1.!What!are!membranes?!

Lecture'1'–'Single'cell'electrophysiology:'concepts'and'methods'I' ! 1.!What!are!membranes?! Critical(to(all(cells(in(the(body.( Plasma(membrane(is(important(in(separating(extracellular(solutions(from(cytoplasm(!(vital(for(cell(function.(( Permeability(barriers(in(one(respect(and(also(have(to(consider(membranes(with(individual(organelles.( But(not(just(a(permeability(barrier(–(also(regulates(cell(function(and(factors(associated(with(regulating(solutes.( ( 2.!What!are!membranes!made!of?! Phospholipid(bilayer.(( ~10nm(thick((which(allows(transport(across(and(also(creation(of(electrical(field(via(the(separation(of(charge).( ( 3.!What!is!the!structure!of!this!bilayer?! Polar(heads(and(hydrophobic(tails(on(inside.(( It(is(a(fluid(structure(–(made(of(phospholipids(which(interact(with(each(other.( Fluidity(is(relative(to(how(closely(packed(the(phospholipids(are,(types(of(phospholipids(present,(saturated/unsaturated( fatty(acid(chains,(length(of(fatty(acid(side(chains((effect(thickness(of(membrane),(presence(of(other(structure(e.g.( cholesterol((binds(tight(and(increases(stiffness(of(membrane(as(well(as(integral(proteins).( ( 4.!What!can!pass!through!the!this!phospholipid!bilayer?! • Small(hydrophobic(molecules(can(easily(diffuse(through((fast)( • Small!uncharged!polar!molecules!can!quite!easily!too! • Larger!uncharged!polar!molecules!find!it!difficult! • In!positively!charged!solutes:!presence!of!charge!makes!it!hard!to!passively!diffuse!through.! ! 5.!What!is!the!relationship!between!membrane!permeability!of!a!solute!and!molecular!weight?! More(lipid(solute/smaller(weight(!(easier(passing(through(the(membrane(( ( 6.!How!do!special!solutes!travel!through!though?! Metabolic(substances,(nutrients(etc.( Integral(and(intrinsic(proteins(help(with(this(–( provide(an(alternate(pathway.( Can(be(channel(or(transport(mediated.( ( 7.!What!are!channel!mediated?! Open(like(a(door(down(the(concentration(gradient,( quite(rapid( ( 8.!What!are!transport!mediated?! Protein(requires(binding(of(molecule,(conformational(change(to(protein(to(occur(resulting(in(transport(of(solute.(( Rate(is(severely(mediated(therefore(takes(time.( Shows(a(saturation(limit.(( ( 9.!What!are!the!kinds!of!transport!mediated?! • Uniport:!Movement!of!single!molecule!down!its!CG!to!other!side( • Coupled:!Symport!or!Antiport.!Both!cases!of!coupled!sue!energy!of! CG!for!one!solute!to!move!another!solute!against!its!CG.!Can!be! moving!those!solutes!in!same!direction!or!opposite!e.g.!Sodium! glucose!transporter.!Strong!concentration!of!Na!doesn!its!CG!but!in! order!for!transporter!to!work!you!need!binding!of!Na!and!glucose!molecule!to!generate!conformational! change.!An!example!of!an!Antiport!is!the!sodium!hydrogen!transporter.!( ! 10.!What!are!the!differences!between!active!and!passive!transport?! !Active(usually(moves(against(CG(and(requires(energy,(passive(does(not(require(energy.(( 11.!What!are!the!nature!of!ion!channels!in!a!membrane?! Can(have(leakage(channels((always(open)(or(gated(ion(channels((voltage,(ligand(or(stretched(activated).((

