Week 2 - Ligand Gated Channels + Electrophysiology

EPSP

excitatory post synaptic potential, easier to stimulate membrane

IPSP

inhibitory post synaptic potential, harder to stimulate activity

resting membrane potential

the steady electrical potential across the cell membrane when the neuron is at rest, this is generated by standing ionic gradients, K+ is concentrated inside the cell while Na+ and Cl- are concentrated outside the cell

do channels always become activated?

once they are open, there are conformational changes where they will no longer allow for different ions to flow through, the states include inactivation, desensitization and open channel block

inactivation

this is when electrical stimuli causes a change in the membrane potential creating action potentials

desensitization

this is caused by electrical stimuli like neurotransmitters/drugs, in continued presence, chemical stimulus can desensitize the channel so it doesn’t stay open anymore

open channel block

this is when a large molecule occludes or blocks the pore, for instance Mg2+ ion this causes long term potentiation and long term depression (LTP/LTD), the NDMA receptor is critical for this and the channels is blocked at Mg2+ ions at rest, blocks the pore

nAchR

n-acetylcholine receptor, termed nicotinic because the actions of nicotine mimic the effects of acetylcholine, they are ion channels so it only binds to ion channel receptors

muscarinic Ach receptors

only binds to G protein coupled receptors

nicotinic acetylcholine receptor nAChR

the first channel to be studied in detail, ligand activated channels, they are heavily localized in the postsynaptic membranes of skeletal muscle fibers, they are permeable to cations so when it binds to ACh or nic it causes channel to open, 5 separate subunits are arranged around the central core (receptor stoichiometry), the 2 alpha ones contain a portion of the binding sites for acetylcholine, ACh is the normal physiologic ligand, its heteropentameric so it has an open conformation with a pore region, no ATP required cause uses concentration gradient

nAChR subunits

there is an amino terminal that helps determine where the alpha subunit will tend to bind and a carboxyl terminal both located in the extracellular region, each submunit is composed of 4 membrane spanning regions, the M2 regions of each of the four subunits line the pore and forms the gate that controls ion movement, also has an intracellular loop, Na+ enters once Ach binds

homomeric nAChRs

means they have the same alpha subunits, they are found in the brain (HPC and cortex), presynaptic terminals, immune cells, microglia and astrocytes

heteromeric nAChRs

have minimum 2 alpha subunits but there rest are different, they are found in the CNS, a4b2 receptors are widely expressed in the cortex, thalamus and brainstem, in the peripheral nervous system neuromuscular junction there are muscle type receptors

proposed model of the nAChR structure

an open pore is created by rotating each helix toward the channel wall, this created by the M2 region, when open this means there is tau expression

GABAa

almost always a heteropentamer, ligand gated ion channel, you need two of them binding in order to open channel

GABAb

G protein coupled receptor

GABAc

ligand gated ion channel

GABA receptors

they form a complex with sushi domain, its presynaptic and controls neurotransmitter release, sAPP is the soluble form of amyloid precursor protein and binds to sushi of GABAb, its stimulates it to inhibit neurotransmitter release

electrophysiology

have to be able to amplify small ion channels that flow through, its the science and branch of physiology that describes the flow of ions in biological tissues and in particular to the electrical recording techniques that enable the measurement of this flow and the potential changes related to them, in intracellular recording, a recording electrode is inserted into a cell so that the intracellular potential can be measured against the extracellular potential

glass micropipette

able to make physical contact on one cell membrane, glass makes such a tight seal that ions don’t even flow through, only way they can flow through is through an open channel

voltage clamp

this is when the membrane potential is held or clamped through use of negative feedback circuit, at a level set by the experimenter, main advantage is that you can measure the amount of ionic current crossing a cell’s membrane at any given voltage at a given time, allows measurement of very small amounts of ions, one neuron to get this tip of glass microelectrode to touch this one neuron, glass microelectrode that needs to touch one cell to make a tight enough seal to get different groups of ion channels under it

why is patch clamp used in electrophysiology?

it allows direct measurement of channel activity, measures ionic currents carried by Na+, K+, Ca2+ and Cl-, it resolves currents at the level of single channels, small membrane patches or entire cells, use very small patches of membrane or how all of them in a specific cell work, this method made ion channels experimentally observable

how does patch clamp work?

a small patch of membrane is sealed to the tip of a micropipette, these are all giga-ohm seals that allow no ion leakage, the high resistance seal ensures that currents flow through the amplifier/glass pipette rather than escaping through the rim of the patch, it can be used to measure the flow of ions through single ion channels

cell attached patch clamp

directly though cell membrane tight seal, it snot ruptured, local patch only, the intracellular contents are preserved, voltage control is poor, ion ingredients are preserved, it measures single channel or small patch currents, typical signals include channel openings and kinetics, it has very high physiological realism and its best used to measure channel behavior

whole cell patch clamp

this is when the cell is in continuous contact with the pipette, you break the membrane seal so it bursts open allowing content of the glass pipette to be continous with the cell, allows access to the entire cell, intracelluar contents are dialyzed into the pipette, it has excellent voltage control, ion gradients are altered, measres total membrane currents, typical signals include EPSPs, IPSPs and APs, it allows intracellular manipulation, its very easy, physiological realism is moderate and it is best used for neuronal excitability

