Chloride Channels

Yuanting LuCristian Escobar

09/23/2011

Cells actively transport Cl- across the plasma membrane by transporters that accumulate Cl- intracellularly (Cl- loaders including the Na+-K+-Cl- cotransporters NKCC, Cl--HCO3- exchangers AE, and Na+-Cl- cotransporters NCC) or pump Cl- out of the cell (K+-Cl- cotransporters KCC and Na+-dependent Cl- - HCO3- exchanger NDCBE). Cl- flows passively across a variety of Cl- channels in the plasma membrane including Ca-activated Cl- channels (CaCC), cAMP-activated Cl- channels (CFTR), cell-volume regulated anion channels (VRAC), and ligand-gated anion channels (GABAA and glycine receptors). In addition, Cl - channels and transporters are found in intracellular membranes, such as the endosomallysosomal pathway, and play a role in regulating intra-vesicular pH and Cl - concentration. Intravesicular pH and [Cl-] are important in vesicular trafficking.

Functions

•Stabilization of membrane potential.

•Depolarization

•Cell volume regulation.

•Fluid transport in epithelia.

•Neutralization of H+ in lysosomes.

ClC-1ClC-2

GABAGlycine

BestrophinTMEM16A

VDCC

ClC-Kb

CFTR

ClC-3

Ligand gated Cl- channels

Voltage dependent

Volume sensitive Cl- channels

cAMP

pH

pH

Ca2+

CLC Chloride Channels

CLC Chloride Channels Structure

Myotonia and muscle fiber type changes. Mutations in Na1 (1) or Cl2 (2) channels or the lack of the sarcolemmal chloride channel (ClC-1; 2) can lead to overexcitability of the sarcolemma (myotonia). Normal muscle responds with single action potentials upon single stimuli, whereas myotonic muscle often responds with runs of action potentials. Increased membrane excitation can cause protein kinase C (PKC) activation in the nucleus and changes in the pattern of myogenic regulating factors (MRF). The myogenic factors control gene transcription and therewith couple membrane excitation to the muscle fiber type. A second signaling pathway involves cytoplasmic Ca21. The propagation of action potentials into the transverse tubule system (TT) activates the L-type Ca21 channel and stimulates Ca21 release from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR). A Ca21 signaling pathway into the nucleus is suggested.

Stabilization of membrane potential

CLC Chloride Channels

Influx of Cl- suppresses depolarizing inputs and stabilizes membrane potential

CLC Chloride Channels Functions

Endosomal acidification

• H+ concentration inside of lysosomes require Cl- influx to neutralize positive charges.

• Some ClC channels mediate Cl−:H+ exchange rather than voltage-dependent anion channel activity.

Epithelial transport

CLC Chloride Channels

• ClC-Kb transport Cl- acumulated by NKCC trasporter to the intertisium.

Fluid transport and Cl– channel. Ascending loop of Henle (thick ascending limb, TAL) is an important part of the tubular segment (left panel), playing a cardinal role in construction of high osmolality in the renal medulla by unidirectional transport of NaCl. Luminal Na+, K+ and Cl– are transported via the Na/K/2Cl transporter into cells without water. Na+ and Cl– are transferred into the basolateral space and thereby concentrated in osmolality. K+ is back-leaked by K+ channels resulting in lumen-positive (compare with interstitium) transtubular potential. This lumen-positive potential drives cations, Ca2+ or Mg2+, through the cell junction (claudin) under the electrochemical gradient. Thus, all 3 modes of transport, Na/K/2Cl transporter, K channel and Cl channel (ClCK) are cardinal in the construction of the high osmolality.

Cystic fibrosis transmembrane conductance regulator (CFTR)

Model showing proposed domain structure of cystic fibrosis transmembrane conductance regulator (CFTR). MSD, membrane-spanning domain; NBD, nucleotide-binding domain; R, regulatory domain; PKA, cAMP-dependent protein kinase.

• Localization on apical membranes

• Plays an important role in fluid secretion in airways, intestine, sweat glands.

• Their activity is regulated by cAMP-dependent phosphorylation

current-voltage relationship of channel at 357C with symmetrical 140 mM N-methyl-D-glucamine chloride (n 5 for each data point).

