively long open time. In mature muscle the channel conductance is larger and the average open time is shorter. At intermediate stages of development the population of channel variants is mixed; both brief, large-conductance channel openings and longer-lasting, small-conductance channel openings are evident.
This functional difference results from a developmental switch in subunit composition caused by a change in gene expression. Both immature and mature forms of the receptor have α-, β-, and δ-subunits. However, the γ-subunit expressed early in development is replaced by the ε-subunit as the animal matures.
Describe evidence that variants of a channel with slightly different properties may be expressed in different cell types throughout the brain
Autoradiograms in docs show the expression patterns of mRNA transcripts of the four genes comprising the KV3 subfamily of voltage-gated K+ channel subunits (determined by in situ hybridisation). In each autoradiogram the dark areas represent high densities of expression. The brain was sectioned at the level of the posterior thalamus.
How did Rod MacKinnon and his colleagues provide insight into the molecular architecture of ion-selective channels?
Rod MacKinnon and his colleagues provided the first high-resolution X-ray crystallographic analysis of the molecular architecture of an ion-selective channel.
How did MacKinnon et al overcome the difficulties inherent in obtaining crystals of integral membrane proteins
To overcome the difficulties inherent in obtaining crystals of integral membrane proteins, they initially focused on a bacterial K+ channel, termed KcsA. These channels were useful for crystallography as they can be expressed at high levels for purification, are relatively small, and have a simple transmembrane topology that is similar to inward-rectifier K+ channels present in higher organisms, including mammals. . The structure of the channel was further simplified using molecular engineering to truncate cytoplasmic regions that are not essential for forming the ion-selective pore.
What information was provided by the crystal structure determined from the modified KcsA protein?
The crystal structure determined from the modified KcsA protein provides several important insights into the mechanisms by which the channel facilitates the movement of K+ ions across the hydrophobic lipid bilayer.
Describe the broad structure of the potassium channel determined from crystallography
The channel is made up of four identical subunits arranged symmetrically around a central pore (docs). Each subunit has two membrane- spanning α-helixes, an inner and outer helix, that are connected by the P-loop, which forms the selectivity filter of the channel. At the extracellular end of the channel the two α-helixes tilt away from the central axis of the pore so that the structure resembles an inverted teepee.
Describe the cytoplasmic end of the potassium channel determined from crystallography
The four inner α-helixes from each of the subunits line the cytoplasmic end of the pore. At the intracellular mouth of the channel these four helixes cross, forming a very narrow opening—the “smoke hole” of the teepee. The inner helixes are homologous to the S6 membrane- spanning segment of voltage-gated K+ channels.
Describe the extracellular end of the potassium channel determined from crystallography
At the extracellular end of the channel the pair of transmembrane helixes from each subunit are connected by a region consisting of three elements:
(1) a chain of amino acids that surrounds the mouth of the channel (the turret region);
(2) an abbreviated α-helix (the pore helix) approximately 10 amino acids in length that projects toward the central axis of the pore; and
(3) a stretch of 5 amino acids near the C-terminal end of the P-region that forms the selectivity filter
How does the shape and structure of the pore determine its ion-conducting properties?
Both the inner and outer mouths of the pore are lined by acidic amino acids with negative charges that help attract cations from the bulk solution. Going from inside to outside, the pore consists of a medium wide tunnel 18 Å in length that leads into a wider (10 Å diameter) spherical inner chamber. This chamber is lined predominantly by the side chains of hydrophobic amino acids. These relatively wide regions are followed by the very narrow selectivity filter, only 12 Å in length, which is rate- limiting for the passage of ions. A high ion throughput rate is ensured by the fact that the inner 28 Å of the pore, from the cytoplasmic entrance to the selectivity filter, lacks polar groups that could delay ion passage by binding and unbinding the ion.
Where does an ion passing from the polar solution through the non-polar lipid bilayer encounter the least energetically favourable region?
The middle of the bilayer
How is the high energetic cost for a K+ ion to enter this region minimised?
The high energetic cost for a K+ ion to enter this region is minimised by two details of channel structure. The inner chamber is filled with water, which provides a highly polar environment, and the pore helixes provide a dipole whose electronegative carboxyl ends point toward this inner chamber
How is the high energetic cost incurred as a K+ ion sheds its waters of hydration compensated?
