BN 1.4 Voltage Gated Ion Channels PDF

VOLTAGE GATED ION CHANNELS Basic Neuroscience 1.4 Flow of information in the brain ★ Conveyed by electrical and chemical signals ★ Net flow of charge = current ★ Brain electrical signals: changes in the flow of ions (current) drive electrical pote...

VOLTAGE GATED ION CHANNELS Basic Neuroscience 1.4 Flow of information in the brain ★ Conveyed by electrical and chemical signals ★ Net flow of charge = current ★ Brain electrical signals: changes in the flow of ions (current) drive electrical potential (voltage) changes across the cell membrane Ohm's law: I = V / R -12 -9 ★ I – current – amps (A) – typical range in neuroscience pA (10 ), nA (10 ) -6 -3 ★ V – voltage – volts (V) – typical range in neuroscience µV (10 ), mV (10 ) 6 ★ R – resistance – ohms (Ω) – typical range in neuroscience MΩ (10 ) Conductance (G) measured in Siemens (S) is calculated by: G = 1/R Thus, I = G * V Current flow through any conductor is determined by the potential (voltage) difference across the conductor and the conductance of the conductor (reciprocal of the resistance, i.e. a good conductor is a poor resistor). In the extreme: without a voltage/potential difference and a conductance there is no current. The cell membrane acts as a resistor. ELECTROCHEMICAL GRADIENT ★ Electro – affected by voltage ★ Chemical – diffusion principles  ★ Both the concentration gradient of the ions and the voltage of the cell make up the electrochemical gradient. Concentration gradient – if there is a difference in concentration of an uncharged chemical species across a permeable membrane then there will be a net directional flow of the species ★ Net flow through a permeable membrane: high → low concentration + Concentration gradients can also affect the net flow of charged chemical species (e.g. a Na ion), but, since the ions are charged, the flow also becomes affected by the voltage of the cell. ★ An electrical potential (voltage) acting on an ion can enhance or counteract concentration gradients In this diagram, there is no concentration gradient, but there is a voltage difference; as the cell is negatively charged, the positively charged ions will move into the cell. In this diagram, the voltage is counteracting the concentration gradient. Making the cell positive forces the positively charged ions out of the cell. There was no concentration gradient originally, but by making the cell positive the ions moved out of the cell. EQUILIBRIUM POTENTIAL (NERNST EQUATION) We can calculate the electrical potential (driving force) acting on an ion. However, we first need to know the equilibrium potential (effectively a "zero-point"). Equilibrium potential (Eion) is the electrical potential difference that counteracts the concentration gradient of the ion (sometimes called "Nernst potential" or "reversal potential"). ★ To oppose the concentration gradient – the voltage the cell would have to be at for there to be no net flow of ions. ★ A zero-point is a point which you can compare to the membrane potential If we know the concentration of an ion inside and outside the cell, we can calculate the equilibrium potential using the Nernst equation: Eion – equilibrium potential (mV) -1 1 R – universal gas constant (8.314 J.K.mol- ) T – temperature (K) + + 2+ - z – valence of the ion (+ve or -ve: e.g. K = +1; Na = +1; Ca = +2, Cl = -1) -1 F – Faraday's constant (96485 C.mol ) 10 ln – natural logarithm (2.303 x log ) [X] – concentration of ion For example, for sodium: This means you would have to depolarise the cell to 71mV to stop sodium from moving into o[]\the cell. Imagine an uncharged cell (0mV), which has a high concentration of sodium outside and a low concentration inside. If it is impermeable to sodium (no Na+ conductance) equals no flow. If the cell is made permeable to sodium, in the first instance the sodium will flow down the concentration gradient. This influx of sodium will depolarise the cell, which establishes an electrical field which opposes the influx of sodium. Eventually the chemical (influx) and electrical (efflux) fields are balanced and there is no net flow of sodium. It is in equilibrium at ENa. ★ May be easier to view both as opposing forces (although only voltage is a force) (No exam questions on equilibrium potentials of each ion, this is just to have for notes) Extracellular sodium concentration is high, but low intracellular. Extracellular potassium concentration is low, but high intracellular Intracellular calcium concentration is kept very low. The Eion for chloride is close to resting membrane potential ★ GABA receptors are chloride permeable – GABA causes membrane hyperpolarisation because the chloride Eion is close to RMP and it is the difference between the membrane potential and the equilibrium potential of the ion that determines what the effect is on the cell Typical resting membrane potential = -70mV to -55mV Pumps and transporters establish the chemical concentration gradient of the ions. Concentration gradients of the ions don’t really change – you need very few ions moving