CFTR gating

Fig. 2. A model of secretory epithelial cell and secretory diarrhea. Cholera toxin or heat-stable enterotoxin can increase the intracellular cAMP or cGMP levels by activating the membrane-localized adenylate cyclase (AC) or guanylate cyclase (GC). An increase in the intracellular cAMP or cGMP leads to the phosphorylation of the R-domain of CFTR by PKA or cGMP dependent protein kinase II (cGKII), which, in turn, activates the CFTR chloride channel, resulting in Cl secretion into the lumen. As a consequence, Na+ and water are effuxed into the lumen through the paracellular transport mechanism. Therefore, the net result is the secretion of fluid and lectrolytes across the apical surface into the gut lumen. Cl is taken up from the basolateral (blood) side by the Na+–K+–2Cl cotransporter (NKCC). K+ recycles through basolateral K+ channels, and Na+ is pumped out of the cell by the Na+–K+-ATPase.

CFTR, example of transport

Intestine

Cell models for electrolyte transport in human airways. CFTR is the dominant pathway for luminal Cl− exit in human airways, while TMEM16A (ANO1) is probably the major secretory pathway in mouse airways. (ADE: adenosine).

CFTR, example of transport

Airways

Ca2+ Activated Chloride Channels

Regulation of membrane potential by CaCCs

Ca2+ Activated Chloride Channels

Physiological roles of CaCCs. In epithelial cells, activation of CaCCs by intracellular Ca2þ elevation leads to Cl secretion followed by transepithelial transport of Naþ and water. In smooth muscle cells, activation of CaCCs is part of an amplification echanism. Intracellular Ca2þ increase by extracellular stimuli activates CaCCs and Cl efflux. The resulting membrane depolarization opens voltage-dependent Ca2þ channels that cause a further intracellular Ca2þ increase, thus potentiating contraction. Another amplification mechanism occurs in olfactory receptors, where the initial Ca2þ increase is triggered by cAMP-gated channels. CaCC is also involved in phototransduction and regulation of neuronal excitability

Functions

TMEM16A (ANO1)

Putative topology of TMEM16A channels. TMEM16A (NM 018043) has eight putative transmembrane domains and a p-loop between transmembrane (TM) domain 5 and 6 but no apparent similarity to other ion channels. A number of consensus sites for phosphorylation by kinases are present in the N-terminus, but no binding site for Ca2+ (numbering of amino acids according to splice product a,c,d [13]). Three cysteines (at position 651, 656, 661) are located in the pore forming loop and are accessible to sulfhydryl reagents.

Ca2+ Activated Chloride Channels

ANO1 is activated by intracellular Ca2+ in a voltage-dependent manner. Dose–response relationship of ANO1 activation by Ca2+. Current responses were normalized versus those observed at 10 mM (circles, 260 mV, n58) or 3 mM Ca2+ (triangles, 160 mV, n58). d, An amplitude histogram of single-channel currents activated by Ca2+ at 160 mV.

Ca2+ Activated Chloride Channels

Contribution of Ca2+-dependent Cl− secretion by TMEM16A (ANO1) in mouse and human airways. Cell models for electrolyte transport in mouse airways. CFTR is the dominant pathway for luminal Cl− exit in human airways, while TMEM16A (ANO1) is probably the major secretory pathway in mouse airways. (ADE: adenosine).

Airways

Ligand Activated Chloride Channels

Inhibitory Glycine Receptor

• Glycine is a neurotransmitter in inhibitory synapses

• GlyR are pentamers of α and subunits (2 α : 3 ).

Schematic drawing of the pentameric arrangement of GlyR subunits in homo-oligomeric a1 (left) and heterooligomeric a1b (right) GlyRs. Binding sites for glycine are indicated in yellow, and glycine sites also capable of binding strychnine are shown by a red surround

GABA Receptor

Ligand Activated Chloride Channels

• 2 types of receptor:GABAa = LGCC

GABAb = G-protein couple

Mechanism of cell swelling and a possible role of VDCC in tubulo-glomerular feedback. (A) Mechanism of cell volume regulation and activation of VDCC is shown. The cell under the aniso-osmotic surrounding is illustrated at the top of the panel. The tonicity of extracellular and intracellular solution is expressed on the bottom. Cells swell in hypotonic (indicated as a white column) extracellular solution by an influx of water via aquaporin channels. The influx of water per se or change of volume induces several lines of signal transduction, which then activate Cl– (VDCC) and K+ channels. Efflux of these ions lowers the concentration of ions in the cell interior reaching equilibrium to extracellular tonicity, and then cell volume is restored.