The high energetic cost incurred as a K+ ion sheds its waters of hydration is partially compensated by the presence of 20 oxygen atoms that line the walls of the selectivity filter and form favourable electrostatic interactions with the permeant ion. Each of the four subunits contributes four main-chain carbonyl oxygen atoms from the protein backbone and one side-chain hydroxyl oxygen atom to form a total of four binding sites for K+ ions. Each bound K+ ion is thus stabilized by interactions with a total of eight oxygen atoms, which lie in two planes above and below the bound cation.
How is the structure of the ion channel made so that it is attractive for K+ ions to pass through but not other ions such as Na +?
The amino acid side chains of the selectivity filter, which are directed away from the central axis of the channel, help to stabilise the filter at a critical width, such that it provides optimal electrostatic interactions with K+ ions as they pass but is too wide for smaller Na+ ions to interact effectively with all eight carbonyl oxygens atoms at any point along the length of the filter
In light of the extensive interactions between a K+ ion and the channel, how does the KcsA channel manage its high rate of conduction?
Although the channel contains a total of five potential binding sites for K+ ions—four in the selectivity filter and one in the inner chamber—X-ray analysis shows that the channel can be occupied by at most three K+ ions at any instant. One ion is normally present in the wide inner chamber, and two ions occupy two of the four binding sites within the selectivity filter. Because of electrostatic repulsion, two K+ ions never simultaneously occupy adjacent binding sites within the selectivity filter; rather, a water molecule is always interspersed between K+ ions.
Explain how multiple ions in the selectivity filter enhance the conductance speed
During conduction a pair of K+ ions within the selectivity filter hop in tandem between pairs of binding sites. If only one ion were in the selectivity filter it would be bound rather tightly, and the throughput rate for ion permeation would be compromised. But the mutual electrostatic repulsion between two K+ ions occupying nearby sites ensures that the ions will linger only briefly, thus resulting in a high overall rate of K+ conduction.
X-ray crystallographic analysis has also begun to pro- vide insight into the conformational changes that underlie the opening and closing of K+ channels. Studies by Clay Armstrong in the 1960s suggested that a gate exists at the intracellular mouth of voltage-gated K+ channels of higher organisms.
What evidence was found for this?
Small organic compounds such as tetraethylammonium can enter and block the channel only when this internal gate is opened by depolarisation. We now know that this internal gate is the narrow opening formed by the crossing of the four α-helixes at the intracellular mouth of the channel. The small opening at the helix bundle crossing in KcsA revealed by X-ray crystallography turns out to be too narrow to allow ions to pass. Thus the X-ray crystal structure is that of a closed channel.
What do K+ channels look like when they are open?
Although we do not know the answer for KcsA, MacKinnon and his colleagues determined the open structure for a related bacterial K+ channel, MthK. Each subunit of this channel has two transmembrane segments, similar to KcsA. Unlike KcsA, MthK has a cytoplasmic binding domain for Ca2+ and can be opened by high concentrations of internal Ca 2+.
MacKinnon and colleagues determined the structure of the open state by growing crystals of MthK in the presence of Ca 2+. Remarkably, the inner helixes that form the tight bundle crossing in KcsA are bent in MthK at a flexible glycine residue that causes them to splay outward, forming an internal orifice that is dilated to 20 Å in diameter, wide enough to pass K+ as well as larger compounds such as tetraethylammonium
Why is this MthK ion channel opening mechanism likely a general one?
This mechanism is likely to be a general one because the inner helixes of many K+ channels of bacteria and higher organisms have a conserved glycine residue at this position. The presence of a bend at this glycine gating hinge was recently confirmed in the X-ray crystal structure of a mammalian voltage-gated K+ channel.
In what two ways are ion channels distinuished from ion pumps?
(1) they require a source of energy to actively transport ions against their electrochemical gradients
(2) they transport ions at rates much lower than those of ion channels, too low to support fast neuronal signalling.
How (dis)similar are pump and channel structures?
Nevertheless, some types of transporters and certain ion channels may have a similar structure according to studies of a bacterial membrane protein that transports protons and Cl−. These studies also yield insights into the structural basis for Cl− channel selectivity.
A large family of Cl− channels, the ClC channels, is widely expressed in neurons and other cells of vertebrates. What is their function?
In vertebrate skeletal muscle the ClC-1 channels are important in maintaining the resting potential.
What do mutations in the genes responsible for CIC channels result in?
Mutations in the genes for these channels underlie certain inherited forms of myotonia (a neuromuscular condition in which the relaxation of a muscle is impaired); the loss of Cl− channel activity leads to a depolarising after-potential following an action potential, resulting in repetitive firing of action potentials that produces abnormally prolonged muscle contraction.
