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.     

Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria.

Patch clamp analysis of membranes reconstituted with a fraction isolated from detergent-solubilized mitochondrial membranes by affinity chromatography on immobilized quinine earlier indicated the presence of two classes of ion channels, of about 40- and 140-pS conductance in medium including 150 mM KCl. Now a 57-kDa constituent of the quinine-affinity column eluate has been identified as the 40-pS channel. Protein fractions derived from the quinine-affinity column eluate by preparative isoelectric focusing with a Rotofor cell have been reconstituted into phospholipid vesicle membranes by detergent dialysis, and vesicles have been enlarged for patch clamping by dehydration and rehydration. Voltage clamp analysis has been carried out on excised patches bathed symmetrically in buffered medium containing 150 mM KCl and 100 microM CaCl2. Patches of membrane incorporating the 57-kDa protein exhibit 40-pS conductance transitions. The magnitude of conductance transitions is similar when Na+ replaces K+ in the bathing medium, indicating little selectivity of the 40-pS channel for K+ relative to Na+. Another fraction derived from the quinine-affinity column eluate is found to contain the larger channel, now estimated to have an average conductance of about 130 pS. Patches of control membrane prepared in the same way but without protein exhibit no channel activity.     

Reconstitution of ionic channels from inner and outer membranes of mammalian cardiac nuclei.

Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.     

Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.     

Lysenin forms a voltage-dependent channel in artificial lipid bilayer membranes

Lysenin, a hemolytic protein derived from the body fluid of earthworm, was incorporated into artificial bilayer membranes. Upon insertion, it formed a voltage-dependent large conductance channel in asolectin bilayers in a sphingomyelin-dependent manner. The channel had low ion-selectivity. Single-channel conductance was calculated as approximately 550 pS in 100 mM KCl. The channel in asolectin bilayers closed when the membrane was held at a positive potential. In contrast, the channel showed no voltage dependency in membranes made of pure phosphatidylcholine and sphingomyelin, suggesting some lipid contents included in the asolectin membranes affected channel gating.     

Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.     

Slow conversions among subconductance states of cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel exhibits multiple subconductance states. To study the regulation of conductance states of the CFTR channel, we expressed the wild-type CFTR protein in HEK 293 cells, and isolated microsomal membrane vesicles for reconstitution studies in lipid bilayer membranes. A single CFTR channel had a dominant conductance of 7.8 pS (H), plus two sub-open states with conductances of approximately 6 pS (M) and 2.7 pS (L) in 200 mM KCl with 1 mM MgCl2 (intracellular) and 50 mM KCl with no MgCl2 (extracellular), with pH maintained at 7.4 by 10 mM HEPES-Tris on both sides of the channel. In 200 mM KCl, both H and L states could be measured in stable single-channel recordings, whereas M could not. Spontaneous transitions between H and L were slow; it took 4.5 min for L-->H, and 3.2 min for H-->L. These slow conversions among subconductance states of the CFTR channel were affected by extracellular Mg; in the presence of millimolar Mg, the channel remained stable in the H state. Similar phenomena were also observed with endogenous CFTR channels in T84 cells. In high-salt conditions (1.5 M KCl), all three conductance states of the expressed CFTR channel, 12.1 pS, 8.2 pS, and 3.6 pS, became stable and seemed to gate independently from each other. The existence of multiple stable conductance states associated with the CFTR channel suggests two possibilities: either a single CFTR molecule can exist in multiple configurations with different conductance values, or the CFTR channel may contain multimers of the 170-kDa CFTR protein, and different conductance states are due to different aggregation states of the CFTR protein.     

Electrophysiological evidence that glycoprotein IIb-IIIa complex is involved in calcium channel activation on human platelet plasma membrane.

