Dual Voltage-clamp Method Research Articles

0184 : Distinct ratios of co-expressed Cx40 and Cx43 regulate a fine gap junction channel make-up and properties

Co-expressed connexins (Cxs) can co-assemble to form gap junction channels (GJCs) of mixed Cxs composition characterized by specific electrical properties. To date the detailed GJCs make up in the distinct cardiac tissues that coexpress distinct gradients of Cxs stays poorly understood. We aim to investigate whether and how the GJCs make-up depends on the level and ratios of coexpressed connexin 40 (Cx40) and Cx43. Rat Liver Epithelial cells that endogenously express Cx43 and stably transfected to induce accurate ratios Cx43:Cx40 were used to perform electrical recordings on cell pairs by applying the dual voltage clamp method. We previously showed that the induction of Cx40 decreases the electrical coupling at high ratio Cx43:Cx40 while increasing at low ratios, decreases the voltage-dependence and slows the kinetics of deactivation and of recovery of GJCs. A deeper investigation at the single channel level permits to evaluate the Cxs composition and the distribution of different kinds of GJCs. Whereas non-induced cells, are coupled by homomeric-homotypic Cx43 GJCs, induction of Cx40 modifies the GJCs population in function of the ratio Cx43:Cx40: at high ratio GJCs made of Cx43 dominate the population, whereas lower ratios favor the Cx43/Cx40 heteromerization and the contribution of the Cx40. Our data show that the GJCs make-up is finely regulated in function on the ratio Cx43:Cx40 to form functional homotypic or heteromeric GJCs with distinct contributions of Cx40 and Cx43 properties and function in regulating the impulse propagation in the healthy heart and the pro-arrhythmic Cxs dysfunction. To correlate these results with the relative cardiac Cxs expression profiles, cell pairs of different Cxs expression pattern will be studied.

0167 : Dependence of the ratio of co-expressed connexin43 and connexin45 on the connexin composition of gap junction channels

In the heart, the propagation of the action potential (AP) is regulated by several factors, including gap junction channels (GJCs) made of connexins (Cxs). The ventricular myocardium expresses large amount of Cx43 and traces of Cx45 at distinct co-expression ratios that change in physiological and pathological conditions. This study aims to understand whether and how the ratio Cx43:Cx45 regulates the formation of GJCs. Rat Liver Epithelial cells that express endogenous Cx43 and stably transfected with Cx45 to induce accurate Cx43:Cx45 ratios are used. Electrical macroscopic and unitary properties of GJCs are determined by applying the dual voltage clamp method. We previously showed that induction of Cx45 decrease the electrical coupling, increase the voltage dependence and accelerate or decelerate the kinetics of deactivation and recovery. To date, our single channel recordings permit to estimate the Cxs composition of GJCs. Induction of Cx45 leads to the formation of one dominant GJCs population made of mixed Cx43/Cx45 composition, whereas GJCs composed with only Cx43 or Cx45 are present in minor proportions. Interestingly, this finely regulated GJCs make-up with Cx43 and Cx45 seems to be independent on the ratios Cx43:Cx45. Further experiments will be performed to mimic cardiac border zone which have different ratios of Cxs co-expression. This will provide a better understanding of the regulation of the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior of Cxs dysfunction and remodeling in the diseased heart.

0106: Influence of the ratio of co-expressed cardiac connexins Cx43 and Cx45 in the formation of gap junction channels and their electrical properties

The cardiac action potential (AP) propagation is regulated to permit the coordinated and rhythmic atrial and ventricular contractions. This regulation requires several factors, especially gap junctions, which ensure a direct pathway for electrical and biochemical signaling. They are clusters of few to hundred intercellular gap junction channels (GJC) made of two hemichannels docked in the membrane of adjacent cells, which are composed of six connexins (Cxs). Their distinct electrical properties are a key factor regulating the propagation of the AP. Four cardiac Cxs, Cx40, Cx43, Cx45 and Cx30.2, exhibit specific patterns of expression that change in the healthy and diseased heart, which leads to different possible configurations of GJC. The aim of this study is to investigate the function of the distinct ratio of co-expressed Cxs in regulating the formation and function of GJC. Electrical properties of GJC (junctional coupling, voltage dependence, unitary conductance) are determined by performing electrical recordings on cell pair by applying the dual voltage-clamp method. Rat Liver Epithelial cells stably transfected to induce accurate Cx43:Cx45 ratios of 0 (single Cx43 expression), 0.5, 1 and 2, are used. The ongoing recordings show distinct electrical properties before and after the induction of Cx45: induction of Cx45 decreases the cell-to-cell coupling and rectifies the voltage dependence of GJC. Preliminary unitary recordings suggest a distinct formation of GJC of mixed Cx43/Cx45 composition in function of the Cx43:Cx45 ratio. Further investigations will provide better understanding on the distinct contributions of Cx43 and Cx45 in the GJC make-up, electrical properties and function of the Cx43/Cx45 expression pattern in regulating the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior in the diseased heart.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Long-distance physical connections between chondrocytes; cell-to-cell communication within hyaline cartilage

