Ion Channels Lectures 2024-25 PDF

Summary In these lectures we will be looking at the molecular organisation and biophysical properties of ion channels, responsible for maintenance of the resting membrane potential and for generation of action potentials. We will look at how the function of ion channels can be studied and some impor...

Summary In these lectures we will be looking at the molecular organisation and biophysical properties of ion channels, responsible for maintenance of the resting membrane potential and for generation of action potentials. We will look at how the function of ion channels can be studied and some important terminology. Mechanisms of ion selectivity, activation and inactivation will be discussed. Ion channels S Kasparov 2024 1. What are ion channels and how we study them? Revision of basic biophysics, Nernst and Goldman equations, I/V plots. 2. “Leak” channels, basis of the resting membrane potential, channels, sensitive to homeostatic variables and environmental stimuli. 3. Voltage gated channels, which underpin action potential generation and propagation. How ligand gated channels (ionotropic receptors) initiate action potentials in neurones.  Lecture 1 Why ions move across the membrane (revision). Revision of basic biophysics, Nernst and Goldman equations, I/V plots. Main families of potassium channels. Ions can only cross plasma membrane via some kind of a channel. Ion channels are everywhere, in nervous, muscle, epithelial cells… In these lectures we focus on nerve cells but there will be lectures covering their physiology in other cells and tissues. Electrical activity of a neurone is determined by the flow of ions via ion channels. Combination of these currents is what shapes behaviour of a neurone at rest and during action potential. Amplifier 0 mV AP threshold -60 mV RMP  Concentration of selected solutes in intracellular fluid and extracellular fluid in millimols mM 160 140 120 100 Inside the cell 80 60 In extracellular fluid 40 20 0  Na/K ATPase keeps unequal concentrations of Na+ and K+ across the membrane Na+/K+ ATPase ATPase is mildly electrogenic – pumps 3 Na+ OUT versus 2 K+ ions IN. However, this is not the main reason for generation of membrane potential. The resting membrane potential (RMP) is generated by steady exit of K+ ions via specialised K+ channels. outside inside K+ only! “leaky” K+ channels + + + + + + + + + + + an electric gradient builds up! ++ -- + - RMP is negative inside the cell because positive K+ ions exit, following their concentration gradient If electrical field gets strong enough to completely balance out the chemical gradient, the system will reach an equilibrium. We can calculate it. Chemical driving force _ _ + + _ + + Electrical driving force _ + + _ _ + + + _+ + _ _ + + _ + + + _ + + _ _ + Net flux + + _ + + _ + + + V  The Equilibrium (Reversal) Potential: Potential of the membrane at which the electrical driving force is exactly equal the chemical driving force and therefore THE NET FLUX of this particular ion is NIL. Reversal potential can be calculated using Nernst equation: Eion = 61.5 mV Log10 (Cout /C in) Z  What is the logic behind Nernst equation? Logically at equilibrium the chemical and electrical potential energy differences across the membrane are equal but opposite. Chemical energy Electrical energy THIS is what for a given ion: for a given ion: we are after (Vm) + [Xi] 0= R *T ln F (II -- OO ) Zx * F* [Xo] R – universal gas constant Zx – valence of an ion (-1,+1 or T – temperature in Kelvins +2) Xi – concentration inside F – Faradey’s constant Xo – concentration outside I-o potential difference across membrane Vm = - R *T ln [Xi] = Zx * F [Xo] Eion = 61.5mV Log (C /C ) 10 out in Z  Using this equation: ENa = 61.5/1 * log (145/15) ≈ 60.5 mV EK = 61.5/1 * log (4/140) ≈ -95 mV This means in practical terms, is that in a hypothetical neurone sodium flux through the open channels will tend to bring membrane potential to~+60.5 mV, while potassium flux will aim to bring it to ~ -95 mV. But this could only happen if these ions were allowed to freely flow through the membrane … and they are not!  Goldman Equation describes membrane potential when more than one ion is involved: PK[Ko] + PNa[Nao] + PCl[Cli] Vm = 61.5 * Log10 PK[Ki] + PNa[Nai] + PCl[Clo] Coefficients P are there to account for the “conductances” for these 3 ions. If the conductance is maximal, P=1, if channels for this ion are closed, conductance is 0. At rest K+ conductance is high, sodium is low. NOTE THAT FOR Cl- it is the other way round because it is negatively charged ALSO REMEMBER THAT “61.5” means that this is taken for 37oC.  