ChannelsofintracellularorganellesA vesicularvolumeincreaseis reportedtoaccompanytheexocytosis of secretorygranulesin mastcellsandinpancreaticacinarcells,which was also mediated by the uptake ofpotassiumchloride.Clchannelsplayanimportantroleinmaintainingelectroneutrality.Electrogenic uptakeof protonsorcalciumions intointracellular compartmentswillverysoon createacharge imbalancehampering further uptake.Thisis true for theCa2+-ATPase of endoplasmicandsarcoplasmic reticulumaswellasfortheV-typeH-ATPaseof theGolgilamellae aswellasendosomalandsynapticvesicles.Tobuildupthenecessary calciumorprotongradients,theexcess positive chargeintheseorganelleshastobeneutralized.Inprinciple,this may be achieved either byimportof chloride(viaanion channels)orby export ofpotassium (viacationchannels)
Channels of intracellular organelles A vesicular volume increase is reported to accompany the exocytosis of secretory granules in mast cells and in pancreatic acinar cells, which was also mediated by the uptake of potassium chloride. Cl- channels play an important role in maintaining electroneutrality. Electrogenic uptake of protons or calcium ions into intracellular compartments will very soon create a charge imbalance hampering further uptake. This is true for the Ca2+-ATPase of endoplasmic and sarcoplasmic reticulum as well as for the V-type H-ATPase of the Golgi lamellae as well as endosomal and synaptic vesicles. To build up the necessary calcium or proton gradients, the excess positive charge in these organelles has to be neutralized. In principle, this may be achieved either by import of chloride (via anion channels) or by export of potassium (via cation channels)
ChannelsofintracellularorganellesAcidificationismoreefficientinthepresenceofextravesicularchloride.Insitustudieswith secretory andrecycling endosomes ofthetrans-Golginetwork indicatedadependence of the acidification rate onbothpotassiumand chloride in the cytosol.This demonstrates therequirement for a chloride conductance in the acidificationoftheseintracellularorganelles
Channels of intracellular organelles Acidification is more efficient in the presence of extravesicular chloride. In situ studies with secretory and recycling endosomes of the trans-Golgi network indicated a dependence of the acidification rate on both potassium and chloride in the cytosol. This demonstrates the requirement for a chloride conductance in the acidification of these intracellular organelles
Methods studyingforintracellularchannelsTheisolationofthemembranei.e.intheformofsmallvesicles.Tracer-fluxassaysLipidbilayerforelectrophysiological investigationPatch-clamptechniquesThe purification of the channel protein and subsequent reconstitutionis analternative,butthis entailsthelossof thenativeenvironmentandpossiblyconformationalchangesoftheprotein.The directobservationof intracellularion channelsin intactmembranes has been reported, butthisistechnicallyvery demandingAmuchsimplerwaytostudy intracellularchannelswould betoredirectthemtotheplasma membrane.By overexpressing them,someof the intracellularCiC channels(CiC-3,-4,-5)areincorporatedinto the plasma membrane,butthisdoes not workfor all intracellularchannels
Methods studying for intracellular channels The isolation of the membrane i.e. in the form of small vesicles. Tracer-flux assays Lipid bilayer for electrophysiological investigation Patch-clamp techniques The purification of the channel protein and subsequent reconstitution is an alternative, but this entails the loss of the native environment and possibly conformational changes of the protein. The direct observation of intracellular ion channels in intact membranes has been reported, but this is technically very demanding. A much simpler way to study intracellular channels would be to redirect them to the plasma membrane. By overexpressing them, some of the intracellular ClC channels (ClC-3, -4, -5) are incorporated into the plasma membrane, but this does not work for all intracellular channels
StructureThestructureofthesechannelsarenotlikeotherknown channelsChloridechannelsubunitscontainbetween 1and12transmembranesegments.This family of ion channels contains 10 or12transmembranehelices.Eachproteinformsasingle poreSome members of this family form homodimers.In terms of primarystructure,they areunrelated toknown cation channels or othertypesofanionchannelsThreeCICsubfamiliesarefoundinanimals.CIC-1(UniProtP35523)isinvolved in setting and restoring the resting membranepotential ofskeletal muscle,while other channels play important parts in soluteconcentration mechanisms in thekidney.TheseproteinscontaintwoCBSdomainsSomemembersofthisfamilyareactivatedbyvoltage,whileothersareactivatedbyCa2t,extracellularligands,andpHamongothermodulators
Structure The structure of these channels are not like other known channels. Chloride channel subunits contain between 1 and 12 transmembrane segments. This family of ion channels contains 10 or 12 transmembrane helices. Each protein forms a single pore. Some members of this family form homodimers. In terms of primary structure, they are unrelated to known cation channels or other types of anion channels. Three ClC subfamilies are found in animals. ClC-1 (UniProt P35523) is involved in setting and restoring the resting membrane potential of skeletal muscle, while other channels play important parts in solute concentration mechanisms in the kidney. These proteins contain two CBS domains. Some members of this family are activated by voltage, while others are activated by Ca2+, extracellular ligands, and pH among other modulators
Mechanismsof regulationof ClchannelsPhosphorylationaCFTRExtracellularATP bindingATPCOOHNH2ClosedOpenNBD1NBD2IntracellularR domainCysticfibrosistransmembraneconductanceregulator(CFTR)Shownhereare12membrane-spanningsegmentsofCFTRplustwo nucleotide binding domains (NBDs 1 and 2)and a regulatoryR domainCFTRactivationinvolvescyclic AMP-dependentphosphorylationandbindingofATPmoleculesattheNBDs
Mechanisms of regulation of Cl- channels Cystic fibrosis transmembrane conductance regulator (CFTR). Shown here are 12 membrane-spanning segments of CFTR plus two nucleotide binding domains (NBDs 1 and 2) and a regulatory R domain. CFTR activation involves cyclic AMP-dependent phosphorylation and binding of ATP molecules at the NBDs