Membrane transport Lecture1 by cgg10267

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									Membrane transport

    Lecture 1


                      Cell biology 2008

                    Stine Falsig Pedersen
                                     1
                           Dept. of Biology
                 University of Copenhagen
 Membrane transport is the basis for the ionic differences between cell
  and environment, and therefore, a fundamental prerequisite for life




Boron and Boulpaep (2002)                                            2
    Medical Physiology
         Membrane transporters and disease
•   K+ channels: mutations in KCNQ channels cause cardiac
    arrhythmias, deafness, epilepsy

•   Cl- channels: mutations in CFTR cause cystic fibrosis, mutations
    in ClC channels cause deafness, kidney stones, myotonia,
    neurodegeneration

•   TRP (Na+, Ca2+) channels: mutations cause multiple sensory
    dysfunctions, and polycystic kidney disease

•   Na+/H+ exchangers: excessive activity plays major roles in
    ischemic cell death, and in cancer development; loss of function
    causes epilepsy

•   Aquaporins: loss of function associated with brain edemas, vision
    defects, kidney disease

•   ....... And there are many many more                                3
             The membrane transport lectures


1. The foundations: the membrane, types of transporters, driving
   force for transport, Vm, methods

2. Channels: ion channels and aquaporins

3. Carriers: uniporters, antiporters, cotransporters, and pumps

4. Physiology and pathophysiology of membrane transport


                                                                  4
             The membrane transport lectures


1. The foundations: the membrane, types of transporters, driving
   force for transport, Vm, methods

2. Channels: ion channels and aquaporins

3. Carriers: uniporters, antiporters, cotransporters, and pumps

4. Physiology and pathophysiology of membrane transport


                                                                  5
             Plasma membrane structure and function
All cells are surrounded by a plasma membrane, which:
• maintains differences between environment and cell
    interior
• is a ~3.5 nm thick, mobile lipid bilayer, which is not an
    inert barrier – e.g. important functions in signaling and
    control of membrane transport
• contains membrane proteins with essential roles in cell
    structure/adhesion, membrane transport, and
    communication




                                                            The Singer and Nicholson ”fluid
           Electron micrograph of the plama                 mosaic” model (top) and updated
           membranes of two adjacent cells                  model (Engelman, 2005 Nature)6
    Lipids of the plasma membrane I – phospholipids
•   Most abundant lipids in the plasma membrane of eucaryotic cells
•   Polar, hydrophilic ”head” with a phosphate group
•   Nonpolar ”tail” consisting of two fatty acid chains
•   Generally derived from glycerol-3-phosphate
•   Generally zwitterions - only phosphatidylserine is charged (-) at neutral pH

                                             Variable head
                                             group
Major phospholipids:
• Phosphatidylcholine
• Phosphatidylserine
• Phosphatidylethanolamine
• Sphingomyelin
• Phosphatidylinositol


                                                                           7
    Lipids of the plasma membrane II – cholesterol




•   15-25% of the lipids in eucaryotic plasma membranes
•   Steroid, precursor for steroid hormones
•   Interacts with the fatty acid chains of the phospholipids just below the
    head groups, making the membrane more stiff and impermeable
•   Important component in membrane subdomains (lipid rafts, caveolae)8
     Lipids of the plasma membrane III – glycolipids
•    Typically about 5% of the membrane
•    High concentrations in e.g. myelin
•    Only in the non-cytosolic monolayer
•    Major roles in e.g. cell-cell recognition and glycocalyx formation

Major glycolipids:
• Galactocerebroside
• Ganglioside
• Sialic acid

    Membrane
    composition varies
    widely between
    species, cell types,
    and organelles!
                                                                          9
    The plasma membrane is ”more mosaic than fluid”
•    The two planes of the bilayer are very different in their lipid composition,
     which is crucial for the localization and function of many transport
     proteins, receptors, and signaling molecules
•   The plasma membrane often has distinct compartments, between which
    movement of proteins and lipids is restricted – e.g. the apical and
    basolateral compartments of an epithelial cell

