Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax of Electrophorus electricus.

The tetrodotoxin-binding protein purified from electroplax of Electrophorus electricus has been reincorporated into multilamellar vesicles that were used for patch recording. When excised patches of these reconstituted membranes were voltage clamped in the absence of neurotoxins, voltage-dependent single-channel currents were recorded. These displayed properties qualitatively and quantitatively similar to those reported for Na channels from nerve and muscle cells, including uniform single-channel conductances of the appropriate magnitude (approximately equal to 11 pS in 95 mM Na+), mean open times of approximately equal to 1.9 msec, and 7-fold selectively for Na+ over K+. Currents averaged from many depolarizations showed initial voltage-dependent activation and subsequent inactivation. In the presence of batrachotoxin, channels were observed with markedly different properties, including conductances of 20-25 pS (95 mM Na+), mean open times of approximately equal to 28 msec, and no indication of inactivation. Collectively, these findings indicate that the tetrodotoxin-binding protein of electroplax is a voltage-regulated sodium channel.     

Functional reconstitution of the purified brain sodium channel in planar lipid bilayers.

The ion conduction and voltage dependence of sodium channels purified from rat brain were investigated in planar lipid bilayers in the presence of batrachotoxin. Single channel currents are clearly resolved. Channel opening is voltage dependent and favored by depolarization. The voltage at which the channel is open 50% of the time is -91 +/- 17 mV (SD, n = 22) and the apparent gating charge is approximately 4. Tetrodotoxin reversibly blocks the ionic current through the sodium channels. The Ki for the tetrodotoxin block is 8.3 nM at -50 mV and is voltage dependent with the Ki increasing e-fold for depolarizations of 43 mV. The single channel conductance, gamma, is ohmic. At 0.5 M salt concentrations gamma = 25 pS for Na+, 3.5 pS for K+, and 1.2 pS for Rb+. This study demonstrates that the purified brain sodium channel--which consists of three polypeptide subunits: alpha (Mr approximately 260,000), beta 1 (Mr approximately 39,000), and beta 2 (Mr approximately 37,000)--exhibits the same voltage dependence, neurotoxin sensitivity, and ionic selectivity associated with native sodium channels.     

Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions.

Norepinephrine and carbamoylcholine stimulate accumulation of [3H]inositol phosphates from [3H]inositol-labeled guinea pig cerebral cortical synaptoneurosomes through interaction with alpha 1-adrenergic and muscarinic receptors, respectively. In addition to such agonist, a variety of natural products that affect voltage-dependent sodium channels can markedly stimulate accumulation of [3H]inositol phosphates. These include alkaloids that activate sodium channels, such as batrachotoxin, veratridine, and aconitine; peptide toxins that alter activation or slow inactivation of sodium channels, such as various scorpion toxins from Leiurus, Centruroides, and Tityus species; and agents that cause repetitive firing of sodium channel-dependent action potentials, such as pyrethroids and pumiliotoxin B. Ouabain, and agent that will increase accumulation of internal sodium by inhibition of Na+, K+-ATPase, also stimulates formation of [3H]inositol phosphates, as does monensin, a sodium ionophore. Tetrodotoxin and saxitoxin, specific blockers of voltage-dependent sodium channels, prevent or reduce the stimulatory effects of sodium channel agents and ouabain on phosphatidylinositol turnover, while having lesser or no effect, respectively, on receptor-mediated or monensin-mediated stimulation. Removal of extracellular sodium ions markedly reduces stimulatory effects of sodium channel agents, while removal of extracellular calcium ions with EGTA blocks both receptor-mediated and sodium channel agent-mediated phosphatidylinositol turnover. The results provide evidence for a hitherto unsuspected messenger role for sodium ions in excitable tissue, whereby neuronal activity and the resultant influx of sodium will cause activation of phospholipase systems involved in hydrolysis of phosphatidylinositols, thereby generating two second messengers, the inositol phosphates, which mobilize calcium from internal stores, and the diacylglycerols, which activate protein kinase C.     

Functional reconstitution of the purified sodium channel protein from rat sarcolemma.

