Gap26, a connexin mimetic peptide, inhibits currents carried by connexin43 hemichannels and gap junction channels.

Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca(2+) across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell-cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca(2+) solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations.

Conductive and kinetic properties of connexin45 hemichannels expressed in transfected HeLa cells.

Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca2+]o revealed an extra current. Its sensitivity to extracellular Ca2+ and gap junction channel blockers (18alpha-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, Ihc, were determined adopting a biphasic pulse protocol. Ihc activated at positive Vm and deactivated partially at negative Vm. The analysis of the instantaneous Ihc yielded a linear function ghc,inst = f(Vm) with a hint of a negative slope (ghc,inst: instantaneous conductance). The analysis of the steady-state Ihc revealed a sigmoidal function ghc,ss=f(Vm) best described with the Boltzmann equation: Vm,0= -1.08 mV, ghc,min=0.08 (ghc,ss: steady-state conductance; Vm,0: Vm at which ghc,ss is half-maximally activated; ghc,min: minimal conductance; major charge carriers: K+ and Cl-). The ghc was minimal at negative Vm and maximal at positive Vm. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. Ihc deactivated exponentially with time, giving rise to single time constants, taud. The function taud = f(Vm) was exponential and increased with positive Vm (taud=7.6 s at Vm=0 mV). The activation of Ihc followed the sum of two exponentials giving rise to the time constants, taua1 and taua2. The function taua1=f(Vm) and taua2 = f(Vm) were bell-shaped and yielded a maximum of congruent with 0.6 s at Vm congruent with -20 mV and congruent with 4.9 s at Vm congruent with 15 mV, respectively. Neither taua1 =f(Vm) nor taua2 = f(Vm) coincided with taud=f(Vm). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes.

Characterization of zebrafish Cx43.4 connexin and its channels.

Connexins (Cx) form intercellular junctional channels which are responsible for metabolic and electrical coupling. We report here on the biochemical and immunohistochemical characterization of zebrafish connexin zfCx43.4, an orthologue of mammalian and avian Cx45, and the electrophysiological properties of junctional channels formed by this protein. The investigations were performed on transfected COS-7 cells or HeLa cells. Using site-directed antibodies, zfCx43.4 cDNA (GenBank accession no. X96712) was demonstrated to code for a protein with a M(r) of 45 000. In transfected cells, zfCx43.4 was localized in cell-cell contact areas as expected for a gap junction protein. zfCx43.4 channels were shown to transfer Lucifer Yellow. The multichannel currents were sensitive to the transjunctional voltage (V(j)). Their properties were consistent with a two-state model and yielded the following Boltzmann parameters for negative/positive V(j): V(j,0) = -38.4/41.9 mV; g(j,min) = 0.19/0.18; z = 2.6/2.3. These parameters deviate somewhat from those of zfCx43.4 channels expressed in Xenopus oocytes and from those of Cx45, an orthologue of zfCx43.4, expressed in mammalian cells or Xenopus oocytes. Conceivably, the subtle differences may reflect differences in experimental methods and/or in the expression system. The single channel currents yielded two prominent levels attributable to a main conductance state (gamma(j,main) = 33.2 +/- 1.5 pS) and a residual conductance state (gamma(j,residual) = 11.9 +/- 0.6 pS).

Synthesis of the fully phosphorylated GPI anchor pseudohexasaccharide of Toxoplasma gondii.

Retrosynthesis of the fully phosphorylated glycosylphosphatidyl inositol (GPI) anchor pseudohexasaccharide 1a led to building blocks 2-6, of which 5 and 6 are known. The formation of pseudodisaccharide building block 2 is based on readily available building block 7, which gave, via derivative 11 and its glycosylation with known donor 12, the desired compound 2. Building block 3, with the required access to all hydroxy groups being permitted, was prepared from mannose in five steps. From a readily available precursor, building block 4 was obtained, which on reaction with 3 gave disaccharide 23. The synthesis of the decisive pseudohexasaccharide intermediate 32 was based on the reaction of 23 with 5, then with 6, and finally with 2. To obtain high stereoselectivity and good yields in the glycosylation reactions, anchimeric assistance was employed. To enable regioselective attachment of the two different phosphorus esters, the 6f-O-silyl group of 32 was first removed and the aminoethyl phosphate residue was attached. Then the MPM group was oxidatively removed, and the second phosphate residue was introduced. Unprotected 1a was then liberated in two steps: treatment with sodium methanolate removed the acetyl protecting groups, and finally, catalytic hydrogenation afforded the desired target molecule, which could be fully structurally assigned.

