EXAM#2 Part 2: Cell Neuro

Cellular Neurobiology Exam #2 Content September 11th, 2024 Conductance = tells you how much current is moving through the membrane. You can determine it for each ion. 𝑔= 𝐼 𝑉 , 𝑉 = 𝐼𝑅 , 𝑔 = 1 𝑅 I = current V= driving force G= conductance Toxins and poisons used to study neural function due to their specific actions TTX= tetrodotoxin - from pufferfish and plugs sodium-gated channels but does not affect potassium channels, affects inward current TEA= tetraethylammonium blocks K+ channels —----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- September 16th , 2024 Scorpion betta toxin = causes a shift in voltage gating of sodium channels so they open at more negative values With beta toxin voltage clamp recording: see a larger initial inward current 2 possibilities 1. Sodium channels are opened longer (open sooner) 2. Potassium channels are not opening normally This can be sorted out using TTX and TEA - Repeat the experiment voltage clamp but this time you will apply both the beta toxin and TTX When a neuron is treated with TEA and beta toxin Saxitoxin (STX) = with a very low dose STX applied to a neuron shows - a higher-than-normal threshold Amplitude if the action potential is reduced Options to get this effect : ● Sodium channels cant open ● Potassium channels are opened sooner At higher concentrations of STX you can not get the membrane potential to depolarize Using voltage clamps : 1.) Set your Vm above the threshold AND apply STX 2.) You will see a perfectly normal outward current but no inward current because sodium channels are blocked Aconitine on Neurons - Initial inward current occurs more rapidly - Late inward current is te outward current 1.) Sodium channels open readily and they don't close like they normally do Voltage clamp recording with Aconitine What can you deduce so far? - The action potential is sooner and longer - Maybe potassium channels take longer to open or do not open at all - The inward current is bigger and longer so potassium channels are open So aconitine does not affect potassium channels, they are operating as always, does however affects sodium channels and keeps them from closing Patch Clamp Recording Technique —----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- September 18th, 2024 Patch Clamp Recording Technique Patch clamp records channel activity just below the open end of the recording pipette. This will be a relatively small number of channels. The recording makes about 300 quick recordings in success. Currents are added up to get a greater range of what many channels would be doing . This looks very much like the 2-electrode voltage-clamp recording. Many types of ion channels have identical kinetics as seen in patch clamps - 4major ion-passing channels - Na - K - Cl - Ca Na and Ca channels are identical Both are a single protein with membrane-spanning regions. There are 6 membrane-spanning regions in each of the 4 domains. 4 domains surround a pore that has charged amino acids. Inside this amino acid loop give the channel its selectivity. The channel has a solubility filter that sorts ions by size. Sodium and potassium both attract water molecules, sodium is more attracted to water molecules, sodium is more attractive to water and this water binds more tightly to sodium than to potassium. As a result, the sodium ion with its attracted water is larger than the potassium with its water. Because inside the potassium channel, oxygen will substitute for some of the water it carries so it can shed a few of them and enter the channel with only about 2 water molecules. Voltage-gated channels open when Vm depolarizes because they have helices in their membrane-spanning regions that are full of charged amino acids, which are repelled by depolarization and move within the channel causing it to open. In NA thus has been identified as the S4 helix. Sodium channels open with depolarization but even if the depolarization continues they do close again They have 2 gates Activation gate = S4 helix, the gate opens to depolarization, sodium moves Inactivation gate = depolarization causes this gate to close Explanation - The outside of the cell is positive and the inside is more negative. At rest, the activation gate is closed. And the inactivation gate is open. The cell becomes depolarized where the inside of the cell becomes less negatively charged compared to the outside and the activation gate opens and sodium ions rush in and flow through the inactivation gate. The inactivation gate is triggered to close. When the cell repolarizes, the membrane gradually becomes more negative so the activation gate will close. The closing of the activation gate is followed by the opening of the inactivation gate. An inactivation gate is probably a loop of charged amino acids that when the inside of the cell becomes positive is pushed into the pore to block it. Sodium and calcium channels are all from single games and are single-channel proteins But K+ channels are different, They have four domains but each of them is a separate protein. - $ proteins come together to make the channel. There are a large number of different types of these proteins and a potassium channel can mix and match these to create a wide variety of channel types. The squid axon potassium channel opens with depolarization and stays open for a long time. Some potassium channels will close after opening. A channel has a ball and chain b) Patch clamp forms a high resistance seal around a patch of cell membrane i)allows recording of ion flow through only a few channels under that membrane