Transporters

differences from channels:speed?saturation?concentration?

--alternating access

channel

transporter

Pumps

transporters pumps ionic coupling

pumps generate the ionic gradients that drive flux through channels and through other transporters

energy provided by ATP hydrolysis

relatively small number drive flux at plasma membranesecretory pathway (including endocytic)mitochondria

P-type ATPase: Na+/K+-ATPase

phosphorylated intermediateNa/K-ATPase makes Na+, K+ gradientsH/K-ATPase acidifies stomachphospholipid flippases (maintain asymmetry of bilayer)Ca/H-ATPase removes cytosolic Ca++

(Olesen et al, 2007)

how do ATP binding, phosphorylation drive flux?

crystallization of Ca++-ATPase--in multiple conformations

acidic residues bind ionstwo gates never open togetherboth gates close when ion boundN, P, A control gateseach event triggered by previous

Rotary Club: F0F1 ATPase (ATP synthase)

H+ flux drives unidirectional rotation and ATP synthesis

(Junge et al., 2009)

matrix

cyto

V-type H+-ATPase

V-type

F0F1

uses ATP to make H+ electrochemical gradient in endosomes, lysosomes, synaptic vesicles 3 ATP/rotation (at A/B interface) (both V- and F0F1) what confers difference in direction?

transporters pumps ionic coupling

secondary active transport

big conformational changes as well as faster binding, unbindingmovement of unloaded carrier essential for net fluxmovement of unloaded carrier must be distinct from loaded

exchange reaction

--no net flux

*glucose

glucose

membrane vesicles were loaded with 14C-glucose diluted into medium containing different concentrations of non-radioactive glucose (indicated on the x-axis)

avoids movement of unloaded carriertranslocation steps slow--channel cannot do this

heteroexchangeobligate exchangers cannot do net flux amphetamines may release monoamines this way (exchange-diffusion)

Na+ cotransport

dopamine transporter

impaired dopamine clearance95% decrease in dopamine stores! --role in recycling as well as clearance

striatal slicevoltammetry

stimulation

coupling rules required for active transport--why?AND operation

stoichiometry of 1 Na+ : 1 S and ~12-fold Na+ gradientwill generate ? gradient of S

at equilibrium, equal rates in and out of cell

[Na+]o x [S]o = [Na+]i x [S]i

[Na+]o / [Na+]i = [S]i / [S]o

what if it is an exchanger?

So + Na+o Si + Na+

i

if coupling involves 2 Na+ : 1 S, then

[Na+]o2 x [S]o = [Na+]i

2 x [S]i

([Na+]o / [Na+]I)2 = [S]I / [S]o or

log10 (Sin/Sout) = 2 log10 (Na+out/Na+

in)

why not just make the stoichiometry very high?

and what if net flux involves charge movement?

2 Na+2 Na+

So + 2Na+o Si + 2Na+

i

n Na+n Na+

the negative resting membrane potential augments the chemical gradient for Na+ by

– zTVm/ 60 mV (log units)

where zT = net charge moved

added to the concentration gradient,

log10 (Sin/Sout) = n log10 (Na+out/Na+

in) – zT/ 60 mV

where n = # Na+ ions cotransported

-- the power of membrane potential!

this equation changes for different ionic coupling

electrogenic transport (transport that moves net charge)

Cl-

nNa+

gly

electrogenic transport produces currents--depend on Na+, Cl-

defined by gly addition

can measure charge:fluxusing labeled glycine, Cl

glycine transport

(Roux and Supplisson, 2000)

for electrogenic glycine transport,log10 (glyin/glyout) = m log10 (Na+

out/Na+in) + n log10 (Cl-out/Cl-in) – zT/ 60 mV

zT/ 60 mV = log10 Na+om x Cl-

n x So

Na+im x Cl-i

n x Si

= 60 mV log10 Na+om x Cl-i

n x So = Erev

zT Na+im x Cl-i

n x Si

--like Nernst equation:

ENa = 60 mV log10 Na+o/Na+

i

what are the differences?

why determine coupling?ionic coupling determines direction of flux

magnitude of gradient (can exceed 106:1)regulation by membrane potential

GlyT1 GlyT2

reversal potentials used to determine stoichiometry

GlyT1 KO: excess glycine (excess inhibition)--main role clearanceGlyT2 KO: resembles GlyR KO (startle)--main role packagingdifferences in ionic coupling can also confer transfer between cells

glia neuron

(Gomeza et al, 2003)

excitatory amino acid transporters (EAATs)

stoichiometry predicts huge gradientscontrol activation of perisynaptic receptors, spillover

(Boudker et al, 2007)

bacterial transporter trimer (bowl):

3 Na+:1 H+:1 glu- 1 K+

(Reyes et al., 2009)

transport within monomerHP1,2 the gates?structure in out and inward conformations

rigid body motion

(Wadiche et al, 1995)

glutamate-induced currents reverse for EAAT1 and 3--glutamate-gated chloride channel (receptor)

uncoupled uncoupled

glu

3 Na+:1 H+:1 glu- 1 K+

glutamate added to the outside must trigger inward + charge movement

transport cycle can gate an ion channel?evolutionary intermediate

some transporters also behave like channels

Shakervoltage-gated K+ channel

R365H

no Na+ or K+ (~gating charge)depends on H+

current flow in direction of pH gradient--voltage sensor turned into transporter

(Starace et al., 1997)

protons: the currency of organelles

SV

H+

+

+

+ +

+

+++ ++

+

Cl-

H+

H+

H+

H+

H+

ATP

ADP

H+ concentrations much lower than other ions gradients easier to make, break

--can also be regulated, such asmembrane potential without pH!!(mito have Vm ~ -160 mV, pH < 0.5)

--acridine orange quenches when trapped in acidic membranes:

in absence of Cl-, H+-ATPase makes only membrane potential

--Cl- entry required for acidification--pH, can be regulated

independently

ClC family includes Cl- channels and Cl-/H+ exchangers

intracellular ClCs mediate exchange of 2Cl- : 1 H+

why couple to H+ exchange, which ought to dissipate pH?

H+

++

+

Cl-

H+

ATP

ADP

H+

+

2 Cl-

H+H+

ATP

ADP

H+ exchange mechanism makes bigger pH?!

several crystal structures

ClC transporter is achannel with a lever arm--can count Cl- ions?!--Cl- itself a gate--alternating access without large conformational change

(Feng et al., 2010)

(Faundez and Hartzell, 2004)

what confers the progressive acidification of endosomes?

same stoichiometry of coupling (2 Cl- : 1 H+) predicts same pHsame cytosolic [Cl]

plasma membrane Na+/H+ exchangers regulate cell pHintracellular isoforms mediate K+/H+ exchange, dissipate pHmechanism to make

H+

+

+

+ +

+

+++ +

+

H+

H+

H+

H+

ATP

ADP

Na+/K+

H+

is there an opposing activity that dissipates pH?

Differences from Channels

1) Alternating access rather than poreimportance of unloaded carrier for net flux (active transport)exchange

2) Pumps use ATP to make ionic gradients3) Ionic coupling uses these gradients to drive movement of other solutes

behavior not predicted by Nernst equationdirection of flux, stoichiometry can varyreversal potential can be used to determine couplingcoupling dictates physiological role

4) Ions interact more tightly than with channel5) Transport cycle can gate channel (evolution of channel gating?)6) Bulk H+ concentrations can also change (like Ca++)

pH promoted by anion entry promoted by cation entry

BackgroundAlberts, Chapter 11Nicholls, D.G. and Ferguson, S.J. Bioenergetics 2, Academic Press, 1992. Stein, W. Channels, Carriers and Pumps. Academic Press, 1990.

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