s / Osteoarthritis and Cartilage 20 (2012) S10–S53 S11 3 LONG-DISTANCE PHYSICAL CONNECTIONS BETWEEN CHONDROCYTES; CELL-TO-CELL COMMUNICATION WITHIN HYALINE CARTILAGE M. Mayan , R. Gago , P. Carpintero , P. Filgueira-Fernandez , N. Goyanes , V. Valiunas , P. Brink , F.J. Blanco . Osteoarticular and Aging Res. Lab. INIBIC-CHUAC, A Coruna, Spain; Dept. of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA Purpose: Even chondrocytes are metabolically active cells, responsible of the maintenance of the hyaline cartilage during the whole adult life, it is highly accepted in the field that chondrocytes in cartilage do not connect each other, as they are isolated inside their lacunae separated from each other by a distance between 5 to 60mm. In the same lacuna can co-exit several chondocytes interacting between them. However, how the chondrocytes from different lacuna interact between each other and timely respond to physical or chemicals stimuli, are largely unknown. Thus far, the unique communication between chondrocytes in the superficial layer with chondrocytes in themiddle and deeper layers it is accepted that only occurs through diffusion. Nevertheless, the intercellular communication confers to a tissue the ability to responduniformly to localized stimuli. The purpose of this study was to investigate how chondrocytes communicate each other within the matrix and find for some alteration that could explain the degeneration of the matrix that occurs in patients with OA. Methods: In situ cartilage from human and Sus scrofa were fixed and frozen immediately using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Samples were fixed with acetone for confocal optical microscopy assays. Samples were embedded in cacodylate buffer before dehydratation for Scanning Electron Microscope (SEM). Gold was use for coating. Frozen samples were also used for immunofluorescence and immunohistochemistry assays. For total RNA isolation, the proteins and DNA were removed from cell extracts using TRIZOL Reagent (Invitrogene) before using the RNeasy Kit (Qiagen) which includes DNase treatment (RNAse-Free DNAse Set). Quantifications were done using SYBr green real-time PCR. Virgin Valiunas performed dual voltageclamp method and whole-cell/perforated patch using chondrocytes from healthy donors and patients with OA. Results: By using confocal optical microscopy and Scanning Electron Microscopy (SEM), we have found that chondrocytes are physical connected between each other across the extracellular matrix through large cytoplasmic projections. The projections are between 5 to 150 mm in length in human healthy cartilage and young cartilage from Sus scrofa. However condrocytes projections found in OA cartilage are between 10 to 200 mm in length. High levels of expression for the gap junction protein connexin 43 have been detected by RT-PCR and immunohistochemical experiments. We have also found expression of Cx32 and Cx45, however electrophysiology experiments demonstrated that healthy and OA chondrocytes show voltage-dependent behaviour for channels mainly formed by Cx43. We have performed patch clamp experiments to study the interchange of Lucifer Yellow and small molecules of RNA. Our results demonstrated that healthy and OA chondrocytes are able to interchange Lucifer Yellow and small molecules of RNA through Cx43 channels. In fact, our preliminary results showed that inhibition of channels affect the synthesis of collagen type II, MMP3 andMMP9 suggesting that the activity of channels are actively involved with the maintenance of the cartilage matrix. Conclusions:Wehavefoundcell/cellphysicalconnectionsthroughall layers of cartilage between chondrocytes located at different distance within cartilage. Intercellular communication occurs through channels voltagedependent formedby Cx43. Cytoplasmic projections fromOAchondrocytes are longer than from healthy chondrocytes, therefore the channel activity can be affected by the electrical depolarization of the membrane in OA chondrocytes. We have found that chondrocytes interchange small RNAs and inhibition of channels affect the expression of matrix-related genes. 4 SYNERGISTIC INDUCTION OF ELF3. IN VITRO EFFECT OF LEPTIN AND IL-1 IN HUMAN CHONDROCYTES J. Conde , M. Scotece , M. Otero , R. Gomez , J.J. Gomez-Reino , M.B. Goldring , O. Gualillo . 1 SERGAS, Santiago Univ. Clinical Hosp. NEIRID LAB, Santiago de Compostela, Spain; Hosp. for Special Surgery,