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12.!What!are!ungated!channels?! Also(known(as(leak(channels(–(always(open(and(allow(passive(movement(of(ions(down(CG.( ( 13.!What!are!gated!channels?! • Voltage:(Change(in(voltage(across(membrane(causes(conformational( change(in(ion(channel(causing(movement(down(CG.((Voltage(gated( Na/K(channels(–(molecule(binds(to(protein(causing(conformational( change(therefore(opening(up(ion(channel(e.g.(acetylcholine(receptors( in.( • Ligand:(binding(of(channel(to(ligand(causes(conformational(change.( • Stretch(activated:(e.g.(motor(end(plate.(Mechanical(deformation(of( cytoskeleton(causing(opening(of(ion(channel((common(in(sensory( transduction)(e.g.(muscle(spindles(mechanoreceptors( ! 14.!What!are!the!2!components!in!an!electrochemical!gradient?! Concentration(gradient(and(membrane(potential.( ( 15.!What!does!concentration!gradient!refer!to?! Applied(to(nonTcharged(solutes(too.( • When(2(forces(are(working(in(the(same(direction,(high(outside(and(low( inside(there(is(a(strong(driving(force(from(CG(driving(in(ions.(Both(chemical( and(electrical(forces(working(in(same(direction.(Final(movement(of(ions( depends(on(their(permeability.(Typically(scenario(for(Na+(ions(within(body.(( • When(CG(is(going(the(opposite(way(–(acting(to(move(ions(out(of(the(cell( electrical(force(is(acting(on(them(to(hold(them(within(cell(therefore( competing(forces(therefore(the(amount(of(ions(moving(is(considerably(less( than(example(before(because(of(opposing(forces(e.g.(K+(scenario.( ! 16.!What!cells!are!ion!channels!and!membrane!potentials!important!in?! ALL(cells(not(just(excitable(cells.( Osmotic(balance(in(cells(–(ions(are(most(abundant(dissolved(solutes.( Ion(flows(and(voltages(can(control(fluid(flows(in(specialised(epithelia(–(secretion(and(absorption.( Ion(flows(and(voltages(control(many(any(phenomena:( • Sensory!signalling,!force!generation!Ca,!intracellular!enzyme!cascades!and!gene!expression,!cell!growth!and! cell!death.!(NOT!just!dealing!with!action!potentials)( ! 17.!What!is!osmolarity?! The(cumulative(number(of(all(individual(particles(in(sample(of( water.(( Changes(in(osmolarity(!(swelling/lysis(can(effect(cell(function.(( ( 18.!As!shown!on!the!right,!discuss!membrane!voltages!in!nonZ excitable!cells.! In(renal(tubules(and(small(intestines.( Different(expression(of(different(ion(channels(on(apical(and( basolateral(membranes.( Apical(–(Na+(pump(and(cotransporting(glucose(against(CG( Basolateral(–(Na/K(pump((2(Na(out(and(3(K(in)(.(Constant(movement(of(Na(out(helps(keep(the(concentration(of(Na(low( allowing(movement(into(the(cell(on(apical(side.(( !(This(all(develops(a(trans(epithelial(voltage( Build(up(of(cations(causes(attraction(of(chloride(therefore(diffuses(down(electrochemical(gradient(to(positive(charge(until( balanced.(But(increase(in(solutes(on(tight(therefore(increased(osmolarity(therefore(water(movement(down(its(CG.(( ( ! !

