Molecular determinants of membrane potential dependence in vertebrate gap junction channels.

The conductance, g(j), of many gap junctions depends on voltage between the coupled cells (transjunctional voltage, V(j)) with little effect of the absolute membrane potential (V(m)) in the two cells; others show combined V(j) and V(m) dependence. We examined the molecular determinants of V(m) dependence by using rat connexin 43 expressed in paired Xenopus oocytes. These junctions have, in addition to V(j) dependence, V(m) dependence such that equal depolarization of both cells decreases g(j). The dependence of g(j) on V(m) was abolished by truncation of the C-terminal domain (CT) at residue 242 but not at 257. There are two charged residues between 242 and 257. In full-length Cx43, mutations neutralizing either one of these charges, Arg243Gln and Asp245Gln, decreased and increased V(m) dependence, respectively, suggesting that these residues are part of the V(m) sensor. Mutating both residues together abolished V(m) dependence, although there is no net change in charge. The neutralizing mutations, together or separately, had no effect on V(j) dependence. Thus, the voltage sensors must differ. However, V(j) gating was somewhat modulated by V(m), and V(m) gating was reduced when the V(j) gate was closed. These data suggest that the two forms of voltage dependence are mediated by separate but interacting domains.

Acidic fibroblast growth factor inhibits junctional communication of Schwann cells in culture.

The effect of acidic fibroblast growth factor (aFGF) was investigated on junctional communication of rat Schwann cells (SC) in culture. As measured by dye transfer, the incidence of coupling between SC was very low during the phase of proliferation and increased slowly and progressively with time under culture conditions that induced the myelinating phenotype. Treatment with aFGF alone or in combination with heparin decreased markedly coupling between SC in both culture stages. The coupling inhibition was rapid, the earliest effects being apparent 5-15 min after addition of growth factor, and was transient with a slower recovery of coupling at 1-3 h. The uncoupling effect of aFGF could be prevented by an inhibitor of protein-tyrosine kinase. Addition of heparin to cultures decreased the most effective aFGF concentration by 100-fold, from 100 ng ml-1 to 1 ng ml-1. The dose-response curves exhibited a characteristic window-shape. The results suggest that FGF might be involved in the regulation of the junctional communication between rat SC via tyrosine kinases.

Molecular dissection of transjunctional voltage dependence in the connexin-32 and connexin-43 junctions.

Most gap junction channels are sensitive to the voltage difference between the two cellular interiors, termed the transjunctional voltage (V(j)). In several junctions, the conductance transitions induced by V(j) show more than one kinetic component. To elucidate the structural basis of the fast and slow components that characterize the V(j )dependence of connexin-32 (Cx32) and connexin-43 (Cx43) junctions, we created deletions of both connexins, where most of the carboxy-terminal (CT) domain was removed. The wild-type and "tailless" mutants were expressed in paired Xenopus oocytes, and the macroscopic gating properties were analyzed using the dual voltage clamp technique. Truncation of the CT domain of Cx32 and Cx43 abolished the fast mechanism of conductance transitions and induced novel gating properties largely attributable to the slow mechanism of gating. The formation of hybrid junctions comprising wild-type and truncated hemichannels allowed us to infer that the fast and slow components of gating reside in each hemichannel and that both gates close at a negative V(j) on the cytoplasmic side. Thus we conclude that the two kinetic components of V(j)-sensitive conductance are a result of the action of two different gating mechanisms. They constitute separate structures in the Cx32 and Cx43 molecules, the CT domain being an integral part of fast V(j) gating.

Altered formation of hemichannels and gap junction channels caused by C-terminal connexin-32 mutations.