•   The plasma membrane has lateral sub-domains: lipid rafts and caveolae,
    which play major roles in cell function

                                      Control                Cholesterol-depleted




                                    Cav-1 labeling showing caveolae in preadipocytes
                                                                                10
                 Plasma membrane proteins

Membrane proteins constitute about 30% of the genes in the human
 genome, and roughly 50% of the weight of the plasma membrane


                   Membrane protein functions:



     Transport      Structure and adhesion   Signaling: receptors, enzymes




                                                                        11
Lipid bilayers are semi-permeable membranes




                                              12
Lipid bilayers are semi-permeable membranes

The lipid bilayer of the plasma membrane is:

    •   Most permeable to small hydrophobic or neutral molecules

    •   Less, but still very permeable to small uncharged polar
        molecules (e.g. H2O)

    •   Impermeable to large, uncharged polar molecules (e.g.
        glucose, amino acids)

    •   Impermeable to ions

Thus, the plasma membrane a semi-permeable membrane

   Thus, transport mechanisms are needed for those not
   freely diffusible molecules which the cell is dependent
      on being able to exchange with its environment               13
    Transport is mediated by channels and carriers




Channel proteins:
Form aqueous pores across the membrane, through which ions/
molecules (substrates) to be transported can pass when the pore is open

Carrier proteins:
Bind the substrate(s), and undergo conformational changes resulting in
translocation of the substrate(s) across the membrane                14
  Transport is mediated by channels and carriers




What do you suppose is most efficient in terms of the number of
 molecules transported per time unit – channels or carriers?

                            Why?
                                                             15
Thermodynamics determine the direction of transport



 Channel proteins catalyze the rapid and selective transport of
     their substrate(s) down their electrochemical gradients



    Carrier proteins have a slower turnover, but are capable of
   employing energy in various forms to transport solutes against
                  their electrochemical gradients


             To understand transport, we need to
           understand the electrochemical gradient

                                                              16
The electrochemical gradient and the driving force for transport
                                      ∼
 The electrochemical gradient, Δμx, for an ion with respect to a given membrane has
 two components:

 ΔGconcentration: RT · ln [x]i/[x]o
 ΔGelectrical : z · F · Vm,

 Where R is the gas constant; T the temperature in K; [x] the concentration of x;
 z the valence of x; F Faraday’s constant; and Vm the membrane potential


 For the ion x, the driving force (i Joules/mol) for its transport across the membrane
 is its electrochemical gradient across that membrane, i.e.:


               ∼
             Δμx : ΔGconcentration + ΔGelectrical = RT· ln [X]i/[X]o + z · F ·Vm


                                                                                    17
The electrochemical gradient and the driving force for transport

          ∼
        Δμx : ΔGconcentration + ΔGelectrical = RT· ln [X]i/[X]o + z · F ·Vm

                                   ∼
   Important implications of Δμ:
                   ∼
   Whenever Δμx ≠ 0, x is transported in the direction determined by the
   electrochemical gradient, i.e., the direction of negative ΔG
           ∼
   When Δμx (and thus the net rate of transport) is constant, we have the
   steady state condition
           ∼
   When Δμx = 0, there is no net flux, and x is per definition at equilibrium –
   this is used to derive the Nernst equation
               ∼
   Does Δμx = 0 mean that there is no transport of x at all?
                               Why/Why not?                                   18
                                   ∼
From the expression for Δμx, we get the Nernst potential, Ex
                                                      ∼
     When x is at equilibrium (no net transport), Δμx = 0, which means that:

                          ΔGconcentration,x + ΔGelectric,x = 0
                                          ⇔
                            RT· ln [x]i/[x]o = - zx ·F ·Ex
                    (Ex being the Vm at which x is in equilibrium)
                                         ⇔
                  Ex = -RT/zxF· ln [x]i/[x]o = RT/zx F · ln [x]o/[x]i

