Transport mechanisms in chloride channels.

A comparative study of lipids and proteins in sarcoplasmic reticulum (SR) from rabbit and flounder has been undertaken. The protein/phospholipid ratio (w/w) was 3:1 in flounder SR (FSR) and 2.2:1 in rabbit SR (RSR). Both membranes had similar contents of PC (70%) and PI (6%). PE constituted 15% in RSR and 21% in FSR. PS and sphingomyelin were minor components of both SR (less than 4%). There were differences in the unsaturated chains of the total lipid extracts, PC, PE, and PI between FSR and RSR. RSR was high in linoleate and arachidonate while FSR contained substantial amounts of eicosapentaenoate and docosahexaenoate. FTIR spectroscopy revealed that the lipids of both membranes did not undergo a phase transition between 0 and 50 degrees C. The lipids were in the liquid-crystalline state at physiological temperatures and underwent monotonic increases in conformational disorder as the temperature was raised. CD spectra indicated higher content of alpha-helical structure of proteins in RSR than in FSR. Increasing temperature caused diminution of alpha-helix content. Relatively large decreases in ellipticity were observed between 20 degrees C and 40 degrees C for FSR and 30 degrees C and 60 degrees C for RSR. Measurements of intrinsic tryptophan fluorescence as a function of temperature gave similar results for membrane proteins in both FSR and RSR. The rate of change of tryptophan fluorescence and fluorescence lifetimes was constant over the temperature ranges studied, and no abrupt shifts in fluorescence occurred in the temperature regions where ellipticity decreased rapidly.     

Review. Proton-coupled gating in chloride channels

The physiologically indispensable chloride channel (CLC) family is split into two classes of membrane proteins: chloride channels and chloride/proton antiporters. In this article we focus on the relationship between these two groups and specifically review the role of protons in chloride-channel gating. Moreover, we discuss the evidence for proton transport through the chloride channels and explore the possible pathways that the protons could take through the chloride channels. We present results of a mutagenesis study, suggesting the feasibility of one of the pathways, which is closely related to the proton pathway proposed previously for the chloride/proton antiporters. We conclude that the two groups of CLC proteins, although in principle very different, employ similar mechanisms and pathways for ion transport.     

Maxi chloride channels in L6 myoblasts.

The existence of a large conductance voltage sensitive chloride channel is documented in undifferentiated cells (myoblasts) of the L6 rat muscle cell line. At this stage of development the resting membrane conductance is dominated by potassium ions only (Kidokoro 1975). The conductance of the channel in symmetrical 120 mmol/l choline chloride is 331 +/- 4 pS. The probability of the channel being in the open state decreases with the increasing imposed voltage. Due to rapid inactivation at high membrane potential deviations (both negative and positive) from the equilibrium potential the channel can be resolved clearly by pulse technique protocols only. The incidence of the channel in successful patch trials was higher than usually reported. The channel was present after differentiation of the myoblasts into the myotubes. It showed at least one definite substate and pronounced flickerings between the substate and the main open state. The channel was observed in myoblast attached patches as well. It is supposed to belong to the category of maxi chloride channels, and to play probably a role in regulatory volume readjustment or in cell communication during myogenesis, respectively.     

Small GTP-binding proteins on rat liver lysosomal membranes.

GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [alpha-32P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M(r) was 23 kDa in the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 μM), ATR and CAT (5-100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

Presence of detergent-resistant microdomains in lysosomal membranes

We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts.     

ClC chloride channels

Chloride-conducting ion channels of the ClC family are emerging as critical contributors to a host of biological processes. These polytopic membrane proteins form aqueous pathways through which anions are selectively allowed to pass down their concentration gradients. The ClCs are found in nearly all organisms, with members in every mammalian tissue, yet relatively little is known about their mechanism or regulation. It is clear, however, that they are fundamentally different in molecular construction and mechanism from the well-known potassium-, sodium-, and calcium-selective channels. The medical importance of ClC channels - four inherited diseases have been blamed on familial ClC dysfunction to date - highlights their diverse physiological functions and provides strong motivation for further study.     

Sodium-conducting channels in cardiac membranes in low calcium.

With no Ca in the patch electrode, two kinds of channels conduct Na in spontaneously beating embryonic chick heart cells. One channel conducts Na primarily during the upstroke of the action potential and is blocked by tetrodotoxin (TTX). The other channel conducts Na primarily during the late plateau and early repolarization phase of the action potential, but only in Ca concentrations below 10(-6) M. This second channel is TTX-insensitive and has a conductance of 50 to 90 pS, depending upon the interpretation of open-channel flickering. These two Na-conducting channels correspond to the channels that normally carry the fast Na current (INa) and the slow Ca current (Isi).     

Stretch-activated chloride,potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L.

