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

4/27/16 April 27, 2016 Assignment for Weds, May 4 (Mon lab)/11 (Tues Lab) Patch Clamp Pen your poison: http://www.amnh.org/exhibitions/current-exhibitions/the- power-of-poison Power of Poison: an enchanted book- Interactive botanical book Coming Due Dates: Extra credit helping a freshman physiology lab Thursday, April 28- Results for two snail intracellular (BioNB 1445) recording labs Sunday, May 1- 7:30-8:30PM Monday May 2- Snail: Diversity of neuronal Monday, May 2- 7:30-8:30PM excitability lab Tuesday, May 3- 9:05-10:05AM and 7:30-8:30PM 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 Patch recording techniques Analyze Rinout and tau from data emailed I K Voltage drop across electrode- ignore Start tau, Vm drop I Na measure here Voltage drop across cell membrane H&H predicted separate channels for each ion, with no hard evidence Final voltage peak -0.1nA, 3s duration 1

4/27/16 Scale of biological currents Early evidence for single channels Biophysicists found antibiotic channels in black lipid membranes Lipid bilayer currents flowed in steps Patch Clamp The Nobel Prize in Physiology or Medicine 1991 Bert ¡Sakmann Erwin ¡Neher "for ¡their ¡discoveries ¡ concerning ¡ the ¡function ¡of ¡single ¡ ion ¡ channels ¡ in ¡cells" Thermal noise of charge movement 2

4/27/16 Early discoveries 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. ¡ Openings fast (10 µS) , measuring speed of protein conformational changes!!! Hamill ¡et ¡ al ¡1981 Early discoveries Early discoveries Channels follow Ohm’s law V = IR Current amplitudes change in steps 3

4/27/16 Early discoveries Early discoveries Wide range of channel conductances Defining channel kinetics for opening and closing Channel types More experimental options Single channel currents matched macro currents Na + channels K + channels First time recording from small neurons, even dendrites!! Even cell organelles! Also staining, adding experimental compounds: channel blockers, second messengers Hamill ¡et ¡ al ¡1981 4

4/27/16 Na channels don’t Channel States defined need to open to inactivate Twin pulse experiments Hodgkin and Huxley 3 states of Na channel: 4 states of Na channel: closed closed open open inactivated inactivated Mechanisms of channel modulation? Mechanisms of channel modulation Ex.: 5HT reduction of I K , leading to an enhancement of transmitter relea se Example- 5HT closes channels + 5HT: Possibilities: 5

4/27/16 Problems : Problems : Washout of cell metabolites Sometimes need clean membranes Activation of histamine release from mast cells Subconductance states- ACh receptors) Technology can determine results and interpretation- Anesthetic example Subconductance states Perforated patch recordings maintain intracellular integrity Chapman et al 1997 6