GOLAC: an endogenous anion channel of the Golgi complex

The Golgi complex is present in every eukaryotic cell and functions in posttranslational modifications and sorting of proteins and lipids to post-Golgi destinations. Both functions require an acidic lumenal pH and transport of substrates into and by-products out of the Golgi lumen. Endogenous ion channels are expected to be important for these features, but none has been described. Ion channels from an enriched Golgi fraction cleared of transiting proteins were incorporated into planar lipid bilayers. Eighty percent of the single-channel recordings revealed the same anion channel. This channel has novel properties and has been named GOLAC (Golgi anion channel). The channel has six subconductance states with a maximum conductance of 130 pS, is open over 95% of the time, and is not voltage-gated. Significant for Golgi function, the channel conductance is increased by reduction of pH on the lumenal surface. This channel may serve two nonexclusive functions: providing counterions for the acidification of the Golgi lumen by the H(+)-ATPase and removal of inorganic phosphate generated by glycosylation and sulfation of proteins and lipids in the Golgi.     

Multiple conductance states of the purified calcium release channel complex from skeletal sarcoplasmic reticulum.

The CHAPS-solubilized and purified 30S ryanodine receptor protein complex from skeletal sarcoplasmic reticulum (SR) was incorporated into planar lipid bilayers. The resulting electrical activity displayed similar responses to agents such as Ca2+, ATP, ryanodine, or caffeine as the native Ca2+ release channel, confirming the identification of the 30S complex as the Ca2+ release channel. The purified channel was permeable to monovalent ions such as Na+, with the permeability ratio PCa/PNa approximately 5, and was highly selective for cations over anions. The purified channel also showed at least four distinct conductance levels for both Na+ and Ca2+ conducting ions, with the major subconducting level in NaCl buffers possessing half the conductance value of the main conductance state. These levels may be produced by intrinsic subconductances present within the channel oligomer. Several of these conductances may be cooperatively coupled to produce the characteristic 100 +/- 10 pS unitary Ca2+ conductance of the native channel.     

Probing alamethicin channels with water-soluble polymers. Effect on conductance of channel states.

Channel access resistance has been measured to estimate the characteristic size of a single ion channel. We compare channel conductance in the presence of nonpenetrating water-soluble polymers with that obtained for polymer-free electrolyte solution. The contribution of the access resistance to the total alamethicin channel resistance is approximately 10% for first three open channel levels. The open alamethicin channel radii inferred for these first three levels from the access resistance are 6.3, 10.3, and 11.4 A. The dependence of channel conductance on polymer molecular weight also allows evaluation of the channel dimensions from polymer exclusion. Despite varying conductance, it was shown that steric radii of the alamethicin channel at different conductance levels remain approximately unchanged. These results support a model of the alamethicin channel as an array of closely packed parallel pores of nearly uniform diameter.     

Anion channels transport ATP into the Golgi lumen

Anion channels provide a pathway for Cl^– influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H⁺-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K₂ATP added to the cis-chamber directly inhibited the Cl^– subconductance levels of both channels, with K_m values ranging from 16 to 115 µM. Substitution of either K₂ATP or MgATP for Cl^– on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl^– > ATP^4– > MgATP^2–. Single-channel currents were observed at physiological concentrations of Cl^– and ATP, providing evidence for their importance in vivo. Second, transport of [-³²P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent K_m = 25 µM. Finally, after transport of [-³²P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [-³²P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl^– and ATP influx into the Golgi lumen. Golgi complex; Cl channel; mass spectrometry; phosphorylation     

Gramicidin-based channel systems for the detection of protein-ligand interaction

To detect protein-ligand interaction a gramicidin-based sensor was developed. Biotin was tagged to the C-terminus of gramicidin (Gram-bio 1). The biotin-moiety, which faces the electrolyte, gave little effect on single-channel conductance. Streptavidin added to the electrolyte was detected by Gram-bio 1 through the monitoring channel current using the planar bilayer system. The suppression of macroscopic currents and the acceleration of their decaying time course were observed in a concentration dependent manner. In the single-channel level, however, no significant effect on the single-channel conductance and the open dwell time was observed upon addition of streptavidin. Therefore, streptavidin neither blocked the open channel nor changed the stability of the conducting dimer. Insertion of a linker between gramicidin and biotin did not change the streptavidin-sensitivity of the current reduction. We conclude that the binding of streptavidin to the Gram-bio 1 shifted the distribution of the complex from the membrane to the electrolyte and, thus, reduced the formation of conducting dimer of Gram-bio 1 in the membrane. Interaction of biotin with an anti-biotin antibody was also observed using this system, indicating that this system is applicable for the detection of protein-ligand interaction having a binding constant of approximately 10(8-9) M(-1) or more. Both the adamantane-tagged gramicidin for detection of beta-cyclodextrin and the Strep Tag-II-tagged gramicidin for detection of streptavidin (binding constant: approximately 10(5) M(-1) or less) failed to respond. Thus, high-affinity ligands upon tagging to gramicidin render the gramicidin-based sensor able to execute as a real-time monitoring system for protein-ligand interaction.     

Pressure effects on alamethicin conductance in bilayer membranes.