Describe how MacKinnon et al. assessed the structure of CIC ion channels
MacKinnon and his colleagues obtained a high- resolution X-ray crystal structure for a ClC protein from the bacterium Escherichia coli. Based on the close similarity of their amino acid sequences, it seems likely that the three-dimensional structures of the E. coli protein and vertebrate ClC channels would be very similar. Indeed, the E. coli ClC structure is consistent with the findings of a large number of previous studies of the effects of mutagenesis of vertebrate ClC channels.
What came as a surprise when Chris Miller and colleagues were researching CIC channels in E. Coli?
It therefore came as a surprise when Chris Miller and colleagues found that the E. coli ClC is a transporter, not a channel.
Describe the mechanisms involved in the CIC transporters
The E. coli ClC transporter uses the energy stored in a H+ gradient across the cell membrane to move Cl− against its electrochemical gradient from the inside of the cell to the outside in exchange for the transport of H+ from the outside of the cell to the inside, down its electrochemical gradient.
In the E. coli ClC two Cl− ions are transported in exchange for one proton. The rate of Cl− transport through the E. coli ClC by this exchange mechanism is 100- to 1,000-fold slower than that of vertebrate ClC channels.
What is this kind (CIC) of transporter known as?
This type of transporter is termed an ion exchanger.
Describe the structure of CIC proteins (both in vertebrates and ecoli)
The E. coli ClC protein, similar to vertebrate ClC channels, is a homodimer composed of two identical subunits. Each vertebrate ClC channel subunit forms a separate Cl−-conducting pore that gates independently from the other half. The structure of the E. coli ClC transporter is much more complex than that of a K+ channel. Each subunit contains 18 α-helixes divided into related N-terminal and C-terminal halves, each containing nine helixes
Surprisingly, the two halves are found in a head-to-head arrangement so that the helixes in each half have an opposite orientation in the membrane (helix 1 is related to helix 18, not helix 10). Unlike the pore of a K+ channel, which is widest in the central region, each pore of the E. coli ClC has an hourglass profile. The neck of the hourglass, a tunnel 12 Å in length that forms the selectivity filter, is just wide enough to contain fully dehydrated Cl− ions.
Although the structures of the E. coli ClC transporter and K+ channel differ in significant respects, how are they similar in manners which help with functioning?
First, both can be occupied by multiple ions. The E. coli ClC contains three binding sites for Cl− ions within each selectivity filter. It appears that they can all be occupied simultaneously, thus creating a metastable state that ensures that the ions pass through the pore quickly.
Second, in both cases the ion binding sites are formed by polar, partially charged atoms, not by fully ionised atoms. Thus the Cl− binding sites are formed by main chain amide nitrogen atoms, which bear a partial positive charge, and by side-chain hydroxyl groups. The binding energy provided by these polar groups is relatively weak, ensuring that the Cl− ions do not become too tightly bound.
Third, in both structures permeant ions are stabilised in the centre of the membrane by the partial charges of α helixes. In E. coli ClC the positively polarised (N-terminal) ends of two α-helixes dip partway into the membrane to lower the energetic barrier for Cl− ions within the non-polar environment of the membrane.
Fourth, in both cases wide water-filled vestibules at either end of the selectivity filter allow ions to approach the filter in a partially hydrated state
These features have been conserved with surprising fidelity from prokaryotes through humans.
Why does the E. coli ClC function as a H+/Cl− exchanger, whereas the vertebrate ClC proteins function as conventional channels?
A likely explanation is that, unlike ion channels, the E. coli ClC does not have a continuous open pathway for ion movement from the outside of the membrane to the inside. Rather, like other pumps it is thought to have two gates, one external and one internal. Importantly, the two gates would never open simultaneously. Rather, ion movements and gate movements are presumed to be highly coupled in a tight cycle of reactions
What feature of ion pumps explains why the rate of ion transfer through a transporter is several orders of magnitude slower than that of an ion channel?
The relatively slow opening and closing of two gates in each cycle of ion transport, which takes place on a scale of milliseconds, explains why the rate of ion transfer through a transporter is several orders of mag- nitude slower than that of an ion channel.
Describe the presumed gate observed in the crystal structure of the E. coli ClC transporter
The Cl− ion in the selectivity filter is trapped by a negatively charged side chain of a nearby glutamate residue on one side and an electronegative hydroxyl group from a serine residue on the other side. Protonation of this glutamate is likely to cause its side chain to rotate out of the way, permitting Cl− movement.