Influence of dynamic gap junction resistance on impulse propagation in ventricular myocardium: a computer simulation study.

The gap junction connecting cardiac myocytes is voltage and time dependent. This simulation study investigated the effects of dynamic gap junctions on both the shape and conduction velocity of a propagating action potential. The dynamic gap junction model is based on that described by Vogel and Weingart (J. Physiol. (Lond.). 1998, 510:177-189) for the voltage- and time-dependent conductance changes measured in cell pairs. The model assumes that the conductive gap junction channels have four conformational states. The gap junction model was used to couple 300 cells in a linear strand with membrane dynamics of the cells defined by the Luo-Rudy I model. The results show that, when the cells are tightly coupled (6700 channels), little change occurs in the gap junction resistance during propagation. Thus, for tight coupling, there are negligible differences in the waveshape and propagation velocity when comparing the dynamic and static gap junction representations. For poor coupling (85 channels), the gap junction resistance increases 33 MOmega during propagation. This transient change in resistance resulted in increased transjunctional conduction delays, changes in action potential upstroke, and block of conduction at a lower junction resting resistance relative to a static gap junction model. The results suggest that the dynamics of the gap junction enhance cellular decoupling as a possible protective mechanism of isolating injured cells from their neighbors.

Intracellular domains of mouse connexin26 and -30 affect diffusional and electrical properties of gap junction channels.

To evaluate the influence of intracellular domains of connexin (Cx) on channel transfer properties, we analyzed mouse connexin (Cx) Cx26 and Cx30, which show the most similar amino acid sequence identities within the family of gap junction proteins. These connexin genes are tightly linked on mouse chromosome 14. Functional studies were performed on transfected HeLa cells stably expressing both mouse connexins. When we examined homotypic intercellular transfer of microinjected neurobiotin and Lucifer yellow, we found that gap junctions in Cx30-transfected cells, in contrast to Cx26 cells, were impermeable to Lucifer yellow. Furthermore, we observed heterotypic transfer of neurobiotin between Cx30-transfectants and HeLa cells expressing mouse Cx30.3, Cx40, Cx43 or Cx45, but not between Cx26 transfectants and HeLa cells of the latter group. The main differences in amino acid sequence between Cx26 and Cx30 are located in the presumptive cytoplasmic loop and C-terminal region of these integral membrane proteins. By exchanging one or both of these domains, using PCR-based mutagenesis, we constructed Cx26/30 chimeric cDNAs, which were also expressed in HeLa cells after transfection. Homotypic intercellular transfer of injected Lucifer yellow was observed exclusively with those chimeric constructs that coded for both cytoplasmic domains of Cx26 in the Cx30 backbone polypeptide chain. In contrast, cells transfected with a construct that coded for the Cx26 backbone with the Cx30 cytoplasmic loop and C-terminal region did not show transfer of Lucifer yellow. Thus, Lucifer yellow transfer can be conferred onto chimeric Cx30 channels by exchanging the cytoplasmic loop and the C-terminal region of these connexins. In turn, the cytoplasmic loop and C-terminal domain of Cx30 prevent Lucifer yellow transfer when swapped with the corresponding domains of Cx26. In chimeric Cx30/Cx26 channels where the cytoplasmic loop and C-terminal domains had been exchanged, the unitary channel conductance was intermediate between those of the parental channels. Moreover, the voltage sensitivity was slightly reduced. This suggests that these cytoplasmic domains interfere directly or indirectly with the diffusivity, the conductance and voltage gating of the channels.

Co-operativity between mouse connexin30 gap junction channels.