Making sense of IV plots IV plot is a relationship between current flow and membrane voltage V outward current (A) 0 intracellular -100 -50 50 100 voltage (mV) inward current (A) 1. Current is displayed as “movement of positive charges” for historical reasons. 2. By convention, influx of positive ions INTO the cell is shown as INWARD current looking DOWN. For a negative ion, logic is revered. Influx of a negative ion will be displayed as an “outward” current.  The i/v PLOT (curve) is a relationship between current flow and membrane voltage V Idealized leak K+ current (leak channels open) out +- in ++ x outward + + current (A) EK+ x x intracellular -100 -50 50 100 voltage (mV) inward current (A) Q1. Draw the expected graph. Q2. What would happen if concentration on both sides of the membrane was the same?  The i/v PLOT (curve) V x outward current (A) EK+ x intracellular -100 -50 50 100 voltage (mV) inward current (A) Q3. What would change if we added 15 mM into the extracellular solution? Q4. Will the slope of this line change? Q5. How would that impact on the excitability of neuronal networks (the ability to generate action potentials)  The slope on the IV plot can be used to calculate channel conductance (assuming it reflects just ONE TYPE of channel) V I= R= V G= I R I V G is the reverse of Resistance Unit of conductance is Siemens, typically pS for neuronal recordings How we study ion channels using electrophysiology Range: From macro currents, integrating activity of millions of neurones to pico-currents from individual ion channels. The origins of intracellular recording and voltage clamp The famous giant axon of squeed Recording membrane currents: classic 2-electrode voltage clamp Similar to Hodgkin-Huxley setup, suitable for any large cell but here an oocyte. Xenopus oocytes were particularly useful because of relative ease of expression of various genetic constructs, for example naïve or mutated ion channels.  How do we study individual neurones? Extracellular of cell-attached mode Stimulus Your readout: frequency of action potentials and their patterns Usually just one electrode Microelectrode 1 Microelectrode 2 In whole cell mode you can detect small changes in the membrane potential (sub-threshold events) and also the action potentials. Importantly, you can “inject current” into the cell via your pipette. By doing so you can trigger various events or force the cell to stay at one specific membrane potential and measure currents. This mode is known as “voltage clamp”. Patches of membrane are so small, that only a few channels are present. This allows isolation of the individual acts of opening and closing. Drugs can be also applied to outer surface of the patch.  One very important accroach used in these experiments is manipulation of extra and intracellular solutions. For example, we can change concentration of K+ or Na+ or Ca2+ and observe changes in ion currents. Ions can even be substituted (for example Mg2+ instead of Ca2+ or choline+ instead of Na+). Blockers of ion channels can be added to extra- or intracellular side etc.  In this configuration we can “hold” the neurone at a desired potential (-60 mV in this case) and then initiate “steps” of potential (to -65 mV in this case). While doing that we can monitor the current, flowing via the electrode. This allows us to study various processes, usually using pharmacology to “isolate” the currents which we are interested in. Resistance of the solution in the pipette (usually ignored because it is small compared to Rmem) R of the seal is very large (GigaOhms) and we usually AMP – milliA – microA – nanoA – picoA assume that all current flows via the Rmem. Ohm’s Law: Current is directly proportional to the difference in potentials (VOLTAGE) and inversely proportional to the resistance (R). Units: I = Amp, V = Volt, R = Ohm I= V Calculate R mem in the example above R  Phenomenon of Rectification. The i/v PLOT (curve) of an “inward rectifier” in symmetrical K+ solutions outward current (A) intracellular -100 -50 50 100 voltage (mV) EK+ x inward current (A) “Inward recrifiers” pass K+ better into the cell then out of the cell. When first discovered this was called “anomalous” rectification because it actually makes little sense in terms of cell function as long as the cell stays above K+ reversal potential. Question: how do we know this is a “symmetrical” K+ solution (equal K+ on both sides)?  