The purified saxitoxin (STX) binding component of the rat sarcolemmal sodium channel (SBC) has been reconstituted into phospholipid vesicles. The reconstituted SBC displays the pharmacological properties and the ability to control sodium fluxes expected of a functional sodium channel. Batrachotoxin (BTX) increases 22Na+ influx into reconstituted SBC vesicles by greater than 100% over control at early time points. The BTX-stimulated 22Na+ influx is specifically and quantitatively blocked by STX. Veratridine and aconitine also stimulate Na+-flux--although less effectively than BTX--in the order: BTX greater than veratridine greater than aconitine. The logarithmic dose--response curves for BTX and veratridine are sigmoidal with a K0.5 of 1.5 microM and 35 microM, respectively. Vesicles containing the reconstituted SBC demonstrate 3H-labeled STX binding to a single class of high affinity sites witha Kd of 5--7 nM at 0 degrees C; the thermal stability of the STX receptor is markedly enhanced by reconstitution. Our results confirm that the purified STX binding component from rat sarcolemma constitutes the sodium channel itself and contains at least those components sufficient for channel activation, transmembrane ion movement, and inhibition by STX.     

beta-Adrenergic action on wild-type and KPQ mutant human cardiac Na+ channels: shift in gating but no change in Ca2+:Na+ selectivity.

OBJECTIVE: Prior studies of the modulation of the Na+ current by sympathetic stimulation have yielded controversial results. Separation of the Na+ and Ca2+ currents poses a problem in myocyte preparations. The gating of cloned Na+ channels is different in oocytes compared with mammalian expression systems. We have examined the sympathetic modulation of the alpha-subunit of the wild-type human cardiac Na+ channel (hH1) and the long QT-associated mutant, delta KPQ, expressed in human embryonic kidney cells. METHODS: Stable cell lines of hH1 and delta KPQ were established in human embryonic kidney cells. Whole-cell and single-channel currents were measured with the patch-clamp technique. Sympathetic stimulation was effected by exposure to isoproterenol or 8-bromo-cAMP. Na+ channel activation and inactivation were determined using standard voltage clamp protocols. Ca2+:Na+ permeability ratio was determined under bi-ionic conditions. RESULTS: We observed a qualitatively different effect of sympathetic stimulation on the cardiac Na+ current from that reported in frog oocytes: activation and inactivation kinetics were shifted to more negative potentials. This shift was similar for both hH1 and delta KPQ. [delta V0.5 for inactivation: 8.3 +/- 1.7 mV, p < 0.001 (hH1); 6.8 +/- 0.9 mV, p < 0.001 (delta KPQ)]. Increased rate of closed-state inactivation contributed to the shifting of the inactivation-voltage relationship. Open-state inactivation was not affected as mean open times were unchanged. Reversal potential measurement in hH1 suggested a low Ca2+:Na+ permeability ratio of 0.017, uninfluenced by sympathetic stimulation. In delta KPQ, the size of the persistent relative to the peak current was increased with 8-bromo-cAMP from 3.0 +/- 0.7% to 4.3 +/- 0.6% (p = 0.056). CONCLUSIONS: Sympathetic stimulation exerts multiple effects on the gating of hH1. Similar effects are also seen in delta KPQ which may increase arrhythmia susceptibility in long QT syndrome by modifying the Na+ channel contribution to the action potential.     

Purified skeletal muscle 1,4-dihydropyridine receptor forms phosphorylation-dependent oligomeric calcium channels in planar bilayers.

The purified 1,4-dihydropyridine receptor from skeletal muscle has been incorporated into planar bilayers, and its channel characteristics have been investigated. Conductances showed the characteristics of an L-type Ca2+ channel: divalent cation selectivity (PBa/PNa approximately equal to 30), blockage of Na+ conductance by micromolar Ca2+, and blockage of the Ca2+ channel by D890 and by Cd2+. The alpha 1 subunit of the receptor must be phosphorylated by the cAMP-dependent protein kinase to give channel activity. BAY K 8644 did not activate nonphosphorylated channels, and (+)-PN200-110 caused dramatic prolongation of mean open times when applied after phosphorylation. Channel properties were found to be dependent on association of receptor molecules in the bilayer. Single receptor molecules form channels of 0.9 pS (100 mM Ba2+) and show no voltage-dependent gating. Upon association, both voltage-dependent gating and higher conductance events are recovered; stabilized conductance levels assume values of even multiples of 0.9 pS, predominately 7.5 and 15 pS and multiples of these values up to 60 pS. Thus, individual channels become functionally coupled (synchronous opening and closing) with association, reinstating the characteristics of one larger unitary channel. It is concluded that the L-type Ca2+ channel represents an oligomer of 1,4-dihydropyridine-receptor protein complexes, each of which constitutes a channel, where the array of channels (oligochannel) opens and closes in concerted action.     