We report here the first observations of the effects of elevated hydrostatic pressure on the kinetics of bilayer membrane conductance induced by the pore-forming antibiotic, alamethicin. Bacterial phosphatidylethanolamine-squalene bilayer membranes were formed by the apposition of lipid monolayers in a vessel capable of sustaining hydrostatic pressures in the range, 0.1-100 MPa (1-1,000 atm). Principal observations were (a) the lifetimes of discrete conductance states were lengthened with increasing pressure, (b) both the onset and decay of alamethicin conductance accompanying application and removal of supra-threshold voltage pulses were slowed with increasing pressure, (c) the onset of alamethicin conductance at elevated pressure became distinctly sigmoidal, suggesting an electrically silent intermediate state of channel assembly, (d) the magnitudes of the discrete conductance levels observed did not change with pressure, and, (e) the voltage threshold for the onset of alamethicin conductance was not altered by pressure. Apparent activation volumes for both the formation and decay of conducting states were positive and of comparable magnitude, namely, approximately 100 A3/event. Observation d indicates that channel geometry and the kinetics of ion transport through open channels were not affected by pressure in the range employed. The remaining observations indicate that, while the relative positions of free-energy minima characterizing individual conducting states at a given voltage were not modified by pressure, the heights of intervening potential maxima were increased by its application.     

A synthetic prostone activates apical chloride channels in A6 epithelial cells

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br > NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.     

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.     

Rapid sodium channel conductance changes during voltage clamp steps in squid giant axons.

The sodium conductance of the membrane of the giant axon of squid was isolated by the use of potassium-free solutions and voltage-clamped with pulses containing three levels of depolarization. The conductance appears to undergo rapid changes during certain repolarizing clamp steps whose voltage reach at least partially overlaps the gating range. The percentage change in conductance increases with time of depolarization from approximately 0 to approximately 25-30% at 7 ms for a potential step from +70 to -30 mV. Conductance steps were also observed for voltage steps from various depolarized levels to -70 mV. All observed shifts were in the direction of a decreased conductance. The conductance steps appear to be a weak function of the concentration of external calcium, which also acts as a voltage-dependent channel blocker for inwardly directed sodium currents. A number of possible mechanisms are suggested. One of these is discussed in some detail and postulates a voltage- and time-dependent molecular process that does not itself yield open or closed channel conformations, but that affects the magnitude of the rate constants that do connect open and closed state conformations.     

Ion channels from synaptic vesicle membrane fragments reconstituted into lipid bilayers.

Cholinergic synaptic vesicles were isolated from the electric organ of Torpedo californica. Vesicle membrane proteins were reconstituted into planar lipid bilayers by the nystatin/ergosterol fusion technique. After fusion, a variety of ion channels were observed. Here we identify four channels and describe two of them in detail. The two channels share a conductance of 13 pS. The first is anion selective and strongly voltage dependent, with a 50% open probability at membrane potentials of -15 mV. The second channel is slightly cation selective and voltage independent. It has a high open probability and a subconductance state. A third channel has a conductance of 4-7 pS, similar to the subconductance state of the second channel. This channel is fairly nonselective and has gating kinetics different from those of the cation channel. Finally, an approximately 10-pS, slightly cation selective channel was also observed. The data indicate that there are one or two copies of each of the above channels in every synaptic vesicle, for a total of six channels per vesicle. These observations confirm the existence of ion channels in synaptic vesicle membranes. It is hypothesized that these channels are involved in vesicle recycling and filling.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Gating Kinetics of E. coli Poly-3-Hydroxybutyrate/Polyphosphate Channels in Planar Bilayer Membranes

Nonproteinaceous calcium channel complexes from Escherichia coli, composed of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP), exhibit two distinct gating modes (modes 1 and 2) in planar lipid bilayers. Here we report the kinetic characterization of the channel in mode 2, a mode characterized by two well-defined conductance levels, a fully open state (87 ± 3 pS), and a major subconductance state (56 ± 2 pS). Other subconductance states and full closures are rare (<0.5% of total time). Several kinetic properties of the channel showed asymmetric voltage-dependence indicating an asymmetry in the channel structure. Accordingly, single channels responded to potential change in one of two mirror-image patterns, postulated to arise from opposite orientations of the asymmetrical channel complex in the bilayer. The fraction of time spent in each conductance level was strongly voltage-sensitive. For channels reported in this study, presumably all oriented in the same direction, residence time in the fully open state increased as clamping potentials became more positive whereas residence time in the major subconductance state increased at more negative potentials. Analysis of open time distributions revealed existence of two kinetically distinct states for each level. The shorter time constants for both conductance states exhibited weak voltage-sensitivity; however, the longer time constants were strongly voltage-sensitive. A kinetic scheme, consistent with the complex voltage dependence of the channel, is proposed. Received: 1 February 1999/Revised: 2 April 1999     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

The SecY complex forms a channel capable of ionic discrimination

Protein translocation across the bacterial membrane occurs at the SecY complex or channel. The resting SecY channel is impermeable to small molecules owing to a plug domain that creates a seal. Here, we report that a channel loosely sealed, or with a plug locked open, does not, however, lead to general membrane permeability. Instead, strong selectivity towards small monovalent anions, especially chloride, is observed. Mutations in the pore ring‐structure increase both the translocation activity of the channel and its ionic conductance, however the selectivity is maintained. The same ionic specificity also occurs at the onset of protein translocation and across the archaeal SecY complex. Thus, the ion‐conducting characteristic of the channel seems to be conserved as a normal consequence of protein translocation. We propose that the pore ring‐structure forms a selectivity filter, allowing cells to tolerate channels with imperfect plugs.     

