Transcript 19_RobertEdwards_Lec2

Pumps
make ionic gradients that drive
channel currents
transporter flux
energy usually provided by ATP hydrolysis
relatively small number at
plasma membrane
mitochondria
secretory pathway (including endocytic)
P-type ATPase: Na+/K+-ATPase
phosphorylated intermediate
Na/K-ATPase makes Na+, K+ gradients
H/K-ATPase acidifies stomach
phospholipid flippases (maintain asymmetry of bilayer)
Ca/H-ATPase removes cytosolic Ca++
Rotary Club: F0F1 ATPase (ATP synthase)
matrix
(Junge et al., 2009)
cyto
H+ flux drives unidirectional rotation and ATP synthesis
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)
1 H+ per c subunit
Problem of Week: what confers difference in direction? why?
2o active transport
flux involves conformational change
alternating access
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)
ionic coupling: Na+ cotransport
H+
NT
VNT
A
glnase
N
gln
PNT
Na+
Na+
EAAT1,2
NT
gln
synthase
glu
EAAT3
dopamine transporter
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?
ions bound by charged residues
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
– zTVm / 60 mV (log units)
where zT = net charge moved
added to the concentration gradient,
log10 (Sin/Sout) = n log10 (Na+out/Na+in) – zTΔ / 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)
Problem 5: what are the gradients predicted for GlyT1 and GlyT2?
for electrogenic glycine transport,
log10 (glyin/glyout) = m log10 (Na+out/Na+in) + n log10 (Cl-out/Cl-in) – zTΔ / 60 mV
what is log10 (glyin/glyout) for GlyT1 and GlyT2
zTΔ / 60 mV = log10 Na+om x Cl- n x So
Na+im x Cl-in x Si
Δ = 60 mV log10 Na+om x Cl-on x So
zT
Na+im x Cl-in x Si
--like Nernst equation:
ENa = 60 mV log10 K+o/K+i
what are the differences?
= Erev
(Gomeza et al, 2003)
glia
neuron
GlyT1 KO: excess glycine (excess inhibition)--main role clearance
GlyT2 KO: resembles GlyR KO (startle)--main role packaging
differences in ionic coupling can also confer transfer between cells
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
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)
glutamate-induced currents reverse for EAAT1 and 3
--glutamate-gated chloride channel (receptor)
some transporters also behave like channels
transport cycle can gate an ion channel
?evolutionary intermediate
H+ concentrations much lower than other ions
gradients easier to make, break
regulation of H+-ATPase by DµH+
in absence of Cl-, H+-ATPase makes
only membrane potential
Cl- entry required for acidification
--DpH, Dy regulated independently
--acridine orange quenches when
trapped in acidic membranes:
ATP
H+
+ + +
+
H+
H+
H+ +
+
H+ +
+
H
+
+ + +
ADP
ClSV
H+ electrochemical gradient
can also be regulated, such as
membrane potential without DpH!!
(mito Dy ~ -160 mV, DpH < 0.5)
vesicular neurotransmitter transport
DmH+ = DpH + Dy
XAR
bacterial Cl- carrier a member of the ClC family of Cl- channels
--pH-sensitive
bacterial ClC does not follow Nernst equation for Cl--it is a Cl-/H+ exchanger!!
bacterial, endolysosomal 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?!
glutamate can also acidify SVs
(Hnasko et al., 2010)
glutamate (VGLUT) acidifies more than Cl- at low concentrations
Cl- acidifies faster, and more at higher concentrations
what confers the Cl- conductance?
(Bellocchio et al., 2000)
VGLUTs confer a chloride conductance
ATP
H+
ADP
H+
Cl/?glu
H+
Cl-
+
compete for membrane potential?
what is lumenal [Cl-]?
compete for permeation?
1 or 2 pathways?
H+
NHE1
(Ohgaki et al., 2011)
NHE6 mutations cause severe intellectual disability
NHE9 mutations cause autism
Na+
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, promote vesicular glutamate transport
--new methods available to record directly from endosomes
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) Bulk H+ concentrations can also change (like Ca++)
DpH promoted by anion entry
Dy promoted by cation entry
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.
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