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Na+
K+
Ca++
(TC Sudhof, 2008)
pumps
secondary active transporters: ionic coupling
capacitative charge movement
uncoupled currents
regulation of vesicular DµH+ as DpH or Dy
Transporters
differences from channels:
speed?
saturation?
concentration?
channel
transporter
--alternating access
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 membrane
mitochondria
secretory pathway (including endocytic)
P-type ATPase: Na+/K+-ATPase
Na/K-ATPase makes Na+, K+ gradients
H/K-ATPase acidifies stomach
phospholipid flippases (maintain asymmetry of bilayer)
Ca/H-ATPase removes cytosolic Ca++
--all have phosphorylated intermediate
how do ATP binding, phosphorylation
drive flux?
crystallization of Ca++-ATPase
--in multiple conformations
acidic residues bind ions
two gates never open together
N, P, A control gates
each event triggered by previous
(Olesen et al, 2007)
F0/F1-ATPase
vacuolar H+-ATPase
distinct proteins that mediate flux in opposite directions
to synthesize ATP in mitochondria
or to make a H+ electrochemical gradient
in endosomes, lysosomes, synaptic vesicles
what determines the direction of flux?
3 ATP/cycle
1 H+/c subunit
F0/F1 8-15 c subunits (3-5 H+/ATP)
V-ATPase 5-6 c subunits (2 H+/ATP)
secondary active transport: ionic coupling
big conformational changes as well as faster binding, unbinding
movement of unloaded carrier essential for net flux
movement of unloaded carrier must be distinct from loaded
exchange reaction
--no net flux
membrane vesicles were loaded with 14C-glucose
diluted into medium containing different concentrations of
non-radioactive glucose (indicated on the x-axis)
*glucose
glucose
avoids movement of unloaded carrier
translocation steps slow
--channel cannot do this
heteroexchange
obligate exchangers cannot do net flux
amphetamines may release monoamines this way (exchange-diffusion)
reuptake: dopamine transporter (DAT)
striatal slice
voltammetry
stimulation
impaired dopamine clearance
95% decrease in dopamine stores!
--role in recycling as well as clearance
coupling rules required for active transport--why?
AND operation
ionic coupling
So + Na+o
Si + Na+i
stoichiometry of 1 Na+ : 1 S and ~12-fold Na+ gradient
will 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?
2 Na+
2 Na+
So + 2Na+o
if coupling involves 2 Na+ : 1 S, then
[Na+]o2 x [S]o = [Na+]i2 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?
Si + 2Na+i
electrogenic transport (transport that moves net charge)
n Na+
n Na+
the negative resting membrane potential
augments the chemical gradient for Na+ by
– zTD/ 60 mV (log units)
where zT = net charge moved and D is Vm
added to the concentration gradient,
log10 (Sin/Sout) = n log10 (Na+out/Na+in) – zTD/ 60 mV
where n = # Na+ ions cotransported
-- the power of membrane potential!
this equation changes for different ionic coupling
glycine transport
nNa+
gly
Clelectrogenic transport
produces currents
--depend on Na+, Cldefined by gly addition
can measure charge:flux
using labeled glycine, Cl
(Roux and Supplisson, 2000)
why determine coupling?
ionic coupling determines direction of flux
magnitude of gradient (can exceed 106:1)
regulation by membrane potential
for electrogenic glycine transport,
log10 (glyin/glyout) = m log10 (Na+out/Na+in) + n log10 (Cl-out/Cl-in) – zTD/ 60 mV
zTD/ 60 mV = log10 Na+om x Cl- n x So
Na+im x Cl-in x Si
D = 60 mV log10 Na+om x Cl-i n x So
zT
Na+im x Cl-in x Si
--like Nernst equation:
ENa = 60 mV log10 Na+o/Na+i
what are the differences?
