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

Abstract

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 (g_j) at steady state showed a bell-shaped dependence on junctional voltage (V_j; Boltzmann fit: V_j,0= 27 mV, g_j,min= 0.15, z = 4). The instantaneous g_j decreased slightly with increasing V_j.

3. The gap junction currents (I_j) declined with time following a single exponential. The time constants of I_j inactivation (τ_i) decreased with increasing V_j.

4. Single channels exhibited a main state, a residual state and a closed state. The conductances γ_j,main and γ_j,residual were 179 and 48 pS, respectively (pipette solution, potassium aspartate; temperature, 36-37 °C; extrapolated to V_j= 0 mV).

5. The conductances γ_j,residual and γ_j,main showed a slight V_j 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 (P_o) at steady state decreased from 1 to 0 with increasing V_j (Boltzmann fit: V_j,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 V_j 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.

Our knowledge on functional aspects of gap junction channels has evolved quickly over the last decade. Progress has been closely associated with the availability of cDNAs coding for various gap junction proteins of vertebrates, the connexins. Expression studies with injected Xenopus oocytes and transfected mammalian cells have shown that the functional diversity of gap junctions reflects the structural variety of connexins (see Bruzzone et al. 1996). Recently, a new mouse connexin has been identified and cloned by screening of a mouse genomic library (Dahl et al. 1996). This protein has a molecular mass of 30.366 kDa and hence was called connexin30 (Cx30). Northern blots of total RNA from mouse tissues have indicated that Cx30 is strongly expressed in adult brain as well as skin and is less abundant in uterus, lung and eye tissue. Small amounts of transcripts have been detected in testis and sciatic nerve. No Cx30 mRNA has been found in liver. Sequence comparisons of nucleotides and amino acids suggest that mouse Cx30 is a close relative of mouse Cx26.

There are few reports in the literature on functional aspects of Cx30 gap junctions. Injection of mouse Cx30 cRNA into Xenopus oocytes induced the formation of gap junctions susceptible to intercellular current flow and dye diffusion (Dahl et al. 1996). Neurobiotin diffusion experiments with transfected HeLa cells revealed that Cx30 forms heterotypic gap junction channels with Cx26, Cx30.3, Cx31, Cx40, Cx43, Cx45 and Cx50 (D. Manthey et al., in preparation). Electrophysiological studies on pairs of transfected HeLa cells revealed that Cx30 forms heterotypic gap junctions with Cx43 and Cx50 (Weingart et al. 1996b). It is interesting to note that Cx30 co-localises with Cx43 in adult rat astrocytes (Kunzelmann et al. 1999).

The aim of this study has been to provide a comprehensive description of the biophysical properties of Cx30 gap junction channels. All experiments were carried out on HeLa cells transfected with mouse Cx30 cDNA (Kunzelmann et al. 1999; D. Manthey et al., in preparation). Electrical measurements were performed on pairs of cells using the dual voltage-clamp method. This approach allowed us to examine microscopic currents (gap junctions consisting of single channels) and macroscopic currents (gap junctions consisting of many channels) in the same type of preparation. The data collected provide a physico-chemical basis for an understanding of the biological role of gap junction channels in astrocytes, ependymal cells and leptomeningeal cells, where Cx30 is co-expressed with Cx43. Preliminary reports have been published (Valiunas et al. 1996; Weingart et al. 1996b).

METHODS

Cells and culture conditions

Experiments were carried out on human HeLa cells (human cervix carcinoma cells, ATCC code CCL2). Wild-type cells and transfected cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum, 100 μg ml⁻¹ streptomycin and 100 U ml⁻¹ penicillin (code 2212, Seromed, Fakola, Basel, Switzerland). The medium for HeLa transfectants also contained 1 μg ml⁻¹ puromycin (Sigma, Dreisenhofen, Germany). The cells were passaged weekly, diluted 1:10 and kept at 37°C in a CO₂ incubator (5 % CO₂, 95 % ambient air). To perform electrophysiological measurements, the cells were diluted (∼0.2-1 × 10⁶ cells ml⁻¹) and seeded onto sterile glass coverslips placed in multiwell culture dishes (∼10⁴ cells cm⁻²). Experiments were performed 1-3 days after plating.

