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 rachel_a_kaplan@brown.edu• or rakaplan@brown.edu, 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?