The involvement of platelet glycoprotein (GP) IIb-IIIa complex in calcium channel activity on the plasma membrane was investigated using an electrophysiological approach. Plasma membrane vesicles were prepared from thrombin-stimulated platelets and incorporated into planar lipid bilayers. Voltage-independent Ca2+ channel currents with a conductance of about 10 pS (in 53 mM Ba2+) were observed, in membranes derived from thrombin-stimulated, but not unstimulated platelet membranes. These channel activities were markedly reduced by exposure of membranes to EGTA at 37 degrees C. This reduction was specifically related to the dissociation of the GPIIb-IIIa complex since preincubation of the membranes with a monoclonal antibody to the GPIIb-IIIa complex (AP-2) could protect the channel activities from the effect of EGTA. Thrombasthenic platelets, which lack the GPIIb-IIIa complex, showed impaired channel activities characterized by decreased open probability and lowered conductance states. Furthermore, when platelets were stimulated by thrombin in the presence of EGTA, AP2, or the synthetic peptide RGDS, to prevent fibrinogen binding to the GPIIb-IIIa complex, open probabilities of the channel currents in these membrane vesicles were also decreased. These results suggest that the GPIIb-IIIa complex is involved in platelet Ca2+ channel activation and that ligand binding to the complex during platelet activation may modify the activation of Ca2+ channels.     

Evidence for a large conductance voltage gated cationic channel in rough endoplasmic reticulum of rat hepatocytes

In this work, we report the single channel characterization of a voltage gated cationic channel from rough endoplasmic reticulum (RER) membranes of rat hepatocytes incorporated into a planar lipid bilayer. The channel was found to be cation selective with a main conductance of 598+/-20 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage dependent with a voltage for half activation (V(1/2)) of 38 mV and an effective gating charge z of -6.66. Adding either 4-AP (5 mM) or ATP (2.5 mM) to the side corresponding to the cell internal medium caused a strong inhibition of the channel activity. This channel is likely to be involved in maintaining proper cation homeostasis in the RER of hepatocytes.     

A high-conductance cation channel from the inner membrane of the free-living soil bacteria Rhizobium etli

In this communication we reported the study of a cation channel present in the cytoplasmic membrane of the nitrogen fixing bacterium Rhizobium etli. Inner-membrane (IM) vesicles were purified and fused into planar lipid bilayers (PLBs), under voltage clamp conditions. We have found that fusion of IM-enriched vesicle fractions with these model membranes leads, mainly (>30% of 46 experiments), to the reconstitution of high-conductance channels. Following this strategy, the activity of a channel with main open conductance of 198 pS, in symmetrical 100 mM KCl, was recorded. The single-channel conductance increase to 653 pS in the presence of a 5:1 (cis to trans) gradient of KCl. The channel exhibits voltage dependency and a weak selectivity for cations showing a permeability ratios of P _Rb/P _K = 0.96, P _Na/P _K = 0.07, and a conductance ratio of γ_Rb/γ_K = 1.1. The channel here characterized represents a previously undescribed Rhizobium channel although its precise role in rhizobial physiology remains yet to be determined.     

Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation.

Dihydropyridine receptors were purified from rabbit skeletal muscle transverse tubule membranes and incorporated into planar lipid bilayers. Calcium channels from both the purified dihydropyridine receptor preparation and the intact transverse tubule membranes exhibited two sizes of unitary currents, corresponding to conductances of 7 +/- 1 pS and 16 +/- 3 pS in 80 mM BaCl2. Both conductance levels were selective for divalent cations over monovalent cations and anions. Cadmium, an inorganic calcium channel blocker, reduced the single channel conductance of calcium channels from the purified preparation. The organic calcium channel antagonist nifedipine reduced the probability of a single channel being open with little effect on the single channel conductance. The presence of two conductance levels in both the intact transverse tubule membranes and the purified dihydropyridine receptor preparation suggests that the calcium channel may have multiple conductance levels or that multiple types of calcium channels with closely related structures are present in transverse tubule membranes.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Voltage-gated anion channel of the electric organ of Narke japonica incorporated into planar bilayers