Novel arrhythmogenic mechanism revealed by a long-QT syndrome mutation in the cardiac Na(+) channel

Variant 3 of the congenital long-QT syndrome (LQTS-3) is caused by mutations in the gene encoding the alpha subunit of the cardiac Na(+) channel. In the present study, we report a novel LQTS-3 mutation, E1295K (EK), and describe its functional consequences when expressed in HEK293 cells. The clinical phenotype of the proband indicated QT interval prolongation in the absence of T-wave morphological abnormalities and a steep QT/R-R relationship, consistent with an LQTS-3 lesion. However, biophysical analysis of mutant channels indicates that the EK mutation changes channel activity in a manner that is distinct from previously investigated LQTS-3 mutations. The EK mutation causes significant positive shifts in the half-maximal voltage (V(1/2)) of steady-state inactivation and activation (+5.2 and +3.4 mV, respectively). These gating changes shift the window of voltages over which Na(+) channels do not completely inactivate without altering the magnitude of these currents. The change in voltage dependence of window currents suggests that this alteration in the voltage dependence of Na(+) channel gating may cause marked changes in action potential duration because of the unique voltage-dependent rectifying properties of cardiac K(+) channels that underlie the plateau and terminal repolarization phases of the action potential. Na(+) channel window current is likely to have a greater effect on net membrane current at more positive potentials (EK channels) where total K(+) channel conductance is low than at more negative potentials (wild-type channels), where total K(+) channel conductance is high. These findings suggest a fundamentally distinct mechanism of arrhythmogenesis for congenital LQTS-3.     

Ion channels in transit: voltage-gated Na and K channels in axoplasmic organelles of the squid Loligo pealei.

Ion channels that give rise to the excitable properties of the neuronal plasma membrane are synthesized, transported, and degraded in cytoplasmic organelles. To determine whether plasma membrane ion channels from these organelles could be physiologically activated, we extruded axoplasm from squid giant axons, dissociated organelles from the cytoskeletal matrix, and fused the free organelles with planar lipid bilayers. Three classes of ion channels normally associated with the plasma membrane were identified based on conductance, selectivity, and gating properties determined from steady-state single-channel recordings: (i) voltage-dependent Na channels, (ii) voltage-dependent delayed rectifier K channels, and (iii) large, voltage-independent K channels. The identity of the delayed rectifier channels was confirmed by reconstructing the time course of activation from single-channel responses to depolarizing voltage steps applied across the bilayer. These observations suggest that several classes of plasma membrane ion channels are transported in cytoplasmic organelles in physiologically active forms.     

Incorporation of calcium channels from cardiac sarcolemmal membrane vesicles into planar lipid bilayers.

When purified porcine cardiac sarcolemmal membrane vesicles are incorporated into planar lipid bilayers formed at the tip of patch electrode pipettes, individual divalent cation channels can be monitored. Channel activity is increased in the presence of the Ca2+ channel agonist Bay K 8644, is voltage dependent, and selects for divalent cations over anions. The activity does not inactivate because it is maintained during prolonged depolarizations. Determination of divalent cation selectivity from the reversal potential of single-channel currents indicates a relative permeability ratio for Ba/Ca/Mg of 1:0.45:0.08. Mean channel conductance in 0.1 M Ba2+/0.01 M Mg2+ is 8 pS. Channels are reversibly blocked by the Ca2+ channel inhibitor nitrendipine, and inhibition can be competitively antagonized by Bay K 8644. Binding studies with 3H-labeled D-600 demonstrate the presence of high-affinity receptors for D-600 in sarcolemmal membranes (Kd = 6.4 X 10(-9) M; Bmax = 3 pmol per mg of protein). In addition, experiments with resolved D-600 stereoisomers indicate that (-)D-600 is at least 25-fold more potent than (+)D-600 in competing for this aralkyl amine receptor. Consistent with this, (-)D-600 is much more effective than the (+) isomer in inhibiting bilayer-incorporated channels. These results demonstrate that the divalent cation channel that has been reconstituted in planar lipid bilayers possesses many of the characteristics of voltage-regulated Ca2+ channels in heart and suggest that receptors for Ca2+ entry blockers are functionally associated with this channel.     

Inward sodium current at resting potentials in single cardiac myocytes induced by the ischemic metabolite lysophosphatidylcholine.