Hexamers of connexins (Cxs) form hemichannels that dock tightly in series via their extracellular domains to give rise to gap junction channels. Here we examined the ability of a variety of C-terminal Cx32 mutations, most of which have been identified in X-linked Charcot-Marie-Tooth disease, to form hemichannels and to complete gap junction channels using the Xenopus oocyte system. First, we show that undocked wild-type Cx32 hemichannels at the plasma membrane can be detected as opening channels activated by depolarization. We have been able to estimate the efficiency of assembly of complete channels by measuring the time-dependent incorporation of preformed hemichannels into gap junction channels after cell-to-cell contact. These data offer strong evidence that hemichannels are the direct precursors of gap junction channels. Of 11 Cx32 mutants tested, a group of 5 mutations prevented the formation of functional hemichannels at the cell surface, whereas 4 mutations were fully able to form precursors but reduced the ability of hemichannels to assemble into complete channels, and 2 mutants formed channels normally. The data revealed that a minimum length of human Cx32 including the residue Arg-215 is required for the expression of hemichannels at the cell surface and that the efficiency of hemichannel incorporation into complete channels decreased gradually with the progressive shortening of the cytoplasmic C-terminal domain.

Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments.

Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Paris of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.

Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes.

Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.

Participation of voltage-gated conductances on the response succeeding inhibitory synaptic potentials in the crayfish slowly adapting stretch receptor neuron.

1. We examined the contribution of voltage-gated conductances to inhibitory postsynaptic potential (IPSP) effects under current clamp in silent and spiking slowly adapting stretch receptor neurons (SN1s) in the slow receptor muscle of the crayfish Procambarus. The receptor exemplifies the simplest inhibitory neural circuit, with one presynaptic and one postsynaptic neuron. The effects of synaptic inhibition were compared with the outcome of hyperpolarizing current pulses. Because pulse effects were exclusively due to postsynaptic mechanisms, an estimation of the synaptic or extrasynaptic origin of the results of IPSP was possible. 2. Inhibition by single IPSPs increased gradually with the time elapsed from the preceding spike in 60% of the spiking SN1s. However, early IPSP arrivals were exclusively excitatory in the rest of the cases. Inhibition was restricted to a single expanded SN1 interspike interval, but the early excitation and the postinhibitory rebound lasted several intervals. Rebound was invariably present; it was the only consequence of IPSPs in silent receptors and could be extremely long lasting (> 25 s). 3. The membrane potential of the SN1 neuron was clamped at hyperpolarized values (greater than -65 mV) by prolonged IPSP barrages at high rate (> 20/s). A prominent depolarizing sag and a gradual reduction of the IPSP amplitude were observed with prolonged presynaptic stimulation. There were subthreshold IPSP amplitude oscillations consisting of gradual increases and decreases of the post-IPSP peak depolarization at lower presynaptic rates. IPSP amplitude variations (< or = 10 mV) were primarily due to larger local responses. 4. Essentially all IPSP effects were mimicked by hyperpolarizing pulses. Sag was also evoked by pulses and was accompanied by a gradual conductance increase preceded by a brief initial drop. Sag and rebound were markedly reduced by Cs+ (2 mM) and tetrodotoxin (1 microM) and less by Ba2+ (5 mM) or tetraethylammonium (25 mM) superfusion. Both were somewhat decreased by acetylcholine (30 microM), which also markedly depolarized and accelerated firings, results which were usually reduced by atropine (10 microM). 5. In conclusion, IPSP and hyperpolarizing pulse effects were essentially identical, implying that extrasynaptic membrane properties were decisive. Interestingly, net excitatory consequences were usual, effectively increasing sensitivity and reducing the sensory threshold. Pharmacological evidence is provided suggesting that the hyperpolarization-activated current, IQ, and also probably the K+ M-current, the A-current, and the low-threshold, persistent Na+ conductances participate in sag and rebound genesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Bovine connexin44, a lens gap junction protein: molecular cloning, immunologic characterization, and functional expression.