Volume sensitive Cl- channels

• They have been difficult to identify.

• MCLC intracelular Cl- Channel

• Two key properties for ion channels: – Selective ion conduction– Gating

• ClC channels: “fast gating”

Crystal Structure of the EcClC Fab complex

FIG.1 A

Red: Light Fab chainOrange: Heavy Fab chainBlue: ClC subunitGreen: ClC subunit

Each subunit within the dimer forms its own independent pore for Cl- ions.

EcCLC Crystal structure

Selectivity filter

FIG. 1.A

FIG. 1.BFIG. 2.B

+80 mV to -180 mV

-100 mV

Probability to be open

WT

E166 mutants

Single channel measurement

Whole-cell oocyte currents expressing ClC-0 from Torpedo ray

WT E148A E148Q

Close Open

Mutant EcClC crystal structure

FIG. 2.B FIG. 4.B FIG. 4.D

Proposed gating mechanism

FIG. 5

Conclusions:

• When the Sext site is occupied by the glutamate carboxyl group, the pore is closed;When the site is occupied by a Cl- ion, the pore is open.

• Two pores are gated independently.• The conformational change is local.

Ca2+ - activated Cl- currents are dispensable for olfaction

G.M. Billing, B. Pal, P. Fidzinski & T. J. Jentsch

Nature Neuroscience, vol 14(6), 2011

Main olfactory epithelium(MOE) & the vomeronasal organ (VNO)

http://www.neuro.fsu.edu/~mmered/vomer/

Olfactory System

http://en.wikipedia.org/wiki/Olfactory_system

1. Olfactory bulb2. Mitral cells3. Bone4. Nasal epithelium5. Glomerulus6. Olfactory receptor

cells

OSN signal transduction pathwayThe disruption of Ano2 caused no change in G-protein, adenylate cyclase.

Ano2 in MOE and VNOFIG. 1

Ano2 in MOE and VNO•Ano2 co-localized with the cilia marker protein ac tubulin in MOE and the Cnga2 subunit of the olfactory CNG channel.

FIG. 2

Ano1 & Ano2

Olfaction is not grossly impaired in Ano2-/- mice

Tyrosin hydroxylase helps convert tyrosine to dopamine / norepinephrine /epinephrine.

Ca2+ activated Cl- currentsare absent from Ano2-/- neurons

FIG. 5

Disruption of Ano2 moderately reduces electro-olfactograms(EOGs)

Grey: NFABlack:Normal Ringer

No olfactory deficits detected in Ano2-/- mice

Conclusions

• Disruption of Ano2 in mice virtually abolished Ca2+ -activated Cl- currents in MOE and VNO.

• Disruption of Ano2 reduced fluid-phase EOG by only ~40%, did not change air-phase EOG.

• Ca2+ -activated Cl- currents are dispensable for olfaction.

Experimental system

• Colon Carcinoma epithelial cell line T84

• SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium)

• Fluorescence spectrometer configuration:

SPQ emission and excitation spectra

320 nm

344 nm

443 nm

10 µM SPQ in water

SPQ loaded T84 cells

344 nm

443 nm

Ex

Em

SampleEx

Em

Sample

Stern-Volmer constants F0/F = 1 + Ksv [Q]

2.5 µM SPQ in water

SPQ loaded T84 cells

• Nigericin K+/H+ antiporter

• Tributyltin Cl-/OH- antiporter

• KCl and fluorescein gradient

• Fluorescein as internal standar (ex=495 nm em = 519 nm)

SPQ

Fluorescein

Functional Assay on T84 cell

cAMP IBMX isopreterenol

ControlGlibenclamide (100 µM) CFTR(inh)-172 (5 µM)

• Using SPQ as Cl- sensing molecule can be applied to measure it accurately

• This study present an easy method to study CFTR activity.

Conclusions:

Thank you !!