SUMMARY 1.. Na+/K+ ATPase maintains non-equilibrium state of these ions, It is mildly electrogenic but this makes only a small contribution to the maintenance of resting membrane potential. Intracellular concentration of K+ is neurones is high and Na+ is low relative to the extracellular space 2. The reversal (equilibrium) potential is the potential which the membrane would reach if it was freely permeable to this particular ion, and no other ions. At this potential chemical driving force is exactly equal electrical driving force and the net ion flux in nil. 3. Reversal potential for K+ ions in neurones is very negative (~ -95 mV) while for Na+ ions positive (~ +60 mV). 4. Resting membrane potential is generated mainly by a steady flux of K+ ions through ion channels embedded into the membrane of the neurone. 5. Resting membrane potential is not equal to the K+ reversal (equilibrium) potential because K+ channels normally are not all fully open and because there are some Na+ and in some cells also Ca2+ currents which depolarise the membrane slightly, e.g. make it more positive. 6. Ion currents can be studied at different levels, from “macro currents”, such as EEG to single “pico currents” from channel currents. Patch clamp allows to manipulate potential across the membrane of neurone/patch and apply drugs to study how channels behave. 7. Some channels pass current in one direction better than the other, this is called “rectification”. More about it in the next lectures. Lecture 2 Channels, responsible for generation of resting membrane potential and the action potentials. Molecular organisation of ion channels. Selectivity. Gating. Inactivation. In real neurones, “resting membrane potential” is never perfectly stable, at least in vivo. It is being constantly modified by the activity of “leak” K+ currents (many inward rectifiers are controlled by G-protein coupled receptors), “persistent” sodium currents and currents mediated by synaptic inputs on the dendrites and soma of the neurones. If via integration of all inputs membrane reaches the threshold for action potential generation, the information is sent to the target neurones. AP threshold (opening of V-gated sodium channels) RMP Ion channels have an aqueous pore & ions have a shell of water  Ion flow is facilitated by the pore substituting for water K+ ions are hydrated with water molecules Carbonyl residues are surrogates for hydration shell in pore and this interaction is critical for the passage of an ion. rehydration on exit outside pore inside carbonyl residue  The pore is the basis of selectivity Pore dehydrates ions, and substitutes for water - ions must be appropriate size for this interaction. This explains why a SMALLER Na+ ion cannot pass via a K+ pore: inside of the pore in must interact with the carbonyl residues of the amino-acids, which is only possible if the size is exactly right.  Ion channels “Leak” channels: “Gated” channels: Have moderate conductances and are Open and close in response to a specific opened all the time without a specific stimulus, for example change in stimulus. Their activity can change on membrane potential, binding of a ligand relatively slow time scale (seconds- or physical impacts (pressure or minutes-hours) via association with temperature). regulatory proteins or phosphorylation. However, they are not suitable for generation of fast electrical events and provide “baseline” level of excitability. Important: While there are some channels which are extremely selective, there are also many which can conduct more than one ion, for example Na+, K+ and Ca2+ (often called non-selective cation channels). Potassium channels responsible for resting membrane potential generation (“leak potassium channels”)  Homology tree of K channels in higher animals Inward rectifier K+ channel These channels are regulated by intracellular signalling mechanisms. Sensitive to various factors such as pH, temperature as well as many extracellular signalling molecules. Tandem pore (K2P)  Two classes of potassium channels primarily responsible for resting membrane potential generation and regulation Have 2 transmembrane domains. Have 4 transmembrane domains. Often called “inward rectifier K+ Often called TWIK, “Tandem pore”, channels” “K2P channels” Because these channels do not open and close fast during action potentials but operate at a slower scale, they provide baseline “leak” conductances. The terms “leak current” is often used in the literature.  