Lectures 1,2,3Rachel A. Kaplan and Elbert Heng
2.3.14
Announcements
• Detailed Answers for PS1 are on wiki
• Contacting TAso please use piazza, you’ll get the fastest
responseo if you don’t want to, email (not canvas
message) uso if you’re emailing Rachel
her email is [email protected]• or [email protected], either one is fine
• Tell us what you want us to cover! That’s what we’re here for!
Lecture 1: Intro to Electrophysiology
Background
● Neuron Doctrine● Cajal vs. Golgi● Graded vs. Discrete Signals● Lipid bilayer - hydrophobic; need ion channels● Properties of ion channels● Definitions: ions, voltage, membrane potential,
current, depolarization, hyperpolarization
Studying Ion Channels
● Driving force● Current vs. Voltage clamp● Hodgkin & Huxley: squid giant axon● Neher & Sakman: patch clamp
○ inside out patch○ outside out patch
aka On Cell
Cell Attached
Whole Cell
Inside-Out
Outside-Out
Lecture 2: Ion Channel Function
Current Clamp
Voltage Clamp
Single Channel i/V plots
• Shows relationship between (direction and amount of) current (i) flowing through a channel given various membrane potentials (that you have clamped the membrane at to experimentally determine current)• If it’s OHMIC, it’s just a linear relationship
• not so scary, you learned this in middle school!
Drawing i/V Curves
• If a channel is ohmic, relationship between i and V is defined by Ohm’s law
• i = g V• In voltage clamp, we are manipulating the
independent variable: V• And we are measuring the dependent
variable: i• g is the slope
Drawing i/V Curves
If you want to draw a linear curve, what do you need?
Drawing i/V Curves
• That’s right, a slope and a point.• We can find a slope and a point • Slope: conductance• Point: where the curve crosses the x-axis (the
potential axis) – Eion or Vrev
• (these two are the same for channels that pass only one type of ion)
Drawing i/V Curves
• How do I find conductance?• Don’t worry about it, it will be given to you.
• How do I calculate the Eion?• Nernst Equation, duh
What’s all this talk about driving force?
• Driving force = (Vm-Eion)• This is the potential difference that will
actually be doing the work of moving the current through the channel. • This allows you to use your i/V plot,
evaluate the relationship given a certain membrane potential, see how much current is flowing.
• Essentially normalizes the curve so that it’s like Eion was at the origin. • i = (Vm-Eion)g
Questions about this part of the homework?
Macroscopic currents
(Elbert)
Channel Gating on Macroscopic Currents
(Elbert)
Lecture 3: Ion Channel Structure
Basics
• Ion channels are proteins• Can be made of more than one protein (more
than one subunit)
• Ion channels do things• Need special machinery to do things –
domains• If the university was an ion channel, what
would be an example of a subunit and a domain?
What do ion channels need to do?
• Pass ions from the outside (extracellular space) to the inside (intracellular)• Why can’t ions pass by themselves?• Thus, ion channels need to be able to
associate water and lipids• They do because of their amino acid
sequence.
How do we deduce the structure?
• Hydrophobicity Plot – shows regions of the aa sequence that likely are in the membrane, so they likely are the membrane spanning regions.
• Glycosylation – will be on the extracellular part of the protein.
• Immunostaining – will show you if a terminus (the end of the protein) is intra or extracellular• FROM ALL OF THIS DATA, YOU CAN DRAW A
PUTATIVE STRUCTURE.
Other ways you can deduce structure:
• Imaging – really hard, but super cool! EM
• X-Ray Crystallography – also really hard, but that’s how Rod MacKinnon figured out what the Drosophilia Shaker K+ Channel looks like.
Function of ion channels
• Expression systems allow you to selectively express one channel and then measure the properties of that one type of channel. • Frog egg = your canvas• cDNA = your paint, with which you express
your feelings channels
Selectivity of Channels
Most Selective Less Selective Least Selective
VG Ligand Gated Gap Junctions
Calcium Activated K+ Channels
Non-specific cation/anion channels
Cyclic Nucleotide
Determination of Selectivity
• S5-S6 Region – Pore Forming Region• Residues here form an environment
for the ion that is as energetically favorable (comfortable) as it’s sphere of hydration (the water molecules that normally surround an ion)
Gating
• S4 is the voltage sensor• Like charges repel, so this is how it
moves!• The S4 alpha helix has a lot of positively
charged residues• So when the membrane becomes
depolarized enough (there is enough + charge on the intracellular side) it is repelled from that positive charge and moves up/out of the way
Inactivation
• This is the ball and chain model – ball will plug up channel and not let it pass more ions, and then eventually fall out so the channel can close (and then open again)• Remember, this is why we have the absolute
refractory period!
Modulation
• Modulation: changes the open probability of the channel• You are all experts at macroscopic I/V plots,
so you can reason how a modulator would affect the plot.
• MODULATORS:
Modulation
• Modulation: changes the open probability of the channel• You are all experts at macroscopic I/V plots,
so you can reason how a modulator would affect the plot.
• MODULATORS:• Other subunits of the protein (beta
subunits)• Second messengers• Changes in gene expression• Phosphorylation• Allosteric regulators
HAVE A GREAT WEEK!
Questions?