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Lectures 1,2,3 Rachel A. Kaplan and Elbert Heng 2.3.14.

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Presentation on theme: "Lectures 1,2,3 Rachel A. Kaplan and Elbert Heng 2.3.14."— Presentation transcript:

1 Lectures 1,2,3 Rachel A. Kaplan and Elbert Heng

2 Announcements Detailed Answers for PS1 are on wiki Contacting TAs o please use piazza, you’ll get the fastest response o if you don’t want to, (not canvas message) us o if you’re ing Rachel  her is or either one is Tell us what you want us to cover! That’s what we’re here for!

3 Lecture 1: Intro to Electrophysiology

4 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

5 Studying Ion Channels ●Driving force ●Current vs. Voltage clamp ●Hodgkin & Huxley: squid giant axon ●Neher & Sakman: patch clamp ○inside out patch ○outside out patch

6 aka On Cell Cell Attached

7 Whole Cell

8 Inside-Out

9 Outside-Out

10 Lecture 2: Ion Channel Function

11 Current Clamp

12 Voltage Clamp

13 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!

14 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

15 Drawing i/V Curves If you want to draw a linear curve, what do you need?

16 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) – E ion or V rev (these two are the same for channels that pass only one type of ion)

17 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 E ion ? Nernst Equation, duh

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19 What’s all this talk about driving force? Driving force = (V m -E ion ) 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 E ion was at the origin. i = (V m -E ion )g

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21 Questions about this part of the homework?

22 Macroscopic currents (Elbert)

23 Channel Gating on Macroscopic Currents (Elbert)

24 Lecture 3: Ion Channel Structure

25 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?

26 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.

27 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.

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29 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.

30 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

31 Selectivity of Channels Most SelectiveLess SelectiveLeast Selective VGLigand GatedGap Junctions Calcium Activated K+ Channels Non-specific cation/anion channels Cyclic Nucleotide

32 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)

33 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

34 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!

35 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:

36 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

37 HAVE A GREAT WEEK! Questions?


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