VL 39 (Otto Baumann) Flashcards
Ion Inside & Outside of a Mammalian Cell
Note: [negative charges]cytosol ≈ [positive charges]cytosol
Conclusion:
there are huge concentration gradients for various ions across the plasma membrane
Ion Gradients = Stored Energy
Ion gradients
- are actively maintained
- represent stored energy
organisms spend 10-60% of their ATP for the maintenance of ion gradients
Various functions:
* signal transduction (e.g. action potential, Ca2+ entry)
* uptake/efflux of nutrients, metabolites, salts, toxic solutes volume regulation
* pH regulation
* H+ (or Na+)-driven ATP synthesis
* H+-driven flagellar rotation
Permeability of an Artificial Membrane
Carriers & Channels
biological membranes require mechanisms for the transmembrane transport of various ions & molecules
Passive vs. Active Transport
Passive: with concentration gradient (simple diffusion, channel mediated, carrier mediated)
Active: against concentration gradient
(always needs energy)
What is the electrochemical gradient?
Ion Influx:
conc. gradient > electr gradient
Ion efflux:
conc. gradient > electr. gradient
Net movement = 0
conc. gradient = electr. gradient
Use Nernst Equation to determine equilibrium potential
The Nernst Equation
R = gas constant (8,3144 J* mol-1K-1)
T = absolute Temperature (Kelvin)
z = ion charge
F = Farady constant 96485,3365 J* V-1mol-1
Where does the Energy for active transport comes from?
-
Secondary active transport
( coupled carrier) - large protein family
- transport of ions or metabolites
- transport coupled to Na+ or H+
- dimers / pseudodimers
- substrate binding site at the interface between both parts
-
primary active transport
(ATP-.>ADP+P)
What are Co-transport mechanisms?
3 diffrent kinds:
* Uniport
* Symport
* Antiport
Example: for Antiport
NA+/Ca+ Exchanger (NCX)
* localized in the plasma membrane
* α1 and α2 repeats
* ion binding
* cytosolic domains
–> regulatory function
* stoichiometry 3 x Na+ / 1 x Ca2+
–> high capacity Ca2+ extrusion
3 NA+ rein und 1 Ca2+ raus
Ion transport ATPases
Explain the Sodium Pump (P-Type ATPAse)
Na+/K+-ATPase = Sodium pump
general structure:
* α subunit of̴ 105 kDa with 10 transmembrane segments
–> ATP-hydrolysis & ion transport
* glycosylated β subunit
–> required for the transport of
newly synthesized pumps to the PM
general function:
* establishment of K+ and Na+ gradients across the PM
exchanges 3 Na+ for 2 K+
–> electrogenic (inside –> negativ)
- large conformational changes –>
2 distinct enzymatic states:
E1 & E2 - conformational change triggered by autophosphorylation & dephosphorylation on a conserved Asp residue
Picture:
blue: K+
yellow: Na+
Explain ABC Transporters: Floppase
Floppase: (ABC Transporter) moves phospholipids from cytosolic to outer leaflet
Flippase: (P-type ATPase) moves PE and PS from outer to cytosolic leaflet
Scramblase moves lipids in either direction, toward equilibrium
- ATP binding at NBD / NBD interface
- substate binding site faces either outward or inward
- ATP binding –> outward-facing conformation
- ATP hydrolysis –> inward-facing conformation
Explain the V-type ATPase
- ~ 900 kDa
- 14 different subunits / in total ~ 30 subunits
- V1 domain: ~ 650 kDa, A3B3CDE3FG3H
- VO domain: ~ 260 kDa, ac8c´c´´de
Function:
* Vacular membranes in plants, yeast other fungi
* Endosomal ans lysosomal membranes in animal cells
* Plasma membrane of osteoclasts and some kidney cells
Gap junctions
- pore Ø ~1.4 nm
- passage of molecules ≤1000 Da (anorganic ions, incl. Ca2+; H2O; sugars; AA; metabolites; nucleotides incl. ATP & cAMP; IP3)
- no passage of proteins
- can be closed (regulated by e.g. Ca2+)
Ion channels
What is a K+ leak channel?
- Two pore domain channel
- structure:
–> 4 transmembrane helices (M1-M4)
–> 2 pore-forming domains (P1, P2) of each monomer
–> homodimer
Explain the resting potential
provided that the plasma membrane is only permeable to K+
and [K+] in= 140 mM, [K+] out = 7 mM,
the resting membrane potential is -77mV
E(K) = 59 mV / z * loh ([K+] out / [K+] in)
E(K) = -77mV
Picture:
* K+ leaks from inside → outside via leak K+ channel → neg. charge inside vs. outside
* membrane impermeable to Na+ (all Na+ channels closed)
* -77 mV typical resting potential in neurons/muscle cells
Gating of channel ions
- Mechanically: tension → opens
- Phosphorylatio-gated ion channels (not in picture)
e.g Voltage gated K+-Channels
* 4 SU, each SU with 6 transmembrane segments + pore-forming domain (points towards pore center)
* Linked transmembrane domains → monomer
* Similar structure → pseudotetramer
Voltage sensor of Ion channels
- epolarization moves extracellular S4 segment part outward through short hydrophobic gating pore→opening permeation pathway
- most S4 segment surrounded by hydrophilic vestibules
- transmembrane electric field falls mainly across gating pore
What is the neuronal action potential?
- Voltage-gated Na+, K+ channels required
- Opening → sodium in axon → depolarisation → channel closes → inactivated (inactivation domain blocks pore) → potassium channels open → resting potential reestablished → inactivation reversed → channel still closed, but can be activated again
Picture:
* action potential elicited by short voltage pulse → depolarization (B)
* green curve: membrane potential would fall back to resting state after depolarizing stimulus if there were no voltage- controlled sodium channels
* red curve: action potential evoked by opening, subsequent inactivation of voltage-controlled sodium channels.
* membrane cannot establish 2nd action potential until sodium channels have returned from inactivated → closed state → membrane ready for new stimulation
* channel open (< 1 ms) → inactivation
* → hyperpolarisedrestingpotential → restingstate(closed)
Patch Clamp:
Action potential propagation
* open Na channel → Na influx → membrane depolarization → AP
* membrane depolarization propagation passive and with attenuation in both directions o refractory Na channels → no new AP
* unopened Na channels → AP
* →one direction of conduction (decreasing amplitude (with decrement, local response))
Picture 1:
→ study ionic currents inindividual isolated living cells, tissuesections, or patches of cell membrane
* Whole-cell recording: micropipette in tight contact with cell membrane → prevents current leakage; voltage applied → forming: voltage clamp → membrane current measured
* Inside-out recording: attach cell membrane to tube → exposing its cytosolic surface
* This gives access to the surface through the electrolyte solution bath. This method is used when changes
are being made at the intracellular surface of the ion channels.
* Outside-out recording: membrane ruptured → electrode out of cell → original outside is now on the inside
→ enabling studies of the inner membrane surface
Picture2:
* Voltage control across membrane
* → record coloumns
* channels open at depolarisation beginning
* K: channels open delayed
Picture 3:
* Apply voltage → measure current (linear relation; → channel open all the time; voltage-independent)
* Top right: below voltage no current (closed), above (open)
* Bottom right: open channel at neg. voltages, closed at
depolarisation?