PURPOSE: To identify, clone molecularly, characterize immunochemically, and express functionally a bovine lens gap junction protein (connexin). METHODS: The methods used were polymerase chain reaction, genomic cloning, RNA and DNA blotting, bacterial expression of a fusion protein, immunoblotting, alkaline phosphatase treatment, Xenopus oocyte expression, and voltage clamp technique. RESULTS: A bovine genomic clone encoding a polypeptide of 44,424 d, termed connexin44 (Cx44), was isolated. Cx44 was most closely related to the lens connexins rat Cx46 and chicken Cx56. The Cx44 DNA hybridized to a 2.5 kb mRNA detected only in lens RNA. The carboxyl terminal 161 amino acids from Cx44 were expressed in bacteria fused to maltose binding protein (MBP). The Cx44/MBP fusion protein reacted in immunoblots with anti-rat Cx46(411 to 416) antibodies and with the monoclonal antibody 5H1, but not with a monoclonal antibody to MP70 nor with antibodies to other connexins. Cx44 translated in vitro from the cloned DNA showed a single band with an apparent electrophoretic mobility of approximately 50 kd on polyacrylamide gels containing sodium dodecyl sulfate. Multiple bands of 53 to 57 kd were detected by immunoblotting in homogenates of bovine lens; these bands were reduced to a broad band of approximately 50 kd by alkaline phosphatase treatment, suggesting that they represented phosphorylated forms of Cx44. Cx44 RNA injected in single oocytes induced a large and characteristic time- and voltage-dependent current. Overexpression of Cx44 produced depolarization and cell lysis. Junctional currents that could be regulated by transjunctional voltage were induced between paired oocytes injected with Cx44 RNA. Observations in paired oocytes suggested the assembly of hemichannels into junctional channels. CONCLUSIONS: Cx44 is a phosphoprotein component of bovine lens fiber gap junctions. Although it has a relatively distinct sequence, it shares sequence similarity, immunologic cross-reactivity, and electrophysiological properties with rat Cx46. These data suggest that Cx44 is the protein previously identified in several immunohistochemical studies of bovine lens gap junctions that used anti-rat Cx46 antibodies. They also suggest that the formation of intercellular channels by pairing of hemichannels might prevent the cell lysis induced by the opening of unpaired hemichannels.

Membrane potential oscillations in CA1 hippocampal pyramidal neurons in vitro: intrinsic rhythms and fluctuations entrained by sinusoidal injected current.

The mechanisms mediating intrinsic and entrained CA1 pyramidal neuron rhythmic membrane potential oscillations were investigated in rat hippocampal slices. Intrinsic oscillations (6-14 Hz, < 10 mV) were evoked by long duration (2 s), depolarizing current pulses in 42% of the cells. Oscillations were also evoked by imposing sinusoidal transmembrane currents at 2, 7, and 14 Hz, adjusted at 7 Hz to imitate the synaptically mediated in vivo "intracellular theta". Slow all-or-none events (40 mV, 55 ms)--reminiscent of the rhythmic, high threshold slow spikes observed in vivo--were evoked and entrained by the sine wave current cycles with large, imposed depolarization in 35% of the cells. Intrinsic oscillations were insensitive to Ca(2+)-free, Co2+ (2 mM) and Mn2+ (2 mM) solutions, but were blocked by tetrodotoxin (TTX; 5 microM), illustrating that they were Na(+)-mediated. Tetraethylammonium (TEA; 15 mM) unmasked slow all-or-none events (40-50 mV, 20-55 ms) and plateau potentials (40-60 mV, 100-700 ms). Plateaus were Co2+ and Mn2+ resistant and were abolished by TTX, hence suggesting that the underlying persistent conductance was Na(+)-mediated. Plateaus were entrained one-to-one at all sinusoidal current frequencies in Ca(2+)-free, TEA + Co2+, or TEA + Mn2+ solutions. However, the high threshold Ca2+ spikes uncovered in TEA + TTX could only follow sinusoidal currents of less than 7 Hz. In conclusion, the high threshold Ca2+ and persistent Na+ conductances coexist in CA1 pyramidal cells. The persistent Na+ conductance mediated the intrinsic oscillations, and fluctuated at all the sine wave current frequencies used. The more sluggish high-threshold Ca2+ conductance exclusively oscillated at frequencies of less than 7 Hz and did not support the intrinsic rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Gap junctions formed by connexins 26 and 32 alone and in combination are differently affected by applied voltage.