  This is the Nernst equation, and Ex is the Nernst- or equilibrium potential for x

           Since R=8.31 J/mol · K, T=310 K at 37°C, F = 96500 C/mol,
                and ln → log is 2.3, we can calculate that at 37°C:

                          Ex ≅   2.3 RT/zxF · log10 [x]o/[x]i

                             ≅    60 mV/zx· log [x]o/[x]i                        19
                    The membrane potential, Vm

Vm is the difference between the electrical potential in the cytoplasm and
that in the extracellular space

Only permeable ions contribute to Vm – how much they contribute depends
on their permeability, Px, and their intra- and extracellular concentrations

Vm at steady state (no net current) can be described by the Goldman-
Hodgkin-Katz (GHK) voltage equation, a.k.a the constant field equation:



                               PK[K+]o + PNa[Na+]o + PCl[Cl-]i
        Vm = 2.3 RT/zF log10
                                PK[K+]i + PNa[Na+]i + PCl[Cl-]o


     The Nernst equation describes the equilibrium for a given
        ion – the GHK equation describes the ”real world”!
                                                                         20
              Generation of a membrane potential




                                             Cl-




 A membrane potential arises when the membrane is selectively permeable, and
there is a concentration difference for the permeable ion(s) across the membrane
                                                                            21
           Generation of Vm in a real cell

The starting point is a pump - the Na+,K+ATPase -
 which creates the transmembrane K+ gradient

                                 140 mM
                          3Na+
 5-20 mM      ATP
           3Na+
                           2K+
                                   4 mM
           2K+

100-150 mM              Being electrogenic, the Na+,K+ATPase
                        also directly contributes to the negative
                        Vm, however, its most important role is in
                        creating the ion gradients             22
           Generation of Vm in a real cell

 In a cell at rest, PK dominates, because the great
     majority of channels open are K+ channels

                                  140 mM
                           3Na+
 5-20 mM      ATP
           3Na+
                           2K+
                                   4 mM
           2K+

100-150 mM               PK at rest reflects the constitutive
                           activity of several types of K+
                          channels (“leak K+ channels”)
                                                            23
                                Thus, the resting Vm is close to EK
                                    PK[K+]o + PNa[Na+]o + PCl[Cl-]i
Vm = 2.3 RT/zF log10                                                   (The GHK equation)
                                     PK[K+]i + PNa[Na+]i + PCl[Cl-]o


                                                   ENa
                                                                       ≅
                          +50                                                         [K+]o
                                                              EK = 2.3 RT/zF log10
Membrane potential (mV)




                                                                                       [K+]i

                           0                        0 mV       (The Nernst equation for K+)

                                                                -70 mV may not seem like
                                                                much - but the plasma
                          -50                                   membrane is only about
                                                                3.5 nm thick. Thus, the
                                                    ECl
                                                                voltage gradient across it is
                                                    Vm
                                                    EK          0.07 V per 3.5 × 10−7 cm,
                    -100                                        or 200,000 volts per cm!
                                                                                        24
                                Thus, the resting Vm is close to EK
                                    PK[K+]o + PNa[Na+]o + PCl[Cl-]i
Vm = 2.3 RT/zF log10                                                   (The GHK equation)
                                     PK[K+]i + PNa[Na+]i + PCl[Cl-]o


                                                   ENa
                                                                       ≅
                          +50                                                        [K+]o
                                                              EK = 2.3 RT/zF log10
Membrane potential (mV)




                                                                                     [K+]i

                           0                        0 mV       (The Nernst equation for K+)