To investigate possible ionic current mechanisms underlying ischemic arrhythmias, we studied single Na+ channel currents in rat and rabbit cardiac myocytes treated with the ischemic metabolite lysophosphatidylcholine (LPC) using the cell-attached and excised inside-out patch-clamp technique at 22 degrees C. LPC has been reported previously to reduce open probability and to induce sustained open channel activity at depolarized potentials. We now report two new observations for Na+ currents in LPC-treated patches: 1) The activation-voltage relation of the peak of the ensemble currents is shifted in the negative (hyperpolarizing) direction by approximately 20 mV compared with control currents. This effect was observed in all patches for depolarizations from a holding potential of -150 mV to different test potentials. 2) In some LPC-treated patches, Na+ channels exhibited sustained bursting activity at potentials as negative as -150 mV, giving a nondecaying inward current. This bursting activity was accompanied by double and triple simultaneous openings and closings, suggesting tight cooperativity in channel gating. These LPC-modified channels were identified as Na+ channels, because their unitary conductance was the same as Na+ channels in control solutions, because the single channel current-voltage relation was extrapolated to reverse at the Na+ Nernst potential, and because the current was blocked by the local anesthetic QX-222. This novel depolarizing current may play a role in the electrophysiological abnormalities in ischemia, including abnormal automaticity and reentrant arrhythmias, and could be a target for antiarrhythmic drugs.     

Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.

Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16 +/- 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35 +/- 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.     

Modification of single Na+ channels by batrachotoxin.

The modifications in the properties of voltage-gated Na+ channels caused by batrachotoxin were studied by using the patch clamp method for measuring single channel currents from excised membranes of N1E-115 neuroblastoma cells. The toxin-modified open state of the Na+ channel has a decreased conductance in comparison to that of normal Na+ channels. The lifetime of the modified open state is drastically prolonged, and channels now continue to open during a maintained depolarization so that the probability of a channel being open becomes constant. Modified and normal open states of Na+ channels coexist in batrachotoxin-exposed membrane patches. Unlike the normal condition, Na+ channels exposed to batrachotoxin open spontaneously at large negative potentials. These spontaneous openings apparently cause the toxin-induced increase in Na+ permeability which, in turn, causes membrane depolarization.     

Modulation of brain Na+ channels by a G-protein-coupled pathway.

Na+ channels in acutely dissociated rat hippocampal neurons and in Chinese hamster ovary (CHO) cells transfected with a cDNA encoding the alpha subunit of rat brain type IIA Na+ channel (CNaIIA-1 cells) are modulated by guanine nucleotide binding protein (G protein)-coupled pathways under conditions of whole-cell voltage clamp. Activation of G proteins by 0.2-0.5 mM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, increased Na+ currents recorded in both cell types. The increase in current amplitude was caused by an 8- to 10-mV negative shift in the voltage dependence of both activation and inactivation. The effects of G-protein activators were blocked by treatment with pertussis toxin or guanosine 5'-[beta-thio]diphosphate (GDP[beta S]), a nonhydrolyzable GDP analog, but not by cholera toxin. GDP[beta S] (2 mM) alone had effects opposite those of GTP[gamma S], shifting Na(+)-channel gating 8-10 mV toward more-positive membrane potentials and suggesting that basal activation of G proteins in the absence of stimulation is sufficient to modulate Na+ channels. In CNaIIA-1 cells, thrombin, which activates pertussis toxin-sensitive G proteins in CHO cells, caused a further negative shift in the voltage dependence of Na(+)-channel activation and inactivation beyond that observed with GTP alone. The results in CNaIIA-1 cells indicate that the alpha subunit of the Na+ channel alone is sufficient to mediate G protein effects on gating. The modulation of Na+ channels via a G-protein-coupled pathway acting on Na(+)-channel alpha subunits may regulate electrical excitability through integration of different G-protein-coupled synaptic inputs.     

Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA.

The voltage-dependent gating mechanism of single A-type potassium channels coded for by the Shaker locus of Drosophila was studied by single-channel recording. A-type channels expressed in Xenopus oocytes injected with Shaker B and Shaker D mRNA exhibited gating and voltage dependence that were qualitatively similar to those of the native Shaker A-types channels from embryonic myotubes. In all three channel types the molecular transition rates leading to the first opening were voltage-dependent, whereas all transitions after the first opening, including inactivation, were independent of voltage. While these channels exhibit some quantitative differences in their transition rates that account for the observed differences in macroscopic currents, in all three cases the voltage dependence of the macroscopic currents is determined by a voltage dependence in the time to first opening. This gating mechanism is similar to that of the vertebrate voltage-gated sodium channel and, together with the sequence similarities in the S4 region of the proteins, suggests a conserved mechanism for activation and inactivation.     