to cause a physiologically relevant change in the membrane potential, so the concentration gradient doesn’t really change much. Resting membrane potential is determined by the Eions and their conductance. RMP is calculated using the Goldman–Hodgkin–Katz equation: This formula basically states that the resting membrane potential is dependent on the concentration gradient of each ion and the membrane's permeability to these ions. The cell is at RMP when net current flow is zero. Neurons are permeable to different ions, and each ion drives the neuron towards its Eion. RMP is determined by the relative influence of each ion on how conductive (permeable) the membrane is to their flow. At RMP none of the ions are at Eion so no ions are at equilibrium at RMP. Thus, a change in conductance (permeability) of an ion will alter RMP (driving it towards the Eion ). RMP is closer to EK than ENa because K+ permeability is higher than Na+ at rest. DRIVING FORCE For a current flow across a membrane we need 1). a driving force (potential difference between the equilibrium potential of the ion and the membrane potential) and 2). conductance. The electrical potential difference acting on the ion (the driving force) is determined by the difference between the membrane potential (Vm) and the equilibrium potential (Eion ). Driving force = Vm – Eion And because I = V/R and G = 1/R… Ionic current (I) = G * (Vm-Eion ) For example, At RMP, sodium has a much larger driving force than potassium (blue/Na+ arrow is bigger than the red/K+ arrow). A negative driving force results in an inward current (inward movement of a positive ion, or an outward movement of a negative ion) – this causes depolarisation. A positive driving force results in an outward current (outward movement of positive ion, or inward movement of negative ion) – this causes hyperpolarisation. Conductance is necessary for ion flow across the membrane. At RMP there is a large driving force on Na+ (-70mV - 70.5mV), however conductance is low; conductance must be increased for ions to flow across the membrane. Conductance can be increased by using ion channels. ION CHANNELS The lipid bilayer is an electrical insulator. Ion channels are transmembrane proteins that allow ions to flow across the cell membrane. i.e. they provide conductance (G) in a lipid membrane, which allows ions to flow (I) down their electrochemical gradient (Vm-Eion ). Ion channels have three important properties: 1. They open and close in response to specific stimuli 1. Chemical (e.g. lipid gated ion channel) 2. Second messenger 3. Mechanical (e.g. PIEZO1 and PIEZO2) and thermal changes (e.g. TRPV1) 4. Voltage across the membrane 2. They recognise and select specific ions 3. They conduct ions across the membrane VOLTAGE GATED ION CHANNELS There are many different voltage-gated ion channels working in concert, opening and closing, to shape electrical signals in the nervous system. At least 143 genes in the human genome encode ion channels. They have diversity in structure (but common themes), as well as differences in ion-selectivity, activation and inactivation kinetics. + Na channels (Nav, 10 genes, 1 family & last to evolve) 2+ Ca channels (Cav, 10 genes, 3 families) + 2+ Na and Ca are similar in structure. Many Ka+ channels (>12 families), not all are voltage gated. ★ Gene therapies for epilepsy are making use of potassium channels  + 2+ Na and Ca channel structure Sodium and calcium channels are formed from a single poly-peptide chain (so it is just a single protein). ★ There is a repeating structure which is similar across four different homologous domains (motifs I-IV) ★ Each of these domains has six-membrane spanning alpha helices (S1-S6) ★ The four domains are arranged around the central channel pore ★ Each of the S-domains (one of the six-membrane spanning alpha helices) have an important role in the channel biophysics (e.g. S4 segments contain the voltage sensor, so they determine whether the activation threshold has been crossed) ★ S5 and S6 are connected by an extended looped string of amino acids which forms the p-region which dips in and out of the external surface and forms an external selectivity filter for the channel. K+ channel structure K+ channels are not assembled from a single poly-peptide chain containing four domains. Instead, K+ channels have four separate protein subunits that assemble to form a functional channel. Each K+ subunit corresponds to one of the domains of the voltage-gated Na+ or Ca2+ channels. Each subunit has six transmembrane segments (S1–S6), and a pore forming P-region. Because K+ channel subunits are separate proteins, each domain could be encoded for by different genes, meaning K+ channels can assemble as homomers (four of the same K+ channel subunits, e.g. 4 x Kv1.1), or heteromers (assembled of different subunits, e.g. Kv1.1 + Kv1.4). ★ This allows for a range of diversity in K+ channels in the proteins within, and also the arrangement of the proteins. During sustained depolarisation, the channel does not remain open indefinitely. The ion