Interplay between Cystic Fibrosis Transmembrane Regulator and Gap Junction Channels Made of Connexins 45, 40, 32 and 50 Expressed in Oocytes

The cystic fibrosis transmembrane regulator (CFTR) is a Cl(-) channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G (j)) and its sensitivity to transjunctional voltage (V (j)) by the dual voltage-clamp method. Application of forskolin induced a Cl(-) current; increased G (j) approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V (j ) gating, monitored by a change in the ratio between G (j) steady state and G (j) peak (G (j)SS/G (j)PK) at the pulse. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G (j) and decreased the sensitivity to V (j) gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G (j) and V (j) changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.

Subconductance States of Cx30 Gap Junction Channels: Data from Transfected HeLa Cells versus Data from a Mathematical Model

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, γ j,substate, were unevenly distributed between the main-state and the residual-state conductance ( γ j,main state = 141 pS, γ j,residual state = 21 pS). Activation of the first subgate reduced the channel conductance by ∼30%, and activation of subsequent subgates resulted in conductance decrements of 10–15% each. Current transitions between the states were fast (<2 ms). Substate events were usually demarcated by transitions from and back to the main state; transitions among substates were rare. Hence, subgates are recruited simultaneously rather than sequentially. The incidence of substate events was larger at larger gradients of V j. Frequency and duration of substate events increased with increasing number of synchronously activated subgates. Our mathematical model, which describes the operation of gap junction channels, was expanded to include channel substates. Based on the established V j-sensitivity of γ j,main state and γ j,residual state, the simulation yielded unique functions γ j,substate = f( V j) for each substate. Hence, the spacing of subconductance levels between the channel main state and residual state were uneven and characteristic for each V j.

The electrical behaviour of rat connexin46 gap junction channels expressed in transfected HeLa cells.

Pairs of human HeLa cells expressing rat connexin46 were used to study the electrical properties of gap junction channels with the dual voltage-clamp method. The steady-state conductance ( g(j,ss)) had a bell-shaped dependence on transjunctional voltage ( V(j)). The parameters of the Boltzmann fit were: V(j,0)=42 mV, g(j,min)=0.12, z=2.5 (pipette solution: K(+) aspartate(-); 27 degrees C). The Boltzmann parameters were sensitive to the ionic composition of the pipette solution (KCl, K(+) aspartate(-), TEA(+) Cl(-), TEA(+) aspartate(-)). The V(j)-dependent inactivation of the junctional current I(j) was approximated by single exponentials (exceptions: two exponentials with KCl at V(j)>or=75 mV and K(+) aspartate(-) at V(j)=125 mV). The time constant of inactivation (tau(i)) decreased with increasing V(j) and was sensitive to the pipette solution. The larger the ions, the slower the inactivation. Recovery from inactivation followed a single exponential. The time constant of recovery (tau(r)) increased with increasing V(j). Single-channel currents showed a main state, several substates and a residual state. The corresponding conductances gamma(j,main) and gamma(j,residual) decreased slightly with increasing V(j); extrapolation to V(j)=0 mV yielded values of 152 and 28 pS, respectively (K(+) aspartate(-); 37 degrees C). The values of gamma(j,main) and gamma(j,residual) were dependent on pipette solution. The ratio gamma(j,main)/gamma(j,residual) increased with increasing ionic size, suggesting that the residual state impairs ion permeation more severely than the main state. The gamma(j,main) data suggest that the ionic selectivity of Cx46 channels may be controlled primarily by ionic size. Compared with hemichannel results, docking of connexons may modify the channel structure and thereby affect the ionic selectivity of gap junction channels. The open channel probability at steady state ( P(o)) decreased with increasing V(j). The parameters of the Boltzmann fit were: V(j,0)=41 mV, z=2.2 (K(+) aspartate(-); 27 degrees C).

