4/27/16 April 27, 2016 Assignment for Weds, May 4 (Mon lab)/11 - - PDF document

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April 27, 2016

Coming Due Dates: Thursday, April 28- Results for two snail intracellular recording labs Monday May 2- Snail: Diversity of neuronal excitability lab Friday, May 6, Snail: Ionic basis of excitability lab Weds, May 4, Monday lab-Pen your Poison people Thursday, May 5, Fly results Monday, May 9- Fly report Weds, May 11, Tuesday lab- Pen your Poison Extra credit helping a freshman physiology lab (BioNB 1445) Sunday, May 1- 7:30-8:30PM Monday, May 2- 7:30-8:30PM Tuesday, May 3- 9:05-10:05AM and 7:30-8:30PM

Patch Clamp

Assignment for Weds, May 4 (Mon lab)/11 (Tues Lab) Pen your poison: http://www.amnh.org/exhibitions/current-exhibitions/the- power-of-poison Power of Poison: an enchanted book- Interactive botanical book

Voltage drop across electrode- ignore Voltage drop across cell membrane Start tau, Vm drop measure here Final voltage peak

• 0.1nA, 3s duration

Analyze Rinout and tau from data emailed H&H predicted separate channels for each ion, with no hard evidence INa IK Patch recording techniques

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Scale of biological currents Biophysicists found antibiotic channels in black lipid membranes Lipid bilayer currents flowed in steps Early evidence for single channels

Bert ¡Sakmann Erwin ¡Neher

"for ¡their ¡discoveries ¡ concerning ¡ the ¡function ¡of ¡single ¡ ion ¡ channels ¡ in ¡cells"

The Nobel Prize in Physiology or Medicine 1991

Thermal noise of charge movement

Patch Clamp

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The ¡Gohm seal ¡is ¡possible: 1) ¡the ¡membrane ¡lipid ¡bilayer is ¡ attracted ¡to ¡the ¡surface ¡of ¡electrode ¡glass. 2) ¡the ¡membrane ¡is ¡fluid, ¡ allowing ¡it ¡to ¡ be ¡pulled ¡into ¡the ¡electrode ¡tip. ¡

Hamill ¡et ¡ al ¡1981

Openings fast (10 µS) , measuring speed of protein conformational changes!!!

Early discoveries

Current amplitudes change in steps

Early discoveries

Channels follow Ohm’s law V = IR

Early discoveries

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Wide range of channel conductances

Early discoveries

Channel types Defining channel kinetics for

• pening and closing

Early discoveries Na+ channels K+ channels Single channel currents matched macro currents

Also staining, adding experimental compounds: channel blockers, second messengers

More experimental options

First time recording from small neurons, even dendrites!! Even cell organelles!

Hamill ¡et ¡ al ¡1981

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Channel States defined

Twin pulse experiments

Hodgkin and Huxley 3 states of Na channel: closed

• pen

inactivated Na channels don’t need to open to inactivate 4 states of Na channel: closed

• pen

inactivated Mechanisms of channel modulation?

Ex.: 5HT reduction of IK, leading to an enhancement of transmitter relea se

Possibilities: + 5HT: Example- 5HT closes channels Mechanisms of channel modulation

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Sometimes need clean membranes Subconductance states- ACh receptors) Technology can determine results and interpretation- Anesthetic example Problems:

Chapman et al 1997

Subconductance states Perforated patch recordings maintain intracellular integrity

Problems: Washout of cell metabolites

Activation of histamine release from mast cells