HeLa cells stably transfected with mouse cDNA coding for connexin30 (Cx30) were used to study the electrical properties of gap junction channels. The experiments involved the measurement of intercellular currents (Ij) from cell pairs using dual whole-cell recording with the patch-clamp method. The aim was to compare Ij from cell pairs whose gap junctions consisted of a single channel and cell pairs whose gap junctions consisted of many channels. We found that both the ensemble average currents gained from single-channel records and the currents obtained from multichannel records inactivated exponentially with time. However, the former inactivated significantly slower than the latter. At ajunctional voltage (Vj) of 50 mV, the time constants of inactivation (tau(i)) were 8.1 s and 1.6 s, respectively. Moreover, the ratio tau(i)(single-channel)/tau(i)(multichannel) turned out to be voltage sensitive, i.e. it decreased with increasing V(j) These observations suggest that the operation of Cx30 gap junction channels in the multichannel configuration involves co-operative interactions.

The kinetics of gap junction currents are sensitive to the ionic composition of the pipette solution.

Myocytes were isolated from neonatal rat hearts using an enzymatic procedure. Cell pairs were used to control the junctional voltage, V(j), and to measure the transjunctional current, I(j), using the dual voltage-clamp method. V(j) gradients provoked I(j) signals with voltage-dependent inactivation. During voltage pulses, I(j) remained virtually constant at ¿V(j)¿ <40 mV. At ¿V(j)¿>40 mV, it inactivated with time to a residual level. The inactivation followed a single exponential. The time constant of I(j) inactivation, taui, and the size of I(j) at steady state, I(j,ss), were both sensitive to the ions in the pipette solution. I(j,ss) was smaller in the presence of tetraethylammonium aspartate (TEA+ aspartate-) than KC1, while taui was smaller in the presence of KC1 than TEA+ aspartate-. The modification of I(j,ss) is readily explained by a change in the residual conductance of the gap junction channels, gammaj,residual x The alterations in taui are correlated with a change in beta, the rate constant that describes the transition of the channel from the main state to the residual state. Pipette solutions may affect the kinetics of gap junction currents by altering the conductive and/or kinetic parameters. Computer simulations revealed a substantial influence of the latter, but only a marginal effect of the former. Conceivably, ions of the pipette solution may affect the kinetics of gap junction channels by screening surface charges of the channel wall.

Electrical properties of gap junction hemichannels identified in transfected HeLa cells.

Human HeLa cells transfected with mouse connexin Cx30, Cx46 or Cx50 were used to study the electrical properties of gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels. Multichannel currents showed a time-dependent inactivation sensitive to voltage, Vm. Plots of the instantaneous conductance, ghc,inst, versus Vm were constant; plots of the steady-state conductance, ghc,ss, versus Vm were bell-shaped. Single-channel currents showed two conductances, gammahc,main and gammahc,residual, the latter approximately or approximately equals=1/6 of the former. Single-channel currents exhibited fast transitions (1-2 ms) between the main state and residual state. Late during wash-in and early during wash-out of 2 mM heptanol, single-hemichannel currents showed slow transitions between an open state and closed state. The open channel probability, Po, was Vm-dependent. It declined from approximately =1 at Vm= 0 mV to 0 at large Vm of either polarity. Hemichannel currents showed a voltage-dependent gammahc,main, i.e., it increased/decreased with hyperpolarization/depolarization. Extrapolation to Vm=0 mV led to a gammahc,main of 283, 250 and 352 pS for Cx30, Cx46 and Cx50, respectively. The hemichannels possess two gating mechanisms. Gating with positive voltage reflects Vj-gating of gap junction channels, gating with negative voltage reflects a property inherent to hemichannels, i.e., Vm or "loop" gating. We conclude that Cx30, Cx46 and Cx50 form voltage-sensitive hemichannels in single cells which are closed under physiological conditions.

Formation of heterotypic gap junction channels by connexins 40 and 43.

Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, and Cx43-HeLa) revealed a relationship, g(j)=f(V(j)), at steady state, that is typified by a nonsymmetrical behavior similar to that previously reported for other heterotypic channels (gap junction conductance [g(j)]; transjunctional voltage [V(j)]). The unitary conductance of the channels was sensitive to the polarity of V(j). A main state conductance of 61 pS was found when the Cx43 cell was stepped positively or the Cx40 cell negatively (V(j)=70 mV); the reverse polarities yielded a conductance of 100 pS. These heterotypic channels were permeable to carboxyfluorescein. In addition, two other heterotypic forms are illustrated to demonstrate that endogenous Cx45 expression cannot explain the results. The demonstration of heterotypic Cx40-Cx43 channels may have implications for the propagation of the electrical impulse in heart. For example, they may contribute to the slowing of the impulse propagation through the junctions between Purkinje fibers and ventricular muscle.

Voltage gating of Cx43 gap junction channels involves fast and slow current transitions.

RIN cells transfected with mouse cDNA coding for connexin43 (Cx43) were used to further examine the electrical properties of single gap junction channels. The experiments involved measuring intercellular currents from cell pairs using dual whole-cell recording with the patch-clamp method. We found that the single-channel currents exhibit two types of transitions and several conductance states. Besides fast transitions between the main open state and the residual state, the channels underwent slow transitions between an open state (i.e. main open state or residual state) and a closed state. The fast transitions lasted less than 2 ms, the slow ones ranged from 3.5 to 145 ms. The incidence of slow transitions increased with increasing transjunctional voltage. These observations are consistent with the notion that Cx43 gap junction channels possess more than one mechanism of voltage gating.

Biophysical properties of mouse connexin30 gap junction channels studied in transfected human HeLa cells.

1. Human HeLa cells expressing mouse connexin30 (Cx30) were used to study the electrical properties of Cx30 gap junction channels. Experiments were performed on cell pairs with the dual voltage-clamp method. 2. The gap junction conductance (gj) at steady state showed a bell-shaped dependence on junctional voltage (Vj; Boltzmann fit: Vj,0 = 27 mV, gj,min = 0.15, z = 4). The instantaneous gj decreased slightly with increasing Vj. 3. The gap junction currents (Ij) declined with time following a single exponential. The time constants of Ij inactivation (taui) decreased with increasing Vj. 4. Single channels exhibited a main state, a residual state and a closed state. The conductances gammaj,main and gammaj,residual were 179 and 48 pS, respectively (pipette solution, potassium aspartate; temperature, 36-37 degrees C; extrapolated to Vj = 0 mV). 5. The conductances gammaj,residual and gammaj,main showed a slight Vj dependence and were sensitive to temperature (Q10 values of 1.28 and 1.16, respectively). 6. Current transitions between open states (i.e. main state, substates, residual state) were fast (< 2 ms), while those between an open state and the closed state were slow (12 ms). 7. The open channel probability (Po) at steady state decreased from 1 to 0 with increasing Vj (Boltzmann fit: Vj,0 = 37 mV; z = 3). 8. Histograms of channel open times implied the presence of a single main state; histograms of channel closed times suggested the existence of two closed states (i.e. residual states). 9. We conclude that Cx30 channels are controlled by two types of gates, a fast one responsible for Vj gating involving transitions between open states (i.e. residual state, main state), and a slow one correlated with chemical gating involving transitions between the closed state and an open state.

Electrophysiological properties of gap junction channels in hepatocytes isolated from connexin32-deficient and wild-type mice.