Transfection

The mouse connexin30 coding sequence was amplified in a PCR reaction using the genomic mouse Cx30 exon 2 sequence (Dahl et al. 1996) as template (primers: 5′-GAATAAGCCTGCACGATGGAC-3′ and 3′-GCACACCTACACTTGACCTTG-5′. The 827 bp PCR fragment was cloned at the Sma I site of the expression vector pBluescript SK⁺ (Stratagene, La Jolla, CA, USA) and completely sequenced on both strands. No Taq polymerase-induced nucleotide exchanges were detected in comparison with the original mouse Cx30 phage insert DNA (Dahl et al. 1996). A 850 bp Eco RI/Xba I fragment, containing the coding region of mouse Cx30, was cloned at the Eco RI/Xba I site of the transfection vector pBEHpac18 (Horst et al. 1991) that contained the SV-40 early promotor, a polyadenylation signal and a gene conferring resistance to puromycin.

HeLa cells were transfected with 20 μg DNA of the Cx30 coding region in pBEHpac18 by the calcium phosphate transfection protocol, as described by Chen & Okayama (1987). Forty-eight hours after exposure to the DNA-calcium phosphate precipitate, 1 μg ml⁻¹ puromycin was added to the medium. Clones were picked after 3 weeks and grown under selective conditions. The clones were checked by radioactive Northern blot analysis (Hennemann et al. 1992) with total RNA (20 μg) and a Cx30 PCR fragment (590 bp, representing the nucleic acid residues from positions 25 to 615) as specific probes.

Solutions

Electrophysiological experiments were performed in modified Krebs-Ringer solution (mM): NaCl, 140; KCl, 4; CaCl₂, 2; MgCl₂, 1; glucose, 5; pyruvate, 2; Hepes, 5 (pH 7.4). The patch pipettes were filled with saline containing (mM): potassium aspartate, 120; NaCl, 10; MgATP, 3; MgCl₂, 1; CaCl₂, 1; EGTA, 10 (pCa ∼8); Hepes, 5 (pH 7.2); filtered through 0.22 μm pores. SKF-525A hydrochloride (mol. wt. 390; Proadifen) was purchased from Biomed Research Laboratories (Plymouth, PA, USA).

Electrical measurements

Glass coverslips with adherent cells were transferred to an experimental chamber superfused with Krebs-Ringer solution at 20-23°C. The chamber was mounted on the stage of an inverted microscope (Diaphot-TMD, Nikon; Nippon Kogaku, Tokyo, Japan). Patch pipettes were pulled from glass capillaries (GC150TF-10; Clark Electromedical Instruments, Pangbourne, Berks, UK) with a horizontal puller (DMZ-Universal; Zeitz-Instrumente, Augsburg, Germany). When filled, the pipette resistances measured 3-5 MΩ. Experiments were carried out on cell pairs. A dual voltage-clamp method and whole-cell recording were used to control the membrane potential of both cells and to measure currents (Bukauskas et al. 1995a). Initially, the membrane potentials of cells 1 and 2 were clamped to the same value, V₁=V₂. V₂ was then changed to establish a junctional voltage, V_j=V₂ - V₁. Currents recorded from cell 2 represent the sum of two components, a junctional current, I_j, and a membrane current, I_m,2; the current gained from cell 1 corresponds to I_j.

Signal recording and analysis

Voltage and current signals were recorded on chart paper (Gould RS 3400; Gould Instruments, Minneapolis, MN, USA) and videotape (Neurocorder DR-886; Neuro Data Instruments, New York, NY, USA). For off-line analysis the current signals were filtered at 1 kHz (8-pole Bessel; -3 dB) and digitised at 5 kHz with a 12-bit A/D converter (IDA 12120; INDEC Systems, Capitola, CA, USA). Data acquisition and analysis was done with commercial (C-Lab; INDEC Systems) and custom-made software (Krisciukaitis, 1997). Curve fitting and statistical analysis were performed with SigmaPlot and SigmaStat, respectively (Jandel Scientific, Erkrath, Germany). The data are presented as means ± 1 s.e.m.

Wild-type HeLa cells express an intrinsic connexin, conceivably Cx45, at an extremely low level (Hülser et al. 1997). Since the properties of Cx45 channels are so different from those of Cx30 channels, they would be readily detectable in HeLa cells transfected with Cx30. However, we did not see any such signals. For a more complete discussion of this issue, see Bukauskas et al. (1995a).