                                                                 What will happen to
                          -50                                    Vm if we open a lot
                                                    ECl           of Na+ channels?
                                                    Vm
                    -100                            EK                                  25
    The number of ions it takes to create substantial changes in Vm
       is negligible compared to the cellular ion concentrations
On a macroscopic scale, electroneutrality is always maintained!
Let’s say we open a K+ channel:
•   Only a negligible fraction of the cellular K+ moves - nothing happens to net [K+] in the cell,
    even when Vm changes dramatically!
•   What happens is that a microscopic imbalance in charge is created along the membrane
•   The membrane acts as a capacitor, storing charge proportional to the net movement of K+ –
    the number of ions it takes to charge the membrane e.g. 60 mV can be calculated, and is in
    the range of ~ 0.0001% of the cellular K+




                                                                                             26
                             Vm = 0                            Non-zero Vm
                In summary - Vm

   Active transport (the Na+,K+-ATPase) establishes
       the transmembrane K+ and Na+ gradients
         these tend to be stable, while the ion
           permeabilities are rapidly variable

Vm is a consequence of these gradients and the relative
permeability of the membrane to these ions

At “rest” Vm is dominated by the combined influence of:
          1. The outwardly directed K+ gradient
          2. The large PK
                                                          27
..and so what?! (why care about membrane potentials?)


   Transmembrane potentials are a prerequisite for e.g.:

        The directed movement of ions, water, and
      nutrients across between cell and environment,
          and between cytoplasm and organelles


        The signaling processes forming the basis
        for e.g. movement of cells and organisms,
            and for all our cognitive processes


                                                           28
                   Passive and active transport
Passive transport = facilitated diffusion:
       ∼
Down Δμx for the transported molecule - no additional energy required
Can be mediated by pores (always open), channels (”gated”), or carriers

Active transport:
          ∼
Against Δμ for the transported molecule – energy input required
Can only be mediated by carriers

                                                      NB: Figure 2.5 on p. 35
                                                      contains an error


                                                           And coupled
                                                          transporters!!



                                                                        29
Carrier proteins come in several forms, and drive transport in different ways




     Passive


Active transport can be:
Primary active: driven by
ATP hydrolysis (or by light)
Secondary active: driven
                                                 ∼
by transport of another
ion/molecule down its Δμ
                               Passive   Secondary active   Primary active   30
Carrier proteins come in several forms, and drive transport in different ways




                                     ∼



                Passive      Secondary active       Primary active

    Some common points of confusion:
      • Uniport, symport, antiport, and pump are the mechanisms of transport
      • Passive, primary active, and secondary active are the ways in which
        transport is driven (where the energy for transport comes from)
      • Even though some pumps may transport two molecules in opposite
                                                                               31
        directions, we still call them pumps, and not antiporters
 Carrier proteins come in several forms, and drive transport in different ways
            ∼
   Since Δμx : ΔGconcentration + ΔGelectrical = RT· ln [X]i/[X]o + z · F ·Vm,
   the direction of transport is determined by [X]i, [X]o, and Vm



                                                          If Vm is -70 mV, which
                                                          way do you expect these
                                                          ions to move if a channel
                                                          for them is opened?



                               ∼                   ∼
For sym- and antiporters, Δμ is the sum of the Δμ’s                      ∼
                                                         A favorable Δμ for one
in the direction of transport:
                                                         molecule can be used to
                    ∼        ∼         ∼
Na+/H+ exchanger: ΔμNa/H = ΔμNa+ - ΔμH+                  drive the transport of another
                             ∼         ∼       ∼         molecule with an
Na+-glucose cotransporter: ΔμSGLT = ΔμNa+ + Δμglucose                    ∼
                                                         unfavorable Δμ as long as
                         ∼         ∼       ∼                        ∼
3Na+/1Ca2+ exchanger: ΔμNCX = 3ΔμNa+ - ΔμCa2+            the total Δμ is favorable!
                                                                                  32
             How do we study channels and carriers?