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19.!How!can!membrane!potential!be!measured?! Can(record(using(intracellular(microelectrode(recording(e.g.(motor(neuron(T(put(tip(of(microelectrode(into(cell(body( without(damaging(membrane(–(compare(voltage(inside(and(outside(the(cell(therefore(getting(voltage(across(membrane.( Can(also(get(resting(MP(from(this(kind(of(recording((~80mV).( Also(changes(in(MP(when(injecting(current(into(cell.( • This(will(reduce(negativity(of(cell,(becoming(more(positive(therefore(getting(depolarisation(of(membrane(and( if(it(reaches(a(critical(point(we(will(get(opening(of(voltage(gated(channels(( ( 20.!How!is!a!membrane!potential!established?! Net(negativity(inside(cell(!(caused(by(3(things( 1. Trapped(organic(ions(T(Because(of(size(they(cannot(diffuse(out( 2. Na/K(pump(–(Na/K(CG(give(net(electrical(negativity,(also(enzyme(Na+/K+(ATPAase(pumps(3Na(out(and(2(K(in( therefore(builds(up(a(negative(charge(inside(cell( 3. Cell(membrane(preferentially(permeable(to(K+(T(presence(of(selectively(permeable(ion(channels(in(membrane( have(dominance(currently(to(membrane(permeability(of(K( ( 21.!How!does!selective!permeability!lead!to!a!separation!of!charge!and!voltage!difference?! Channel(permeable(only(to(K+(resting(potential(is(generated(by(the(efflux(of(K+(down(its(CG.( Continued(efflux(of(K+(builds(up(an(excess(of(positive(charge(on(outside(of(cell(and(excess(of(negative(charge(on(inside(of( cell.( Build(up(of(charge(impedes(further(efflux(of(K+.( Eventually(an(equilibrium(is(reached(–(electrical(and(chemical(driving(forces(are(equal(and(opposite.(( ( 22.!What!are!diffusion!potentials?! Voltage(generated(is(proportional(to(concentration(gradient.( Because(amount(of(charge(movement(is(small(voltage(is(generated(without(significant(changes(in(CG.( Voltage(is(not(directly(dependent(on(metabolic(energy.(( No(special(pumps/channels(are(involved(in(generation(of(diffusion(potential,(no(input(of(metabolic(energy.( ( 23.!What!is!membrane!capacitance?! Property(of(an(electric(conductor(that(characterises(its(ability((to(store(an(electric(charge.(( Voltage(developed(is(proportional(to(amount(of(charge(separated((Q)(and(inversely(proportional(to(the(membrane( capacitance((C).( V(=(Q/C( The(large(the(cell(membrane(area(the(larger(the(total(capacitance(and(hence(the(more(charge(has(to(be(separated(to( develop(a(given(voltage.(( ( 24.!How!does!the!separation!of!a!very!small! number!of!charges!generate!a!large!voltage?! As(shown(by(the(picture(on(the(right.( On(right(–(the(number(of(anions(needed(to(be( separated(in(order(to(build(up(90mV(of(charge( required.( On(left(–(amount(of(ions(available.(( ! 25.!What!is!the!Nernst!equation?! At(equilibrium,(chemical(potential(is(equal(to( electrical(potential.( Concentration(gradient(balanced(by(the( voltage(gradient.(( ! 25.!How!do!you!calculate!electrochemical! potential!energy!difference?! ! !

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26.!What!is!Ex?!What!is!it!dependent!on?! The(value(Vm(would(have(to(have(for(ion(X(to(be(in( equilibrium.( Dependent(on(temperature(and(valance.( It(is(the(theoretical(Nernst(potential(for(ion(X(whereas( Vm(is(the(actual(membrane(potential.(( ( 27.!What!does!it!mean!if!Vm=Ex?! Then(ion(x(must(be(in(electrochemical(equilibrium.(( No(net(flow.( ( 28.!What!does!it!mean!if!Vm!does!not!equal!Ex?! Then(ion(x(cannot(be(in(electrochemical(equilibrium(and(the(net(flow(measured(as(a(flow(of( ions(or(current((I).( ( 29.!How!do!you!calculate!net!ion!flow/current?!! ! Gx!=!membrane!conductance!(number!and!type!of!ion!channels)! Ex!=!driving!force!(Nernst!potential)! ! 30.!What!is!an!IV!curve?! Demonstrates(relationship(between(membrane(potential(and(current(flow.(( ( 31.!What!does!it!mean!when!Vm!=!Ex!on!an!IV!curve?! =(ion(is(in(electrochemical(equilibrium(and(there(is(no(net(ion(flow.( Not(=(net(current(flow(either(into(of(out(of(the(cell.( For(a(given(transmembrane(voltage,(the(lower(the(resistance( (higher(conductance)(the(greater(the(current((steeper(slope)( !

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