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.

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.

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 10μm AA produced reversible uncoupling. Pretreatment with 10μm POCA (sodium-2-[5-(4-chlorophenyl)-pentyl]- oxirane-2-carboxylate; which inhibits mitochondrialβ-oxidation) did not prevent AA-dependent uncoupling. Thus, it seems that metabolites ofβ-oxidation are not involved in AA-induced impairment ofgj. Pre-exposure to 10μm 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 ingjmodulation. Exposure to 5μm NDGA (nordihydroguaiaretic acid; which inhibits the 5-lipoxygenase pathway) or 10μm ETYA (5,8,11,14-eicosatetrynoic acid; which inhibits the 12- and 15-lipoxygenase pathway) led to a reversible decrease ingj. Pre-treatment with 4-BPB (4-bromophenacyl bromide; which inhibits phospholipase A2) did not prevent the effects ongjby NDGA or ETYA. This renders it unlikely thatgjis regulated by eicosanoids. Also, accumulation of endogenous AA cannot be responsible for NDGA- and ETYA-dependent uncoupling. Exposure to 75μm SKF-525A (inhibits the epoxygenase pathway) reversibly impairedgj. This is consistent with a direct action of SKF-525A ongj, 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 withgjby disturbing the lipid-protein interface of the cell membranes and thereby impair gap junction channels.

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.

Modification of gap junction conductance by divalent cations and protons in neonatal rat heart cells

Myocytes were isolated from neonatal rat hearts and grown in culture dishes. Pairs of cells were selected to study the effect of divalent cations and protons on the conductance of gap junctions, gj. The experimental approach involved the dual voltage-clamp method and cell dialysis via patch pipette, i.e. gj was monitored while the cytosolic level of Ca2+, Mg2+, Sr2+, Ba2+ or H+ was modified in one of the cells. A dose-dependent decrease in gj developed when pCa of the pipette solution was lowered (range: pCa = 7.7-2.42, equivalent to a [Ca2+] of 20 nM-3.8 mM). The gj/pCa-relationship revealed a Hill coefficient n of 0.87 and a half-maximal concentration pKCa of 3.5. Pretreatment with 3 mM NiCl2 and 1 micron ryanodine to minimize the removal of cytosolic Ca2+ did not significantly affect the response to gj. Similarly, gj was decreased in a dose-dependent fashion when pHi in the pipette solution was lowered (range: pH = 7.2-5.0, corresponding to a [H+] of 63 nM-10 microns). The gj/pH-relationship yielded an n of 0.92 and a pKH of 5.85. Pretreatment with 1 mM amiloride to minimize the extrusion of protons enhanced the effects of pH on gj. Simultaneous alterations in pCa and pH demonstrated an additive type of action of Ca2+ and H+ on gj. This is consistent with the existence of two types of sensors which contribute separately to the functional state of gj. No significant decrease in gj was detectable when the pipette solution contained Mg2+ or Ba2+ (up to 5 mM). Partial uncoupling was observed with pipette solution containing 5 mM Sr2+. We conclude that gj of neonatal and adult cardiomyocytes exhibit different ionic sensitivities. This discrepancy may reflect differences in connexin expression and/or molecular intermediates involved in regulating gj.

Biophysical properties of gap junction channels formed by mouse connexin40 in induced pairs of transfected human HeLa cells

A clone of human HeLa cells stably transfected with mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ("induced cell pair") to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j (residual state)), and a closed state (gamma j(closed state)). The gamma j(main state) was 198 pS, and gamma j(residual state) was 36 pS (temperature, 36-37 degrees C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, Vj. The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, Po, decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: Vj,o = -44 mV; z = 6). The temperature coefficient, Q10, for gamma j(main state) and gamma j(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40 degrees C). This difference suggests interactions between ions and channel structure in case of gamma j(residual state). In cell pairs with many channels, the gap junction conductance at steady state, gj, exhibited a bell-shaped dependency from Vj (Boltzmann fit, negative Vj, Vj,o = -45 mV, gj(min) = 0.24; positive Vj, Vj,o = 49 mV, gj(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for Vj gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state.