Currents flowing via TWIK channels and Kir channels (K+ inward rectifiers) 2-P domain TWIK leaks Inward rectifiers (KCNH group, Kir 1,2,4 etc) Tandem of P-domains weak inward pass K+ ions into the cell better, than out. The rectifying K+ channel (TWIK): little or no biological reason for this is not entirely clear. inward rectification In the membrane Kir (inward rectifiers) assemble into tetramers (4 subunits). TWIK channels are dimers (2 subunits). One needs 4P domains! NOTE that 4 P domains are needed to create the pore. Either one needs 4 protein molecules each contributing 1 P domain. Or 2 protein molecules each containing 2 P domains. Anaesthetics, particularly halogenated volatile anaesthetics such as isoflurane and halothane, open various TWIK (K2P) channels.  RMP and is not always stable 2 key factors affect RMP: 1. Some of the “leak channels” are actually regulated by various factors, for example second messengers and even directly by G-proteins 2. Presence of persistent sodium currents INaP  Additional Information (Supplementary) Persistent sodium currents are thought to underlay oscillatory behaviour of the respiratory circuits in the brain stem  Additional Information (Supplementary) Astrocytes (the main family of glia cells) have strong K+ conductances and a very negative membrane potential, which is typically around -80 mV. They express: - 4TM (leaks) from the TWIK (KCNK sub-family, especially KCNK1) - Inward rectifiers (2TM family) are expressed at low level in astrocytes except Kir4.1 (KCNJ10) which is highly expressed and plays a major role in setting MP in astrocytes. High potassium conductivity of astrocytes makes them unable to propagate electrical charges on their membranes. Summary 1. Resting membrane potential is mediated by currents via “leak” potassium channels. However it is always more positive than Nernst equilibrium potential for K+ because K+ channels are never fully opened and because of concomitant influx of Na+ via persistent sodium conductances. 2. Pore is formed by special P domains present on individual subunits, which form tetramers (Kir) or dimers (TWIK). Total number of P domains = 4 in both cases. 3. Ions a filtered by the selectivity filter formed by aminoacids with carbonyl oxygen atoms which substitute water, normally surrounding ions. The match must be exact, otherwise an ion cannot pass via the pore. 4. Kir channels are characterised by “inward rectification” whereby inward currents are stronger than outward. Several such channels can be modulated by G-proteins, which are the signalling partners of G-protein coupled receptors. 5. TWIK channels can be modulated by various factors, including external pH and also by volatile anesthetics. 6. Persistent sodium currents in some neuronal populations mediate rhythmical action potentials, this is likely mechanism of respiratory rhythm generation.  Lecture 3 Gated channels. Voltage-gated channels, responsible for action potential generation. Other gated channels. Channelopaties. 10min 10mV AP threshold (opening of V-gated -52mV sodium channels) 5min 1min RMP Key points about single APs Influx of sodium mediates the depolarisation during AP Traces 1-6 : different concentrations of extracellular Na+. Results just as we would expect, as [ion] important in gradient. Also note time course of the process.  Na+ and K+ currents which underpin AP Based on their recordings, Hodgkin & Huxley used a numerical model to recreate the properties of the AP – how currents change with voltage and time Na+ channels are controlled by the potential of the membrane Na+ Voltage sensor and Na+ Na+ Outside of the cell activation mechanism Na+ Na+ Aqueous pore Extracellular domain Lipid membrane Intracellular domain narrow selectivity filter “Inactivation gate” Inside the cell (negative)  “Positive feedback” is responsible for the very fast dynamics of AP 1. Depolarisation 2. Opening of voltage-gated Na+ channels 3. Sodium currents depolarise membrane further Something has to happen for this process to terminate! At positive potentials voltage-gated Na+ channels become “inactivated” Voltage sensor and Outside of the cell activation mechanism Na+ Na+ Na+ Aqueous pore Extracellular domain Lipid membrane Intracellular domain narrow selectivity filter “Inactivation gate” Na+ Na+ Na+ It takes time and re-polarisation to reverse inactivation  Can you predict the full I/V curve for a typical Nav? Outward current ? ? RMP x x x V (mV) -100 -50 50 100 extra +- intra + + Inward current  Outward current The voltage- gated Na+ channel RMP activation ENa+ x x x V (mV) -100 -50 50 100 extra +- intra + + Inward current  Voltage-gated Na+ channel (NaV) top view cross section bottom view Sato et al. (2001) Nature 409, 1047–1051 ‘ball & chain’ model Yu et al. (2005) Pharmacological Reviews 57: 4387-395 Inactivation of V-gated Na+ channels limits the maximal frequency of action potentials. This is the basis of absolute “refractory” periods. Together with delayed rectifiers (K+ currents) this helps to prevent oscillations in the network. Refractoriness prevents retrograde propagation of action potentials. Sodium currents before and after chemical removal normal of the inactivation gate “Textbook” Real traces: representation mutation  Additional information Sodium channels consist of a large α subunit, which is associated with small β subunits of 33-39 kDa. Sodium channels in the adult central nervous system (CNS) and heart contain a mixture of β1 - β4 subunits, while sodium channels in adult skeletal muscle have only the β1 subunit. Note that different voltage-gated sodium channels are expressed in different populations of neurones. For example, NaV1.1 is mainly expressed in GABAergic neurones in the brain. NaV1.7 is mainly expressed in dorsal root ganglion cells and sensory afferents. Therefore, mutations in different NaV have different functional consequences. Additional Information (Supplementary) Conus marine snalis. Omega conotixin is a v- gated Ca2+ channel blocker, used for pain management after intrathecal injection. Colombian poison-dart frog: Irreversible activation of Nav, causes delolarisation of nerves and muscles.  “Shaker” K+ channels which mediate repolarisation (6TM) Shaker family of K+ channels Delayed rectifiers typically exhibit “outward” rectification, meaning that exit of K+ is easier than its entry. Extracellular levels of K+ increase during active firing of AP.  In Kv channels, subunits arrange to form a tetramer The key are “α” subunits, sometimes β subunits are added which confer additional features, such as anchoring or intracellular localisation To form a functional pore one needs 4 “P” domains. We saw the same requirements for V-gated Na+ channels (previous slides). If a subunit has 1 P domain, there have to be 4 subunits. If there are 2 P domains, 2 subunits are enough.  Mammalian KV channel is operated by a voltage sensor, comprised of charged amino-acids. IMPORTANT: these channels open with a delay, which makes sense. Hence sometimes called “delayed rectifiers”. In contrast to the “inward rectifiers”, they pass K+ outward better than in, or at least equally well. Additional information: There are also other voltage-gated channels, but we are not looking at them in these lectures.  Additional information Many channels are gated by binding of a ligand, which can happen on outer of inner side of the membrane. Note that often they are not selective for Na+ vs K+ Glutamate, Acetylcholine, GABA This will be discussed in other lectures  Example of a ligand-gated ion channel: Nicotinic acetylcholine receptor is a non-selective cation channel activated by ACh. As a result of the mixed conductance, it moves membrane potential to ~-40 mV which then can trigger an action potential by opening V-gated Na+ channels. (This information was previously given in the lecture on neuro-muscular transmission in Y1, revise it!) + out in +  Additional information You do not need to remember this Nav1.1 and Nav 1.2 Take home message: there are many known human mutations in ion channels which can lead to both, hypo- and hyperfunction. Many of mutations in voltage-gated Na+ and K+ channels lead to epilepsy and related syndromes. There are also syndromes caused by mutations in different channels, expressed in peripheral tissues, for example “long QT syndrome” – in the heart or mutations in CFTR, causing lung, kidney and intestinal disease. Summary: 1. Gated channels are normally closed almost completely, but open when a specific physiological stimulus is applied. 2. Voltage-gated sodium channels are opened by depolarisation of the membrane because of translocation of the voltage-sensing domains. This results in a strong influx of Na+ ions which further depolarise the membrane and open more channels in a positive feedback loop. 3. V-gated Na+ channels rapidly inactivate, this is a function of a separate domain (inactivation domain) which blocks the channel and cuts the flux of ions. To remove the block, membrane has to repolarise and it takes a bit of time, limiting maximal frequency of action potentials. 4. V-gated K+ channels open with a delay, helping repolarisation and if they are powerful enough can cause “afterhyperpolarisation”, which also limits the excitability. 5. In the membrane V-gated channels are assembled of several subunits, some of which are responsible for modulation, rather than ion movements. Each alpha subunit contains 4 P domains. 6. There are numerous loss- and gain-of function mutations of ion channels. 7. Ion channels are targets for multiple toxins from nature and also for some drugs (local anaesthetics)