RESULTS

Expression of Cx30 DNA in HeLa transfectants

The HeLa Cx30 transfectants together with HeLa wild-type cells were characterised by Northern blot analysis as shown in Fig. 1. The Cx30-transfected clone TD0 2 showed the expected hybridisation signal of 1.1 kb. The expression of Cx30 protein in this clone was verified by immunoblot (Kunzelmann et al. 1999) and immunofluorescence analysis (D. Manthey et al. in preparation). Functional communication through homotypic Cx30 gap junction channels in clone TD0 2 was demonstrated after microinjection of neurobiotin (D. Manthey et al., in preparation). For the subsequent electrophysiological experiments we used clone 2.

Figure 1. Northern blot analysis of total RNA from HeLa cells.

Total RNA was extracted from Cx30-transfected human HeLa cells and wild-type cells, electrophoresed, blotted onto nylon membrane, hybridised to a ³²P-labelled Cx30 probe (PCR fragment, nucleotide position 25-616) under high stringency conditions and autoradiographed. Lines 1-5 correspond to different clones of Cx30-transfected HeLa cells and wild-type HeLa cells (wt). The different transcripts in lane 3 may have resulted from different transcriptional termination sites of several copies of the inserted plasmid DNA. The bar marks the location of the 1.1 kb band of Cx30 mRNA.

Voltage dependence of gap junction currents

To perform this set of experiments, macroscopic intercellular currents, I_j, were examined in 26 cell pairs. In 17 preparations the cells were coupled by gap junctions. They yielded a gap junction conductance, g_j, of 2.7 ± 0.7 nS. The remaining nine cell pairs contained cytoplasmic bridges (Bukauskas et al. 1992). In these cases, g_j was 18.0 ± 5.5 nS. To distinguish between gap junctions and cytoplasmic bridges, the preparations were routinely exposed to 75 μm SKF-525A (Schmilinsky-Fluri et al. 1997). This impaired the current across gap junctions but did not affect the cytoplasmic bridges.

The relationship between V_j and g_j was studied systematically in eight cell pairs with gap junctions. To minimise series resistance problems, preparations with moderate coupling were selected (g_j < 5 nS; see Bukauskas & Weingart, 1993). The protocol adopted was as follows. Both cells were clamped to the same holding potential, V₁=V₂= -20 mV. Voltage pulses of long duration (10-40 s), different amplitudes (up to 110 mV) and either polarity were applied to cell 2 to impose a V_j. Figure 2A shows selected records from such an experiment. Hyperpolarisation of V₂ by 40 mV gave rise to an I_j exhibiting a time-dependent decay (I₁, left-hand side). Subsequent depolarisation by the same amount led to a similar I_j of opposite polarity (I₁, right-hand side). In between, small test pulses (12.5 mV; 200 ms; 1 Hz) were delivered to cell 2 (spikes in V₂) to monitor the electrical stability of the preparation (spikes in I₁).

Figure 2. Dependence of intercellular coupling on transjunctional voltage (V_j).

A, responses of gap junction currents (I_j) to V_j. Records illustrate the membrane potential of cell 1 (V₁) and cell 2 (V₂) and the current measured from cell 1 (I₁). Deflections in V₂ and I₁ correspond to V_j and I_j, respectively. Long V_j pulses of ±40 mV gave rise to a time-dependent I_j. Short V_j pulses (downward deflections in V₂) served to monitor the stability of I_j (upward deflections in I₁). Holding potential, V_h= -20 mV. B, plot of normalised g_j determined at the make (g_j,inst; ○) and break (g_j,ss; •) of V_j pulses versus V_j. Each symbol corresponds to a single determination. Data from 8 cell pairs. ○, instantaneous data; for curve fitting procedure, see text. •, steady-state data; continuous curve represents the best fit of data to the Boltzmann equation; V_j,0= -28.5 and 26.4 mV, g_j,min= 0.15 and 0.14, z = 4.1 and 3.9 for negative and positive V_j, respectively.

Figure 2B summarises the results from these experiments. The amplitude of I_j was determined at the beginning (I_j,inst; inst = instantaneous) and end (I_j,ss; ss = steady state) of each V_j pulse. The values of I_j,inst were normalised with respect to the amplitude of I_j associated with the small test pulse immediately before each V_j pulse. The corrected I_j,inst current was used to calculate the conductance, g_j,inst=I_j,inst/V_j. The values of g_j,inst were then plotted versus V_j (○) to obtain the relationship g_j,inst= f(V_j). The g_j,inst data followed a curved relationship symmetrical with respect to V_j. The smooth curve represents the best fit of data to a function derived from our gap junction model (Vogel & Weingart, 1998; see eqn (29), adapted for a multi-channel case and normalised with respect to the conductance at V_j= 0 mV):

(1)

G_j is a dimensionless fitting parameter and V_H is a decay constant (for a detailed description, see Vogel & Weingart, 1998). The analysis yielded the following values: G_j= 1.98; V_H= 201.5 mV. Starting from a maximum of 0.99 at V_j= 0 mV, g_j,inst decreased with increasing V_j. At V_j=±100 mV, it reached a value of about 0.9.