Mechanosensitive ion channels in the plasma membrane of Vicia faba guard cell protoplasts were studied by use of the patch clamp technique. Stretch-activated (SA) channels in outside-out patches were analyzed for channel conductance, kinetics and ion selectivity. We found three distinct SA channels, permeable to Cl^–, K⁺ and Ca²⁺ and distinguishable from spontaneous (non-SA) channels for these ions on the basis of conductance, kinetics, and voltage-dependence, as well as sensitivity to membrane stretch. In the attached patch configuration, light suction (2 to 10 kPa) reversibly induced channel opening with multiple amplitudes and complex kinetics. The open probability for SA channels increased nonlinearly with pipette suction. In guard cells in situ, these SA channels may mediate ion transport across the plasma membrane directly, as well as influence the activity of non-SA channels via effects on membrane voltage and cytoplasmic calcium. Through such effects, SA channels likely influence volume and turgor regulation of guard cells, and thereby control of leaf gas exchange.Abbreviations EK equilibrium potential for potassium transport - E_Cl equilibrium potential for chloride transport - SA stretchactivated Dedicated to the 80. birthday of Franz HedrichSupported by a grant from the Deutsche Forschungsgemeinschaft to R.H. and a Department of Energy grant to D.J.C. gratefully acknowledges a John S. Guggenheim Fellowship and Fulbright Kommission Senior Professor Award. We thank Ingrid Baumann and Angela Schön for technical assistance, and Klaus Raschke and Heiner Busch for spirited discussions and support.     

Mechanisms of loss of latency of lysosomal enzymes. Effects of incubation on the properties of lysosomal membranes.

1. The effects of sucrose and KCl on the loss of latency of lysosomal enzymes caused by incubation at 37 degrees C, pH 7.4, were examined by using Triton-filled lysosomes from rat liver and two fractions from livers of rats not injected with Triton. 2. After incubation, the percentage free activity of lysosomal enzymes was measured before and after cooling to 0 degrees C in order to determine the amount of latency lost at 37 degrees C without cooling and the additional amount lost on cooling the incubated lysosomes to 0 degrees C. 3. The latency that is lost without cooling is first decreased and then increased by increasing the osmotic strength of the incubation medium with KCl, or with sucrose in the presence of KCl. However, if the osmotic strength is increased with sucrose alone, loss of latency is decreased up to 0.25M-sucrose, but is increased only slightly at higher sucrose concentrations. Apparently the lysosome is permeated by hyperosmolar KCl but not by sucrose during incubation. 4. If the osmotic strength of the assay medium is increased with KCl, the loss of latency caused by incubation for 60 min in hyperosmolar KCl is repressed. Thus it appears that a KCl-permeated lysosome can be obtained which is relatively stable until exposure to lower osmolarities. 5. The loss of latency caused by cooling incubated lysosomes to 0 degrees C is largely eliminated if the osmotic strength of the medium in which the lysosomes are cooled is raised sufficiently with either sucrose or KCl. 6. Osmotic-fragility curves were obtained after incubation for 1 and 60 min at iso-osmoticity (0.2M-KCl or 0.25 M-sucrose). Although little loss of latency occurs at iso-osmoticity, lysosomes incubated for 60 min display greatly increased fragility on exposure to hypo-osmolar KCl, hypo-osmolar sucrose or hyperosmolar KCl. 7. It is suggested that permeability to KCl at 37 degrees C and the increase in fragility on exposure to hypo-osmolar conditions are both consequences of injury, probably from enzymic action, sustained by the lysosomal membrane during incubation at 37 degrees C.     

Mechanism of voltage-dependent gating in skeletal muscle chloride channels.

Voltage-dependent gating was investigated in a recombinant human skeletal muscle Cl- channel, hCIC-1, heterologously expressed in human embryonic kidney (HEK-293) cells. Gating was found to be mediated by two qualitatively distinct processes. One gating step operates on a microsecond time scale and involves the rapid rearrangement of two identical intramembranous voltage sensors, each consisting of a single titratable residue. The second process occurs on a millisecond time scale and is due to a blocking-unblocking reaction mediated by a cytoplasmic gate that interacts with the ion pore of the channel. These results illustrate a rather simple structural basis for voltage sensing that has evolved in skeletal muscle Cl- channels and provides evidence for the existence of a cytoplasmic gating mechanism in an anion channel analogous to the "ball and chain" mechanism of voltage-gated cation channels.     

A role for chloride transport in lysosomal protein degradation

Loss of the lysosomal chloride transport protein ClC-7 leads to complex phenotypes in mice and man, including osteopetrosis, accumulation of lysosomal storage material and neurodegeneration. Using novel tissue-specific ClC-7 knockout mice, we have shown that upon loss of ClC-7, lysosomal degradation of endocytosed protein is slowed down and accumulation of autophagosomes occurs.