Flashcards
Chloride channels 2
Flashcards do conjunto (10)
- 1Describe the chlorine atomChlorine (Cl) is an element/atom and a member of the halide series (including fluorine, bromine and iodine)
Exists as two stable isotopes, with atomic masses of either 35 (35Cl), or 37 (37Cl), dependent upon the number of neutrons (17, or 20), within the nucleus. Mean atomic mass 35.5 occurs because the isotopic composition (mole fraction) is about 75% 35Cl and 25% 37Cl)
Unstable isotopes are frequently used in biomedical research as radioisotopes, the most commonly used being 36Cl, a predominantly β- emitter (i.e. electron) - 2Describe the chloride ionChloride is a negatively charged ion (anion) formed when the third shell of chlorine (a gas) accepts an electron
Chloride is Surrounded by hydration shells (like other ions)
At least partial dehydration of chloride is required for translocation and conduction of the ion by transporters and ion channels - 3Describe the distribution of chloride ions across the plasma membraneThe concentration of Cl- in the extracellular fluid (ECF) is greater than the intracellular fluid (ICF). In the ECF, Cl- is the most abundant anion, bicarbonate (HCO3-) being second
In man, the normal plasma concentration of Cl- is approximately in the range 95 – 110 mM (or mEq/L as often reported clinically). Below and above this range are referred to as hypo- and hyper-chloraemia, respectively
The ICF concentration of Cl- varies widely (5 – 40 mM) between cell types (e.g. skeletal muscle versus epithelial cells) and is regulated by numerous transporters and ion channels. The direction of movement of Cl- by facilitated diffusion through an ion channel is dictated by the relationship between the resting membrane potential (Vm) and the equilibrium potential for Cl- (ECl) – the driving force (i.e. Vm – ECl)
ECl = -61log10([Cl-]o/[Cl-]i)
If ECl is negative to Vm the driving force is positive and Cl- moves in to the cell (influx - e.g. skeletal muscle) – electrophysiologically an outward current
If ECl is positive to Vm the driving force is negative (efflux e.g. epithelial cells) - 4Describe the ClC familyAlthough closely related in structure, the ClCs are functionally divided into:
anion channels expressed at the plasma membrane (ClC-1, ClC2 + regulatory subunit glialCAM), ClC-Ka + obligatory subunit barttin, ClC-Kb + obligatory subunit barttin
Cl-/H+ exchangers (antiporters) expressed in the membranes of intracellular organelles (endosomes/lysosomes) (ClC-3, ClC-4, ClC-5, ClC-6, ClC-7 + obligatory subunit Ostm1 - 5How was Torpedo elextroplax used to characterise ClC-0characterised an anion conductance in membranes from Torpedo electroplax by isolating electroplax vesicles (small unilamellar vesicles; SUVs). Following a freeze-thawing protocol SUVs fuse into large liposomes amenable to single channel recording techniques (e.g., the inside-out patch configuration)
Single channel records of anion channel activity in isolated membrane patches
the anion channel [later coined ClC-0 by Jentsch et al. (1990)] to be open at physiological (resting) membrane potentials but with opening probability increasing further with membrane depolarization
the channel occupying three conductance states: (i) a zero conductance (closed/inactivated) state (D, I), (ii) an intermediate conductance state (M) and (iii) a maximal conductance state (U)
the intermediate state (M) to be exactly half of the maximum conductance state (U)
the anion channel to contain two identical conduction pathways, termed ‘protopores’
the protopores to operate (‘gate’) independently during the period of a burst
a likely dimeric structure of the channel, with each subunit contributing a pore - 6How do you interpret a single channel recording for an inside-out patch?the recording is made in the equivalent of the inside out configuration of the patch-clamp technique
Cl- flows through the channel from the bathing medium to the pipette driven by a positive voltage applied to the pipette (which is the equivalent of a negative membrane potential)
negative charge from inside to outside is an inward current by convention and is a downward deflection in a current trace
the amplitude of the downward deflection indicates the single channel current (in pA = 10-12 A)
the channel cycles between long closed periods and bursts of openings wherein rapid transitions between two identical conducting states occurs - 7What evidence is there for a double-barrel structure of ClC-1 from single channel recordings?Single channel recordings of ClC-1 activity at three different membrane potentials (C, O1 and O2 correspond to D, M and U, respectively in Miller’s terminology
Amplitude histogram obtained at a membrane potential of -140 mV. Single channel current amplitudes are well described by the sum of three equidistant Gaussian (normal) distributions
Opening probability (p) to three different current levels at a membrane potential of -140 mV - 8Describe the ClC protomerClC proteins are homodimers, each with a separate Cl- conduction pathway
Each protomer consists of
18 helical domains (A-R) most of which do not cross the membrane and which are remarkably tilted
2 intracellular cystathionine-β-synthase (CBS) domains
In the 3D structure (next slide)
helices D, F, N and R contribute to the Cl- conduction pathway
helices H, I, P and Q are part of the interface between the two protomers
helix R connects the transmembrane element of the structure to the intracellular domain
structure of the CLC-ec1 dimer in the plane of the membrane shows an internal symmetry of repeats
surface representation of a CLC-ec1 protomer viewed from the intracellular side of the membrane emphasises the contribution of internal symmetry to the conduction pathway - 9Describe the Cl ion conduction pathway in ClC protein monomersEach ClC protomer forms three anionic binding sites, denoted Sex, Scen and Sin. Sex and Sin are exposed to extra- and intra-cellular solutions, respectively, but Scen is cut off from water below by a constriction provided by the side chains of Sercen and Tyrcen and above by a conserved glutamate residue, Gluex. The deprotonated side chain of Gluex can alternatively occupy Sex, or Scen and competes with a Cl- ion for binding
D) Gluex occupies Sex, the channel is non-conducting
E) Mutation of Gluex to glutamine, mimicking protonation of the Gluex side chain allows the binding of a Cl- ion, the channel is conducting
F) Gluex occupies Scen, the channel is non-conducting - 10Give a summary of fast gating of ClC-1The side chain of Gluex acts as the main feature of the gate
For ClC-1 to conduct, all Cl- binding (co-ordination) sites must be occupied by Cl- ions
When deprotonated (i.e. negatively charged), Gluex occupies either Sex, or Scen, preventing the binding (co-ordination) of a Cl- ion at those sites. The channel is non-conducting
When protonated (i.e. neutral) Gluex moves out of the conduction pathway allowing Cl- ions to bind simultaneously to all three binding (co-ordination) sites. The channel is conducting
Each protomer gates independently (the fast gate) in this manner
The model explains why the opening of the fast gates of ClC-1 is facilitated by:
extracellular Cl- (competes with Gluex for binding)
low extracellular pH (promotes the protonated species of Gluex)
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- Biomembranes FlashCards sobre Chloride channels 2, criado por Megan MacLeod em 18-06-2019.
- Criado por
- Megan MacLeod · jun 2019
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- 18 jun 2019
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