Hepatocytes were isolated from wild-type and connexin32-deficient (Cx32-deficient) mice. Pairs of cells were chosen to study the electrical properties of gap junction channels using the dual voltage-clamp method. The total gap junction currents revealed that Cx32-deficient hepatocytes express one type of connexin (Cx26) and wild-type hepatocytes express two types of connexins (Cx26 and Cx32). The unitary gap junction currents suggest that Cx32-deficient cells have homotypic channels (Cx26-Cx26) while wild-type cells form homotypic (Cx26-Cx26, Cx32-Cx32) and heterotypic channels (Cx26-Cx32). Homotypic channels exhibited a main conductance and a residual conductance, both virtually insensitive to gap junction voltage (Vj) (Cx32-Cx32: gammaj,main=31 pS, gammaj,residual=9 pS; Cx26-Cx26: gammaj,main=102 pS, gammaj,residual=17 pS). Residual states were regularly seen in Cx32-Cx32 channels, but rarely in Cx26-Cx26 channels. Heterotypic channels showed a main conductance and a residual conductance. The former was sensitive to Vj (average gammaj,main=52 pS). The electrophysiological data suggest that Cx32 hemichannels are more abundant than Cx26 hemichannels in prenatal (ratio 4:1) and adult wild-type hepatocytes (ratio 23:1) and that the total number of gap junction channels is larger in prenatal cells than in adult cells. The diversity of the relationship gj, ss/gj,inst=f(Vj) (gj,ss: gap junction conductance at steady state; gj,inst: instantaneous gap junction conductance; Vj: transjunctional voltage) seen in wild-type cells suggests that the ratio Cx26/Cx32 hemichannels is variable among hepatocytes. A comparison of total and unitary conductances implies that Cx26 hemichannels are down-regulated in Cx32-deficient cells and that docking between Cx26 and Cx32 hemichannels occurs randomly. While the gap junction currents are compatible with homotypic and heterotypic channels, the presence of heteromeric channels cannot be excluded.

Mathematical model of vertebrate gap junctions derived from electrical measurements on homotypic and heterotypic channels.

1. A mathematical model has been developed which describes the conductive and kinetic properties of homotypic and heterotypic gap junction channels of vertebrates. 2. The model consists of two submodels connected in series. Each submodel simulates a hemichannel and consists of two conductances corresponding to a high (H) and low (L) conductance state and a switch, which simulates the voltage-dependent channel gating. 3. It has been assumed that the conductances of the high state and low state vary exponentially with the voltage across the hemichannel. 4. The parameters of the exponentials can be derived from data of heterotypic or homotypic channels. As a result, the behaviour of heterotypic channels can be predicted from homotypic channel data and vice versa. 5. The two switches of a channel are governed by the voltage drop across the respective hemichannel. The switches of a channel work independently, thus giving rise to four conformational states, i.e. HH, LH, HL and LL. 6. The computations show that the dogma of a constant conductance for homotypic channels results from the limited physiological range of transjunctional voltages (Vj) and the kinetic properties of the channel, so a new fitting procedure is presented. 7. Simulation of the kinetic properties at the multichannel level revealed current time courses which are consistent with a contingent gating. 8. The calculations have also shown that the channel state LL is rare and of short duration, and hence easy to miss experimentally. 9. The design of the model has been kept flexible. It can be easily expanded to include additional features, such as channel substates or a closed state.

Long-chain n-alkanols and arachidonic acid interfere with the Vm-sensitive gating mechanism of gap junction channels.

Experiments were carried out on preformed cell pairs and induced cell pairs of an insect cell line (mosquito Aedes albopictus, clone C6/36). The coupling conductance, gj, was determined with the dual voltage-clamp method. Exposure of preformed cell pairs to lipophilic agents, such as long-chain n-alkanols (n-hexanol, n-heptanol, n-octanol, n-nonanol, n-decanol) or arachidonic acid, provoked a decrease in gj. Hyperpolarization of both cells led to a recovery of gj. Systematic studies revealed that this phenomenon is caused by a shift of the sigmoidal relationship gj(ss) = f(Vm) towards more negative values of Vm (where gj(ss) = conductance at steady-state; Vm = membrane potential). The shift was dose dependent, it developed with time and was reversible. The longer the hydrocarbon chain of n-alkanols, the lower was the concentration required to produce a given shift. Besides shifting the function gj(ss) = f(Vm), arachidonic acid decreased the maximal conductance, gj(max). Single-channel records gained from induced cell pairs revealed that the lipophilic agents interfere with the Vm-sensitive slow channel gating mechanism. Application provoked slow current transitions (transition time: 5-40 ms) between an open state of the channel (i.e. main state or residual state) and the closed state; subsequently, fast channel transitions (transition time: < 2 ms) involving the main state and the residual state ceased completely. Hyperpolarization of Vm or washout of the lipophilic agents gave rise to the inverse sequence of events. The single-channel conductances gammaj(main state) and gammaj(residual state) were not affected by n-heptanol. We conclude that long-chain n-alkanols and arachidonic acid interact with the Vm-sensitive gating mechanism.