Heterotypic gap junction channels (connexin26 ? connexin32) violate the paradigm of unitary conductance

Human HeLa cells transfected with mouse DNA coding for connexin26 (Cx26) or connexin32 (Cx32) were used to examine the properties of heterotypic Cx26-Cx32 gap junction channels. Intercellular current flow was examined in induced cell pairs by means of the dual voltage-clamp method. We found that Cx26-Cx32 channels exhibit voltage-dependent conductances, gamma j: gamma j(main state) increases with increasing positivity at the cytoplasmic aspect of the Cx26 connexon and decreases with increasing negativity (slope: 32 pS/100 mV; gamma j(main state) reaches 48 pS as Vj approaches 0 mV); gamma j(residual state) with a similar Vj-dependence is present when the cytoplasmic end of Cx26 connexon is positive, but absent when it is negative. The single channel data provide an explanation for the asymmetric relationships between the gap junction conductance, gj, and Vj. The results are consistent with the notion that docking of two connexons co-determines the biophysical properties of a gap junction channel.

Voltage-dependent gating of single gap junction channels in an insect cell line

De novo formation of cell pairs was used to examine the gating properties of single gap junction channels. Two separate cells of an insect cell line (clone C6/36, derived from the mosquito Aedes albopictus) were pushed against each other to provoke formation of gap junction channels. A dual voltage-clamp method was used to control the voltage gradient between the cells (Vj) and measure the intercellular current (Ij). The first sign of channel activity was apparent 4.7 min after cell contact. Steady-state coupling reached after 30 min revealed a conductance of 8.7 nS. Channel formation involved no leak between the intra- and extracellular space. The first opening of a newly formed channel was slow (25-28 ms). Each preparation passed through a phase with only one operational gap junction channel. This period was exploited to examine the single channel properties. We found that single channels exhibit several conductance states with different conductances gamma j; a fully open state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j(residual)) and a closed state (gamma j(closed)). The gamma j(main state) was 375 pS, and gamma j(residual) ranged from 30 to 90 pS. The transitions between adjacent substates were 1/7-1/4 of gamma j(main state). Vj had no effect on gamma j(main state), but slightly affected gamma j (residual). The lj transitions involving gamma j(closed) were slow (15-60 ms), whereas those not involving gamma j(closed) were fast (< 2 ms). An increase in Vj led to a decrease in open channel probability. Depolarization of the membrane potential (Vm) increased the incidence of slow transitions leading to gamma j(closed). We conclude that insect gap junctions possess two gates, a fast gate controlled by Vj and giving rise to gamma j(substates) and gamma j(residual), and a slow gate sensitive to Vm and able to close the channel completely.

Early Events of Semliki Forest Virus-Induced Cell-Cell Fusion

Insect cells( Aedes albopictus) were infected with Semliki Forest Virus. Cell-cell fusion was then induced by lowering the extracellular pH. The underlying processes were examined by monitoring the intercellular current flow, I j. Experimentally, this involved the use of cell pairs in conjunction with a dual voltage-clamp method. This approach allowed us to monitor the kinetics of fusion at high temporal resolution. The fusion process began shortly after acidification (delay: 3-138 sec). Initially, I j increased in a stepwise manner, later on it developed more gradually. Fusion between two cells reached a steady state within 7-70 sec. The steps in I j are attributable to the formation of cytoplasmic connections between the cells, presumably involving proteinaceous fusion pores. The mean amplitude of I j steps corresponds to a conductance of 300 pS, consistent with a pore radius of 1 nm. Cytoplasmic connections developed rapidly, i.e., I j steps occurred within less than 1 msec. The absence of I j flickering implies that formation of cytoplasmic connections, and hence SFV induced cell-cell fusion, is irreversible.

Gap junction channels of insects exhibit a residual conductance.

Formation of gap junction coupled cell pairs was used to assess the basic properties of single gap junction channels. For this purpose, two single cells (clone C6/36, derived from larvae of an insect, Aedes albopictus) were maneuvered against each other to provoke gap junction channel insertion. Intercellular current flow was measured with a dual voltage-clamp method. Utilizing this approach, we were able to demonstrate that gap junction channels, after formation, do not close completely upon application of a transjunctional voltage gradient, Vj. Instead, they exhibit a residual conductance, gamma j(residual). On average, gamma j(residual) was 64 +/- 4 pS (n = 40). This corresponds to about 1/6 of the conductance of a fully open channel. The existence of gamma j(residual) explains the observation that the conductance of the entire gap junction, gj, decreases only partially at large Vj.