= Erev
mechanism: chloride dependence (GAT-1)
Cl-dependent
(Zomot et al., 2007)
Na+ requires anion (Cl- or acidic residue)
unlike pore of channel, ions interact with charged residues
excitatory amino acid transporters (EAATs)
bacterial transporter
trimer (bowl):
3 Na+:1 H+:1 glu-
1 K+
(Boudker et al, 2007)
stoichiometry predicts huge gradients
control activation of perisynaptic receptors, spillover
mechanism
transport in monomer
HP1,2 the gates?
structure in out and
inward conformations
rigid body motion
(Reyes et al., 2009)
3 Na+:1 H+:1 glu-
1 K+
glutamate added to the outside must trigger inward + charge movement
glu
(Wadiche et al, 1995)
stoichiometrically coupled charge movement
capacitative charge movement
sound amplification by outer hair cells
membrane potential changes OHC shape
prestin selectively expressed by OHCs
sequence suggests a transporter
3 Na+:1 H+:1 glu-
1 K+
glutamate added to the outside must trigger inward + charge movement
glu
uncoupled
uncoupled
(Wadiche et al, 1995)
uncoupled charge movement
some transporters also behave like channels
transport cycle can gate an ion channel
?evolutionary intermediate
Shaker
voltage-gated K+ channel
R365H
no Na+ or K+ (~gating charge)
but not capacitative
persists due to net flow of ions
depends on H+
current flow in direction of pH gradient
--voltage sensor turned into transporter
(Starace et al., 1997)
(TC Sudhof, 2008)
ATP
regulation of DµH+: DpH
--can be expressed as
membrane potential without DpH!!
(mito have Dy ~ -160 mV, DpH < 0.5)
SV
+ + +
+
H+
H+
H+
+
unlike Na+, K+, Cl- gradients,
H+ gradients can be regulated
H+
+
H+
H+
ADP
+
+
+ + +
Cl-
--acridine orange self-quenches
when trapped in acidic membranes
in absence of Cl-, H+-ATPase makes
only membrane potential
--Cl- entry required for acidification
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 DpH?
ATP
+
+
+
ATP
H+
+ + +
H+
+
ADP
+
+
+ + +
H+
ADP
+
H+
+
H+
+
+
Cl-
2 Cl-
H+ exchange mechanism makes bigger DpH?!
several crystal structures
ClC transporter is a
channel with a lever arm
--can count Cl- ions?!
--Cl- itself a gate
--alternating access without
large conformational change
(Feng et al., 2010)
glutamate also acidifies (VGLUTs)
VGLUTs recognize glutamate, not aspartate
glutamate acidifies more than Cl- at low concentrations
Cl- acidifies faster, and more at higher concentrations
glutamate and Cl- have additive effects on DpH
vesicles acidified with glu retain DpH longer
accounts for dopamine storage promoted by glu
ratchet confers unidirectionality
H+
glu-
SVs acidified with glutamate make bigger
and more stable DpH due to
1) buffering better by glutamate than Cl2) failure of glutamate to efflux
(due to DpH and H+ exchange mechanism)
glutamate “locks” H+ and hence monoamine inside SV
mechanism may be general
ATP
H+
H+
H+
ADP
H+
H+
H+
+baf
-
H+
-
H+
H+
H+
H+
H+
-
H+
Cl-
H+
H+
H+
-
H+ glu-
H+
why do SVs retain DpH longer with glutamate?
--anion must leave SV to maintain D
what confers the progressive acidification of endosomes?
(Faundez and Hartzell, 2004)
same stoichiometry of coupling (2 Cl- : 1 H+) predicts same DpH
is there an opposing activity that dissipates DpH?
ATP
+
+
+
H+
+
H+
H+
+
ADP
H+
H+
H+
+ + +
+
+
Na+/K+
plasma membrane Na+/H+ exchangers regulate cell pH
intracellular isoforms mediate K+/H+ exchange, dissipate DpH
mechanism to make Dy
vesicular neurotransmitter transporters
DpH > Vm
Vm > DpH
transport of all classical transmitters into synaptic vesicles
depends on H+ e-c gradient
BUT different transmitters depend on different components (Dy and DpH)
Differences from Channels
1) Alternating access rather than pore
importance of unloaded carrier for net flux (active transport)
exchange
2) Pumps use ATP to make ionic gradients
3) Ionic coupling uses these gradients to drive movement of other solutes
behavior not predicted by Nernst equation
direction of flux, stoichiometry can vary
reversal potential can be used to determine coupling
coupling dictates physiological role
4) Ions interact more tightly than with channel
5) Transport cycle can gate channel (evolution of channel gating?)
6) Regulation of DµH+ by chloride (ClCs?), glutamate (VGLUT)
and cations (?NHEs)
Background
Alberts, Chapter 11
Nicholls, D.G. and Ferguson, S.J. Bioenergetics 2, Academic Press, 1992.
Stein, W. Channels, Carriers and Pumps. Academic Press, 1990.
References
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