Gap junctions are formed by a family of homologous proteins termed connexins. Their channels are dodecamers, and homomeric forms differ in their properties with respect to control by voltage and other gating stimuli. We report here the properties of coupling from expression of connexin complementary RNAs (cRNAs; sense to mRNA, antisense to cDNA) in Xenopus oocyte pairs in which endogenous coupling was blocked by injection of DNA oligonucleotides antisense to the mRNA of Cx38, the principal endogenous connexin. We found that a connexin recently sequenced from rat liver, Cx26, formed functional gap junctions whose conductance exhibited voltage dependence with unusual characteristics suggestive of two gating mechanisms. Junctional conductance (gj) was increased to a small degree by depolarization and decreased by hyperpolarization of either cell in a coupled pair, indicating dependence on the potential between the inside and outside of the cells (Vi-o). These changes were fast compared with the resolution of their measurement (ca. 10 ms). On a slower timescale, large transjunctional potentials (Vj) of either sign caused a more substantial decrease in conductance similar to that previously reported for several other gap junctions. Homotypic junctions formed of another connexin, Cx32, exhibited a similar slow dependence on Vj but no dependence on Vi-o. In contrast, heterotypic junctions between an oocyte expressing Cx26 and one expressing Cx32 were electrically asymmetric; they exhibited a greater fast change in gj, which depended, however, on Vj, such that gj increased with relative positivity on the Cx26 side and decreased with relative negativity on the Cx26 side. There was also a large slow decrease in gj in response to Vj for relative positivity on the Cx26 side but not for Vj of the opposite sign. These data indicate that properties of the hemichannels contributed by the two connexins in the heterotypic case were changed from their properties in homotypic junctions. The fast change in gj may involve a mechanism analogous to that at fast rectifying electrical synapses. Experiments in which oocytes expressing Cx32 were paired with oocytes expressing both Cx26 and Cx32 demonstrated that asymmetric junctions would form between oocytes expressing both connexins, thereby confirming their potential relevance in vivo, where the same coupled cells are known to express both proteins.

Intracellular analysis of excitatory-inhibitory synaptic interactions in crayfish stretch receptors.

1. To determine the membrane mechanisms underlying the interactions between inhibitory postsynaptic potentials (IPSPs) and excitatory inputs, we investigated, at the membrane potential level, the combined influences of low-frequency (0.05-0.50 Hz) imposed sinusoidal transmembrane currents (termed sine currents), representing the excitatory drive, and trains of regular (3-30/s) IPSPs. The two simplest possible neuron systems exemplified by the slowly and rapidly adapting stretch receptors of crayfish (RM1 and RM2, respectively) were used. 2. At constant elongation the RM1 and RM2 behaved as a pacemaker and a neuron without self-sustained oscillations, respectively, but in dynamic conditions uninhibited controls and IPSP sine current interactions were essentially identical in both RMs. Controls showed the usual smooth variation of the RM firing rate in response to the gradually varying excitatory input. IPSP effects were characterized by the expected overall reduction of the postsynaptic firing rate. More important, special effects were also present, such as the simple fixed alternations of IPSP and postsynaptic spikes (e.g., 1 IPSP, 1 postsynaptic or 1:1; 1 IPSP, 2 postsynaptic or 1:2; 2 IPSPs, 1 postsynaptic spike or 2:1), where interspike intervals were more constant than uninhibited controls and where the sensitivity to the excitatory input was reduced to small values, and the sudden firing rate discontinuities consisting of instantaneous discharge accelerations or decelerations (termed "jumps") between successive alteration ratios, where sensitivity increased to large values. Therefore with inhibition the RM firing rate varied discontinuously in response to the gradually changing input, and the discharge rate could take one of several discrete values by switching between different alteration ratios. 3. At the alternations the times elapsed between an IPSP and the closest spike before (phase, phi) or after it (cophase, theta) increased and decreased, respectively, with increasing excitation. The major membrane potential modification that accompanied the interactions at the alternations was the gradual increase of the post-IPSP slope as a function of excitatory drive, which reduced the time to reach the firing level or theta. 4. Inhibition introduced subtle and complex nonlinear modifications in the coding of convergent excitatory input. The most notable nonlinearity was the discontinuous variations of the firing rate as a function of the gradually changing excitatory input. Effects were due to voltage interactions occurring at the extrasynaptic membrane, with a decisive involvement of the spike generator and insignificant participation of the shunting action of IPSPs. The results provide yet another example of the predominant influence of intrinsic membrane properties in determining the effects of synaptic-evoked activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Dynamic analysis of sensory-inhibitory interactions in crayfish stretch receptor neurons.