Function:
•   Electrophysiology
•   Measurements of cellular ion concentrations and fluxes
•   Inhibitors and activators: natural toxins, pharmacology
•   Knockdown (siRNA) or knockout (mice)
•   Expression of wild type or recombinant/mutant transporter
•   Reconstitution studies

Structure:
•   Topology (from biochemistry and hydropathy analyses)
•   2D crystals and cryo electron microscopy (EM)
•   3D crystals and X-ray crystallography
•   Solution nuclear magnetic resonance (NMR)
•   Electron spin resonance

Expression and localization:
•   Gene/mRNA level: standard and Real time PCR, in situ hybridization
•   Protein level: Western blotting, immunolocalization and microscopy 33
             How do we study channels and carriers?

Function:
•   Electrophysiology
•   Measurements of cellular ion concentrations and fluxes
•   Inhibitors and activators: natural toxins, pharmacology
•   Knockdown (siRNA) or knockout (mice)
•   Expression of wild type or recombinant/mutant transporter
•   Reconstitution studies

Structure:
•   Topology (from biochemistry and hydropathy analyses)
•   2D crystals and cryo electron microscopy (EM)
•   3D crystals and X-ray crystallography
•   Solution nuclear magnetic resonance (NMR)
•   Electron spin resonance

Expression and localization:
•   Gene/mRNA level: standard and Real time PCR, in situ hybridization
•   Protein level: Western blotting, immunolocalization and microscopy 34
Red blood cells were an early model system for membrane
  analysis by gel electrophoresis and Western blotting




Robust bands in SDS-gel elektroforesis of
membranes from red blood cells:
Spectrin
Ankyrin
Band 4.1        Cytoskeletal proteins
Actin
Band 3          Cl-/HCO3- exchanger (AE)
Glycophorin     Unknown function                          35
                The patch clamp technique revolutionized our
                   understanding of ion channel function
                                                 Sakmann and Neher, Nobel prize 1991




The cell is an electrical circuit, with a capacitor (the membrane), a resistance (the
inverse of the total conductance, G), and an electrical potential, Vm

From U = R · I, and R = I/G we get the current across the membrane: I = U · G

Through a given channel x the current is Ix = Gx (Vm-Ex) – thus, we can determine
Gx from measuring the current (when we alter Vm, there will also be a capacitance
current, but this decays rapidly).                                               36
          Immunofluorescence can be used to study the
          localization of transporters in cells and tissues
Immunofluorescence
images show aquaporin-
2 in the kidney moving
away from the apical
membrane after
inhibition of vasopressin
signaling




                                                                              37
                                        Christensen et al 2003 Am J Physiol Renal
     High-resolution 3D structures of membrane proteins are
         difficult to obtain, but the number is increasing

Aquaporin-1 structures
at different resolutions
by cryo-EM of 2D
crystals (a-c) and
compared with the
model based on X-ray
crystallography of 3D
crystals (d)                          6Å                         4.5 Å




                                     3.8 Å                       2.2 Å38
                                              Werten et al 2002 FEBS Letters
                          Take-home points
The lipid bilayer is not just an inert solvent for the membrane proteins, but a
complex, non-homogeneous structure, with roles in signaling and in control of
membrane transport
The lipid bilayer is a semipermeable membrane, and the transport of most
ions/molecules therefore requires transport proteins
Transport proteins are either channels or carriers – carriers can be uniporters,
symporters, antiporters, or pumps
                                                                 ∼
The driving force for transport is the electrochemical gradient, Δμ: the sum of the
free energy from the relevant chemical and electrical gradients across the
membrane
                  ∼
Transport down Δμ - passive transport - can be mediated by either channels or
transporters (uniporters)
                      ∼
Transport against Δμ - active transport – requires energy input, and can therefore
only be mediated by carriers (symporters, antiporters, or pumps)
The resting membrane potential, Vm (often ~ -50 -70 mV) results mainly from the
concerted action of a pump – the Na+,K+ATPase - and leak K+ channels          39

								
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