perforated patch clamp

this is the same thing as the other methods but in the pipette instead of having different electrolyte components, we would also get a perforating agent, for example an antibiotic they would punch holes in the membrane at the level of the pipette but not throughout the whole thing allowing 4 different ions to flow through, there for there is access to the whole cell through the pores, the intracellular contents are largely preserved, voltage control is moderate, ion gradients are preserved via gramicidin, it measures near physiological whole cell currents, signals include synaptic currents and membrane potential, physiological realism is high, its the most challenging method to execute and its best used for physiological inhibition

cell attached cell recording

always start with the tight giga ohm seal via gentle suction applied to the glass pipette, tight contact between the pipette and membrane is what forms the seal, careful refraction of the pipette removes patch of membrane (inside out mode)

whole cell mode recording

strong pulse of suction opens up the membrane, making the cytoplasm continuous with the pipette, careful refraction of the pipette causes the ends of the broken plasma membrane to anneal, reattaches to itself to actual cell membrane that has channel will flip around (outside-out mode)

gramicidin

permeable for Na+ and K+ (monovalent cations only), it preserves Cl-, speed of access is slow, main advantage is the preservation of physiological Cl- gradients, main limitation is its technically demanding, slower, its best used for GABA(A) glycine receptor studies since these use Cl- movement

nyastatin

permeable to small molecules (Na+ K+ Cl-), it allows for faster speed of access, main advantage is quick and easy access but limited by Cl- equilibration, best used for general excitability (non-Cl- studies)

amphotericin B

also permeable to small molecules (Na+, K+ and Cl-), faster speed of access, stable and widely used, limited cause Cl- is not preserved, best used for cardiac, endocrine, long recordings

B-escin/saponin

permeable to ions, small molecules (cholesterol dependent pores), its the fastest, main advantage is speed of access and it preserves structure better than whole cell, main limitation is its not Cl- safe, its best used for large cells, Ca2+ signaling and long recordings

effect of potential on currents

at 0 mv, the concentration of K+ inside and outside the cell is the same so there is no net movement of ion so no current cause no concentration gradient, if we add +20mv it makes the cell more positive inside, so if we voltage clamp at +20 there is a net outward flow of K+ cause the inside of the cell is more positive, follows the concentration gradient, but if we clamp at -20mV it makes the inside of the cell more negative so K+ net flow moves inward

current-voltage relationship (I-V curve)

at 0mV there is no current, if we go above the X axis the net movement of ions is outward and if we go below the X axis the net movement of ions is inward, it is a graphical description of how ion current changes as a function of membrane potential for a fixed channel population under fixed ionic conditions, the current for any ion is determined by the difference between the membrane potential and the equilibrium potential for that ion

equilibrium potential

membrane potential where there is no net movement of an ion species, no current so nothing goes up or down, doesnt necessarily have to be zero just equal inside and outside the cell,

linear I-V curve relationship

this means that the ion channel is following Ohm law so if voltage increases so does the size of the current/conductance so basically how open the channel is

K+ equilibrium

if the concentration inside of K+ is 90mM and outside is 3mM, the K+ will flow out the cell moving down the concentration gradient, the positive charge accumulates on outer surface of the membrane, leaving an excess of negative charge on the inner surface creating a small potential difference across the membrane, the electrical gradient slows the efflux of positively charged K+ ions

electrical gradient

shows the rate ions are moving, balancing concentration gradient at some point

equilibrium potential K+

electrical potential that balances K+ concentration difference that would normally move that ion, there is no net flow of K+ ions at this potential, individual ions may still enter and leave the cell, ion flow is equal to current

experiment B I-V curve

the IV curve is curvilinear/rectified which indicates its a voltage gated channel, the channel is closed until we reach the reversal potential where there is no net flow/current, once the reversal potential is reached the ions will start to flow outward

experiment A I-V curve

the IV curve is linear indicating that its a leak channel because it follows Ohm’s law, this means you can predict the direction of the curve, the steeper the slope of the curve is the more ions flow in

rectify

this is when the opening and permeability of an ion channel is voltage dependent, so ions move through their pores much more rapidly in one direction than another

nerst equation

calculates the potential required to balance the concentration gradient, the equilibrium potential calculated by nerst is equal to the experimental one

what happens when both the recording and bath electrodes are outside the cell?

there is no electrical potential difference recorded, but as soon as one electrode is inserted into the cell, the oscilloscope displays a steady deflection of -65mV being the resting membrane potential, measure this by making steady contact with glass electrode to make a tight seal

equilibrium potential/reversal potential

membrane potential at which there is no net passive movement of an ion into or out of the cell, at +50mV there is 0 net flow/current right at X axis

squid giant axons

used for first experiments examining how changes in ion concentrations affect the membrane potential, this is because they have a large diameter + internal and external ion compositions can be controlled, they squeezed the axon out and filled it with different fluids to see if they could replicate initial fluid measurements

axoplasm

fluid found inside the squid giant axon, very high in Na+