Modulation of cardiac gap junctions: the mode of action of arachidonic acid.

Myocytes isolated from neonatal rat hearts were grown in culture dishes. Cell pairs were selected to examine the mode of action of arachidonic acid (AA) on gap junctions. The dual voltage-clamp method was used to measure intercellular currents and determine the gap junction conductance, gj. Exposure of cell pairs to 10 microM AA produced reversible uncoupling. Pretreatment with 10 microM POCA (sodium-2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate; which inhibits mitochondrial beta-oxidation) did not prevent AA-dependent uncoupling. Thus, it seems that metabolites of beta-oxidation are not involved in AA-induced impairment of gj. Pre-exposure to 10 microM indomethacin (which blocks the cyclooxygenase pathway of the AA-cascade) had no effect on AA-dependent uncoupling. This suggests that cyclooxygenase products such as prostaglandins or thromboxanes play no role in gj modulation. Exposure to 5 microM NDGA (nordihydroguaiaretic acid; which inhibits the 5-lipoxygenase pathway) or 10 microM ETYA (5,8,11,14-eicosatetrynoic acid: which inhibits the 12- and 15-lipoxygenase pathway) led to a reversible decrease in gj. Pre-treatment with 4-BPB (4-bromophenacyl bromide: which inhibits phospholipase A2) did not prevent the effects on gj by NDGA or ETYA. This renders it unlikely that gj is regulated by eicosanoids. Also, accumulation of endogenous AA cannot be responsible for NDGA- and ETYA-dependent uncoupling. Exposure to 75 microM SKF-525A (inhibits the epoxygenase pathway) reversibly impaired gj. This is consistent with a direct action of SKF-525A on gj, but leaves open the possibility of an involvement of epoxides. The data gathered will be discussed in terms of molecular mechanisms. Due to their amphipathic character. AA, NDGA, ETYA and SKF-525A may interfere with gj by disturbing the lipid-protein interface of the cell membranes and thereby impair gap junction channels.

Conductances and selective permeability of connexin43 gap junction channels examined in neonatal rat heart cells.

Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means of the dual voltage-clamp method. The experiments were carried out on three types (groups) of preparations: (1) induced cell pairs, (2) preformed cell pairs with few gap junction channels (1 to 3 channels), and (3) preformed cell pairs with many channels (100 to 200 channels) after treatment with uncoupling agents such as SKF-525A (75 micromol/L), heptanol (3 mmol/L), and arachidonic acid (100 micromol/L). In group 1, the first opening of a newly formed channel was slow (20 to 65 ms) and occurred 7 to 25 minutes after physical cell contact. The rate of channel insertion was 1.3 channels/min. Associated with a junctional voltage gradient (Vj), the channels revealed multiple conductances, a main open state [gamma(j)(main state)], several substates [gamma(j)(substates)], and a residual state [gamma(j)(residual state)]. On rare occasions, the channels closed completely. The same phenomena were observed in groups 2 and 3. The existence of gamma(j)(residual state) provides an explanation for the incomplete inactivation of the junctional current (Ij) at large values of Vj in cell pairs with many gap junction channels. The values of gamma(j)(main state) and gamma(j)(residual state) gained from groups 1, 2, and 3 turned out to be comparable and hence were pooled. The fit of the data to a Gaussian distribution revealed a narrow single peak for both conductances. The values of gamma(j) were dependent on the composition of the pipette solution. Solutions were as follows: (1) KCl solution, gamma(j)(main state)=96 pS and gamma(j)(residual state)=23 pS; (2) Cs+ aspartate solution, gamma(j)(main state)=61 pS and gamma(j)(residual state)=12 pS; and (3) tetraethylammonium+ aspartate solution, gamma(j)(main state)=19 pS and gamma(j)(residual state)=3 pS. The respective gamma(j)(main state)-to-gamma(j)(residual state) ratios were 4.2, 5.1, and 6.3. This indicates that the residual state restricts ion permeation more efficiently than does the main state. Transitions of Ij between open states (main open state, substates, and residual state) were fast (<2 ms), and transitions involving the closed state and an open state were slow (15 to 65 ms). This implies the existence of two gating mechanisms. The residual state may be regarded as the ground state of electrical gating controlled by Vj; the closed state, as the ground state of chemical gating.