channels are very fast activating and deactivating switches. ★ Sodium channels inactivate during sustained depolarisation, which results in no conductance. Even though the activation threshold has been crossed (Vm at -40mV), there is no current. The membrane must be repolarised for the voltage-gated sodium channels to recover from inactivation. A short loop on the intracellular side of the channel linking domains III and IV (nb: domains III and IV, not S3 and S4) forms the inactivation gate (hydrophobic loop of amino acid fills the pore). This hydrophobic protein ball blocks the channel to inactivate it. To remove this, the membrane potential must be repolarised back to RMP. The pore contains an inner chamber and selectivity filter. ★ The filter and pore work by giving the ion charged amino acids to surround it when it is not in water ★ K+ channel is best understood but still some ambiguity about what makes them selective Ion channel selectivity is based on the size, charge and energy of hydration of the ion (the size of the ion+water molecule; not the size of the ion, but its size when bound to water). ★ Ions are bound to water ★ In the ion channel, water is removed from the ion; to do so, water molecules need to bind to amino acid pores, which are structured in a way that only the specific ion bound to water will be able to be stripped of that water ★ E.g. K+ channel pore – amino acids structured in a way that it ca strip water from potassium ions more effectively than other ions Studying voltage-gated ion-channels ★ Activity: electrophysiology (patch-clamp) ○ Very high temporal resolution (~20KHz sampling rate) ○ Allows recording of single channels (cell-attached configuration), or a collection of channels (currents, in a whole-cell configuration) ○ Very good for understanding gating kinetics, permeability, etc. ★ Structure: crystallography and Cryo-EM ○ Very high spatial resolution (~2Å); takes a very detailed "snapshot" of a protein structure ○ Explains how ion selectivity works ○ Allows to infer how the ion channel moves Physiologically: ★ Initial depolarisation (e.g. synaptic activation) activates voltage-gated sodium channels ★ Sodium flows (inward current) down its electrochemical gradient, through the opened sodium channels ★ This further depolarises the neuron, activating more voltage-gated sodium channels. At a point – the action potential threshold – there is a regenerative increase in sodium current. ★ Sodium current increases dramatically to produce the upstroke of the action potential ★ Channels then inactivate ★ Potassium channels are responsibly for repolarisation. Voltage-clamp ★ Experimenter able to control voltage by injecting the equal but opposite current to that which is flowing I the cell to clamp the membrane potential, so experimenters are in control of how it changes ★ Is sodium wants to flow into the cell, it will cause a depolarising current, but the electronics will inject the equal but opposite current to clamp the membrane potential, and what is recorded is the equal but opposite current to the sodium current Physiologically, membrane potential and sodium current are changing, and they change each other (i.e. membrane depolarisation activated more sodium channel currents). Experimenters can clamp the voltage by injecting hyperpolarising currents to oppose the depolarising currents to stabilise the voltage at a set value. They can record the current needed to clamp the voltage. And thus can measure the sodium current that flows as a result given membrane voltage.  ★ Depolarising to -50mV (below the activation threshold of sodium channels), there is no sodium current, so we need negligable current to 'clamp' the voltage at -50mV (purple line and square). ★ At -40mV we activate some sodium channels. Under physiological conditions this would cause a depolarising current that would also change the membrane potential. But in the voltage clamp experiment the opposite current is injected (blue line/square) ★ Further depolarisation increases the number of open sodium channels (increase in Na conductance = increase in Na current). So the amount of opposing current we inject also increases. ★ As clamped voltage increases, the current is not just getting larger but also getting sharper too; ion channel opening is probability based, so for a sodium channel, the more we depolarise, the greater the probability a channel opens over a shorter period of time ○ This means yiu get more conductance over a shorter period of time, while leads to a larger, more quickly rising sodium channel current ○ Inactivation probability also rises with this increase in depolarisation, so conductance goes to zero quicker Peak sodium channel current begins to decrease as we depolarise the neuron further, as the driving force is reduced overtime so the current decreases despite conductance increasing. ★ Membrane potential is approaching equilibrium for sodium, which means the difference between the membrane potential and the equilibrium potential is approaching zero, so the driving force is collapsed.