1. The effects of regular and random inhibition at moderate rates on the sensory response evoked by sinusoidal stretches were investigated in slowly and rapidly adapting stretch receptors of crayfish (RM1 and RM2, respectively). 2. Although the RM1 has pacemaker properties and the RM2 is spontaneously silent, inhibitory postsynaptic potential (IPSP) effects were similar in both mechanosensory neurons. The most common consequence was the expected reduction of the sensory response and the increase of the elongation needed to reach firing threshold. With regular IPSPs there were regions where pre- and postsynaptic spikes alternated at fixed integer ratios, usually 1:1, more rarely 1:2 and 1:3. Increases or decreases of the sensory excitation caused sudden postsynaptic accelerations or decelerations when specific length bounds were crossed and where pre- and postsynaptic alternations changed to lower (e.g., from 1:1 to 1:2) or higher ratios (e.g., from 1:2 to 1:1), respectively. 3. Paradoxical effects were also observed because increasing or decreasing the inhibitory rate for a given alternation ratio (e.g., 1:1) accelerated or decelerated the output rate, respectively. Alternations and paradoxical behaviors disappeared with IPSP pattern irregularization. Random IPSPs strongly irregularized the receptor's output. Inhibition, especially if the pattern was irregular, could excite under special conditions. 4. With regular IPSPs, mechanical sensitivity became zero at the lengths at which receptors were silenced, low during alternations, and maximum at transitions between successive alternation ratios. Irregular IPSPs did not have this delinearizing consequence. 5. In conclusion, inhibition introduced important complex modifications in the coding of mechanosensory information. Effects were similar in both receptor types, indicating that self-sustained oscillations are not fundamental. The observed changes were critically dependent on pre- and postsynaptic rate and pattern. They cannot be explained by simple summation of converging sensory and inhibitory inputs and represent another observation of the complex dynamic behavior of periodically driven nonlinear systems.

Temporal correlations in sensory-synaptic interactions: example in crayfish stretch receptors.

1. The effects of inhibitory postsynaptic potential (IPSP) barrages on coding of sinusoidal length inputs were investigated in slowly and rapidly adapting stretch receptors of crayfish (RM1 and RM2, respectively). To examine the contribution of postsynaptic firings, a description was obtained of the temporal correlations between sensory stimuli, pre- and postsynaptic spikes, plus the relative position of the latter. In this analysis, the times between an IPSP and the closest spike preceding or following it are known, respectively, as phase (phi) and cophase (theta). 2. Interactions were similar in RM1 and RM2. The usual effect was the expected increased postsynaptic interval durations, but special consequences were also evoked. With regular IPSPs there were epochs including inhibitory (S) and postsynaptic (R) spikes and fixed "alternations" at S/R times the IPSP rate. S/R ratios were usually 1:1 and 1:2, more rarely 2:1. There were sudden postsynaptic interval duration changes, or "jumps," between successive alternation ratios where inhibitory effects increased or decreased. With 1:1 and 1:2 ratios there was a gradual "sliding," or phi increase-theta decrease, at the pull and the opposite at the release. The theta and phi roughly paralleled the control postsynaptic intervals and the inhibited-minus-control differences, respectively, when sliding was present. Sliding disappeared, and phi and theta tended to become invariant, with 2:1 ratio. 3. Irregular IPSP increased postsynaptic interval irregularities and eliminated alternations. Phase values were irregularly distributed, but theta and control postsynaptic intervals were similar irrespective of pattern.(ABSTRACT TRUNCATED AT 250 WORDS)