Biophysical properties of heterotypic gap junctions newly formed between two types of insect cells.

1. Cell pairs of the insect cell line Sf9 (Spodoptera frugiperda) were chosen to examine the electrical properties of gap junction channels. The dual voltage-clamp method was used to control the membrane potential of each cell (Vm,1 and Vm,2) and hence the junctional voltage gradient (Vj), and to measure intercellular current. 2. Studies with preformed pairs revealed that the gap junction conductance (gj) is controlled by a Vj- and a Vm-sensitive gate. At steady state, gj = f(Vj) was bell shaped and symmetrical (Boltzmann: Vj.0 = -54 and 55 mV, the normalized minimum conductance at large Vj values (gj,min) = 0.24 and 0.23, z = 5.5 and 6.1 for negative and positive Vj, respectively) and gj = f(Vm) was S shaped (Vm.0 = 13 mV, gj,min = 0, z = 1.5). 3. Single channels exhibited two conductances, a main state (gamma j,main) of 224 pS and a residual state (gamma j,residual) of 42 pS. 4. We conclude that gap junctions in Sf9 cells behave similarly to those in the insect cell line C6/36 (Aedes albopictus). 5. An induced cell pair approach was used to examine heterotypic gap junction channels between Sf9 cells and C3/36 cells. 6. Heterotypic channels showed a gamma j,main of 303 pS and a gamma j,residual of 45 and 64 pS, depending on whether the Sf9 cell or C6/36 cell was positive inside. 7. In heterotypic gap junctions, gj = f(Vj) was bell shaped and asymmetrical (gj was more sensitive to Vj when the C6/36 cell was positive inside) and gj = f(Vm) was S shaped (Vm,0 = 2 mV, gj,min = 0, z = 2.9). 8. We conclude that heterotypic channels possess a Vj- and Vm-sensitive gating mechanism. Vj gating involves two gates, one located in each hemi-channel. Vj gates are operated independently and close when the cytoplasmic aspect is made positive. 9. A comparison of homo- and heterotypic channel data suggests that docking of hemi-channels may affect channel gating, but not channel conductance.

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

Connexin43 gap junctions exhibit asymmetrical gating properties.

A communication-deficient cell line (RIN cells, derived from a rat islet tumour), stably transfected with cDNA coding for rat connexin43 (Cx43), was chosen to further assess the mechanism of voltage gating of Cx43 gap junction channels. The experiments were carried out on preformed cell pairs using a dual whole-cell, voltage-clamp method. The junctional current, Ij, revealed a time- and voltage-dependent inactivation at transjunctional voltages Vj>+/-40 mV. When an asymmetrical pulse protocol was used (in cell 1 the holding potential was maintained, in cell 2 it was altered to establish a variable Vj), the channels exhibited an asymmetrical gating behaviour: Vj,0=-73.7 mV and 65.1 mV for negative and positive Vj, respectively (Vj at which Ij is half-maximally inactivated); gj(min)=0.34 and 0.29 (normalized minimal conductance); tau = 350 ms and 80 ms at Vj=100 mV (time constant of Ij inactivation). Hence, these parameters were more sensitive to positive Vj values. When a symmetrical pulse protocol was used (the holding potentials in cell 1 and cell 2 were altered simultaneously in steps of equal amplitude but of opposite polarity), the Vj -dependent asymmetries were absent: Vj,0=-60.5 and 59.5; gj (min)=0.27 and 0.29; tau =64 ms and 47 ms at 100mV. Putative explanations for these observations are discussed. A possibility is that the number of channels alters with the polarity of Vj.