Purified, modified eel sodium channels are active in planar bilayers in the absence of activating neurotoxins.

A recent study showed that limited trypsin treatment of liposomes containing purified Electrophorus electricus sodium channels activates a sodium radiotracer flux. We now report that similarly treated sodium channels show voltage-gated, tetrodotoxin-sensitive and highly sodium-selective single-channel currents when incorporated into planar lipid membranes. The trypsinized channels opened repeatedly in bursts of several seconds duration, as would be expected for channels whose fast inactivation process had been removed. Furthermore, they have a higher conductance, different voltage-dependence of gating, and a remarkably higher selectivity (PNa/PK = 41) than sodium channels bound by batrachotoxin or other activating neurotoxins; these properties of the trypsinized channels are probably closer to those of channels in intact electrocytes.     

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.     

Voltage-dependent sodium and potassium channels in mammalian cultured Schwann cells.

Cultured Schwann cells from sciatic nerves of newborn rabbits and rats have been examined with patch-clamp techniques. In rabbit cells, single sodium and potassium channels have been detected with single channel conductances of 20 pS and 19 pS, respectively. Single sodium channels have a reversal potential within 15 mV of ENa, are blocked by tetrodotoxin, and have rapid and voltage-independent inactivation kinetics. Single potassium channels show current reversal close to EK and are blocked by 4-aminopyridine. From these results, and from comparisons of single-channel and whole-cell data, we show that these Schwann cells contain voltage-dependent sodium and potassium channels that are similar in most respects to the corresponding channels in mammalian axonal membranes. Cultured rat Schwann cells also have sodium channels, but at a density about 1/10th that of rabbit cells, a result in agreement with saxitoxin binding experiments on axon-free sectioned nerves. Saxitoxin binding to cultured cells suggests that there are up to 25,000 sodium channels in a single rabbit Schwann cell. We speculate that in vivo Schwann cells in myelinated axons might act as a local source for sodium channels at the nodal axolemma.     

Single anion-selective channels in basolateral membrane of a mammalian tight epithelium.

Basolateral membrane chloride permeability of surface cells from rabbit urinary bladder epithelium was studied using the patch-clamp technique. Two types of anion-selective channel were observed. One channel type showed inward rectification and had a conductance of 64 pS at-50 mV when bathed symmetrically by saline solution containing 150 mM chloride; the other resembled high-conductance voltage-dependent anion channels (VDACs). Both channels had the selectivity sequence Cl-approximately equal to Br-approximately equal to I- approximately equal to SCN- approximately equal to NO3- greater than F- greater than acetate greater than gluconate greater than Na+ approximately equal to K+ and were sensitive to the anion exchange inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Basolateral chloride conductance in urinary bladder is apparently due to the 64 pS anion channel, which is active at physiological potentials. Imperfect selectivity of this channel against cations might also account for the low, but finite, sodium permeability of the basolateral membrane.     

Reconstituted voltage-sensitive sodium channel from Electrophorus electricus: chemical modifications that alter regulation of ion permeability.

At equilibrium, voltage-sensitive sodium channels normally are closed at all potentials. They open transiently in response to changes in membrane voltage or chronically under the influence of certain neurotoxins. Covalent modifications that result in chronic opening may help identify molecular domains involved in conductance regulation. Here, the purified sodium channel from electric eel electroplax, reconstituted in artificial liposomes, has been used to screen for such modifications. When the liposomes were treated with the alkaloid neurotoxin batrachotoxin, sodium-selective ion fluxes were produced, with permeability ratios PNa greater than PTl greater than PK greater than PRb greater than PCs. When the liposomes were treated with either of two oxidizing reagents (N-bromoacetamide or N-bromosuccinimide), or with Pronase or trypsin, ion-selective fluxes also were stimulated. These were blocked by tetrodotoxin and the anesthetic QX-314 in a manner suggesting that only modification of the cytoplasmic protein surface resulted in stimulation. Limited exposure to trypsin resulted in strong flux activation, with the concomitant appearance of peptide fragments with masses of approximately equal to 130, 70, and 38 kDa and fragments with masses of 45 and 24 kDa appearing later. We propose that characterization of these fragments may allow identification of channel domains important for inactivation gating.     

Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.