A large, voltage-dependent channel, isolated from mitochondria by water-free chloroform extraction

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >+/-60 mV, conductance fluctuated in the range of approximately 50- approximately 200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V(rev) approximately -15 mV) to ideally cation-selective (V(rev) approximately +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.     

Sodium channel subconductance levels measured with a new variance-mean analysis

The currents through single Na+ channels were recorded from dissociated cells of the flexor digitorum brevis muscle of the mouse. At 15 degrees C the prolonged bursts of Na+ channel openings produced by application of the drug DPI 201-106 had brief sojourns to subconductance levels. The subconductance events were relatively rare and brief, but could be identified using a new technique that sorts amplitude estimates based on their variance. The resulting "levels histogram" had a resolution of the conductance levels during channel activity that was superior to that of standard amplitude histograms. Cooling the preparation to 0 degrees C prolonged the subconductance events, and permitted further quantitative analysis of their amplitudes, as well as clear observations of single-channel subconductance events from untreated Na+ channels. In all cases the results were similar: a subconductance level, with an amplitude of roughly 35% of the fully open conductance and similar reversal potential, was present in both drug-treated and normal Na+ channels. Drug-treated channels spent approximately 3-6% of their total open time in the subconductance state over a range of potentials that caused the open probability to vary between 0.1 and 0.9. The summed levels histograms from many channels had a distinctive form, with broader, asymmetrical open and substate distributions compared with those of the closed state. Individual subconductance events to levels other than the most common 35% were also observed. I conclude that subconductance events are a normal subset of the open state of Na+ channels, whether or not they are drug treated. The subconductance events may represent a conformational alteration of the channel that occurs when it conducts ions.     

Characterizing Single-Channel Behavior of GluA3 Receptors

AMPA receptors play a major role in excitatory neurotransmission in the CNS and are involved in numerous neurological disorders. Agonists bind to each of four bilobed LBDs of this tetrameric receptor, and upon binding, the lobes close to envelope the agonist, leading to channel activation. However, AMPA receptors exhibit complex activation kinetics, the mechanism of which has not yet been determined. We report here single-channel studies of a homomeric AMPA receptor (GluA3) activated by the full agonist, glutamate, and a partial agonist, fluorowillardiine. Both agonists activate the channel to the same three open conductance levels but with different open probabilities in each level. The closed probability (P_c) varied within records, particularly at low agonist concentrations. By sorting discrete segments of the record according to P_c using the X-means algorithm, we defined five modes of activity. The kinetic behavior could then be analyzed for both agonists over a range of agonist concentrations with a relatively simple model (three closed states and two open states for each open conductance level). The structural mechanism underlying the modal behavior is not clear; however, it occurs on a timescale consistent with hydrogen bonding across the lobe interface in the LBD.     

Synthetic analogues of alamethicin: effect of C-terminal residue substitutions and chain length on the ion channel lifetimes.

In a previous study, a synthetic analogue of the peptaibol alamethicin, in the sequence of which all alpha-aminoisobutyric acid (Aib) were substituted by leucine residues and the C-terminal residue modified, was shown to display the same single-channel behaviour as alamethicin in planar lipid bilayer, except that the sublevel lifetimes were much reduced. New analogues differing in their C-terminal residue (Phe-NH2, Pheol, Trp-NH2) have now been tested for their single channel properties in neutral lipid bilayers. The conductance amplitudes and open channel lifetimes do not differ significantly from the previous analogue. Thus, the nature of the last residue, which may be located near the membrane interface, does not seem to play an important role in the destabilisation of the conducting aggregate observed after the Aib substitution by Leu. Since the deletion of one residue (Glu18) in the 14-20 moiety induces a slight decrease of the increment between the conductance levels, but has no effect upon the channel lifetimes, this residue and the length of this segment do not interfer much with the channel lifetime of peptaibols. In conclusion the factors influencing the aggregate stability may be sought in the helix-helix interactions.     

The mitochondrial channel VDAC has a cation-selective open state

The mitochondrial channel VDAC is known to have two major classes of functional states, a large conductance "open" state that is anion selective, and lower conductance substates that are cation selective. The channel can reversibly switch between open and half-open states, with the latter predominant at increasing membrane voltages of either polarity. We report the presence of a new functional state of VDAC, a cation-selective state with conductance approximately equal to that of the canonical open state. This newly described state of VDAC can be reached from either the half-open cation-selective state or from the open anion-selective state. The latter transition implies that a mechanism exists for selectivity gating in VDAC that is separate from partial closure, which may be relevant to the physiological regulation of this channel and mitochondrial outer membrane permeability.