Voltage-gated anion channels in vesicles prepared from the electric organ of Narke japonica were studied using two methods. Ionic permeability was measured by the light scattering method, which could be used to measure the ion permeation of whole vesicles but only at a time scale of slower than about 0.1 s. The single channel conductances and permeability ratios for various ions were measured after fusing the vesicles to phospholipid bilayers. Both sets of results coincided, indicating that the anion channels observed with the planar bilayer method are the major route for anion passage in these vesicles. The channels showed anion selectivity and did not allow the permeation of cations such as K+ and choline+. The single channel conductance was 18 pS in 0.1 M Cl-. SCN- inhibited the conductance in a voltage-dependent reversible manner on both sides of a channel. SCN- may bind to the Cl- binding site in a channel and thus block it. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) blocked a channel on the cis (extracellular) side irreversibly. The number of anion channels per vesicle was estimated to be about 50. It was also shown that all anion channels in the vesicles were open at the very instance of fusion with planar membranes.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Potassium current activated by intracellular sodium in quail trigeminal ganglion neurons

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.     

Single chloride channels from Torpedo electroplax. Activation by protons

Single-channel fluctuations of a chloride-specific channel from Torpedo californica electroplax were studied with high current and time resolution. Channels were incorporated into virtually solvent-free planar bilayer membranes formed from phospholipid monolayers, and the substructure of the open channel was analyzed. The single channel displays three well-defined substates of conductances 0, 10, and 20 pS in 200 mM Cl-. These three substates are interpreted in terms of a dimeric channel complex composed of two identical "protochannels" gating independently in parallel on a time scale of milliseconds, but coupled together by a bursting process on a time scale of seconds. The probability of forming an open protochannel is voltage dependent and is increased strongly as aqueous pH is lowered. Variations of pH are effective only on the same side of the bilayer as the addition of electroplax vesicles. The dependence of single-channel kinetics on pH and voltage lead to a minimal four-state model in which both open and closed states can be protonated on a residue that changes its pK from 6 to 9 upon opening of the protochannel.     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Regulation of the hyperpolarization-activated K+ channel in the lateral membrane of the cortical collecting duct [published erratum appears in J Gen Physiol 1995 Sep;106(3):579]

An intermediate-conductance K+ channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K+ channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCl in the pipette and 5 mM KCl, 140 mM NaCl (NaCl Ringer''s solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KCl solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/Na+ was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann''s equation: Po = 1/[1 + exp(V-V1/2)zF/RT], with z = 1.2 and V1/2 = -40 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K+ channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKAc; 20 U/ml) and 100 microM [corrected] MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKAc increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K+ channel in the basolateral membrane of CCD.     

Two novel cardiac atrial K+ channels, IK.AA and IK.PC

Two K(+)-selective channels in neonatal rat atrial cells activated by lipophilic compounds have been characterized in detail. The arachidonic acid-stimulated channel (IK.AA) had a slope conductance of 124 +/- 17 pS at +30 mV in symmetrical 140 mM potassium and a mean open time of approximately 1 ms, and was relatively voltage independent. IK.AA activity was reversibly increased by lowering pH to 6.0. Arachidonic acid was most effective in activating this channel, although a number of lipophilic compounds resulted in activation. Surprisingly, choline, a polar molecule, also activated the channel. A second K+ channel was activated by 10 microM phosphatidylcholine applied to the intracellular surface of inside-out atrial patches. This channel (IK.PC) had a slope conductance of 60 +/- 6 pS at +40 mV and a mean open time of approximately 0.6 ms, and was also relatively voltage independent. Fatty acids are probably monomeric in the membrane under the conditions of our recording; thus detergent effects are unlikely. Since a number of compounds including fatty acids and prostaglandins activated these two channels, an indirect, channel-specific mechanism may account for activation of these two cardiac K+ channels.     

Calcium-permeable channel in sarcoplasmic reticulum of rabbit skeletal muscle

Membrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50 mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1 pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.