The current I_j,ss was used to calculate the conductance g_j,ss=I_j,ss/V_j. The values of g_j,ss were then normalised with respect to g_j,inst prevailing at each V_j and plotted versus V_j (•) to obtain the relationship g_j,ss/g_j,inst= f(V_j). The g_j,ss/g_j,inst data followed a bell-shaped relationship which was nearly symmetrical. At V_j= 0 mV, g_j,ss/g_j,inst was maximal. Between |V_j| values of 10 and 40 mV, it decreased to a quasi-constant value distinctly different from zero. The smooth curve represents the best fit of data to the Boltzmann equation:

(2)

where g_j,min is the normalised conductance at large V_j and V_j,0 corresponds to the V_j at which g_j,ss/g_j,inst is half-maximally inactivated. A is a constant which expresses the gating charge, zq(kT)⁻¹, where z is the equivalent number of unitary positive charges q moving through the electric field applied, and k and T are the Boltzmann constant and the temperature in Kelvin, respectively. The analysis yielded the following values: V_j,0= -28.5 and 26.4 mV, g_j,min= 0.15 and 0.14, and z = 4.1 and 3.9 for negative and positive V_j values, respectively.

Time-dependent inactivation of gap junction currents

The current records indicated that I_j inactivates with time in a voltage-dependent manner. Inactivation was rapid at large V_j (see Fig. 2A) and slow or virtually absent at small V_j (not shown). To perform a systematic analysis, current records obtained at different V_j gradients were digitised and subjected to a least-squares curve-fitting procedure. It turned out that I_j inactivation obeys a mono-exponential time course:

(3)

where I_j(0) is the current at time t = 0 and τ_i is the time constant of I_j inactivation. Figure 3A summarises the collected τ_i data from eight cell pairs. Individual values of τ_i were averaged and plotted versus V_j. Over the V_j range examined, τ_i decreased with increasing V_j irrespective of the voltage polarity. The smooth curves correspond to the best fit of data to a single exponential:

(4)

where τ_i,0 is the zero V_j intercept and V_τ is the decay constant. The analysis yielded the following values: τ_i,0= 8.5 and 11.4 s, and V_τ= -27.4 and 24.4 mV for negative and positive V_j values, respectively.

Figure 3. Kinetic analysis of junctional currents.

A, plot of time constants of I_j inactivation (τ_i) versus V_j. Each symbol corresponds to a mean value ± 1 s.e.m. Data from 8 cell pairs. The continuous curves represent the best fit of data to single exponentials; τ_i,0= 8.5 and 11.4 s and V_τ= -27.4 and 24.4 mV for negative and positive V_j values, respectively. B, plot of rate constants of channel opening, α (•), and channel closing, β (○), versus V_j. The continuous curves correspond to the best fit of data to single exponentials; λ= 0.21 and 0.18, A_α= -0.069 and 0.102, A_β= -0.042 and 0.049, and V_j,0= -24.1 and 27.8 mV for negative and positive V_j values, respectively.

If one assumes a two-state process (main state residual state) and takes into account that γ_j,residual is different from zero (see ‘Conductances of single channels’), the rate constants of channel opening (α) and closing (β) can be estimated from the formalism proposed by Harris et al. (1981). This analysis was performed using the data presented in Figs 2B and 3A. The values obtained were averaged and plotted versus V_j as shown in Fig. 3B. The graphs α= f(V_j) (•) and β= f(V_j) (○) indicate that at low V_j, τ_i is governed by α whereas at large V_j, it is dominated by β. The continuous curves represent the best fit of data to the equations:

(5)

(6)

where A_α and A_β express the voltage sensitivity of the rate constants and λ is the rate constant at which V_j=V_j,0 (i.e. α=β). The analysis yielded similar values for negative and positive voltages: λ= 0.21 and 0.18; A_α= -0.069 and 0.102; A_β= -0.042 and 0.049; V_j,0= -24.1 and 27.8 mV. If one combines eqns (5) and (6), an independent estimate of τ_i can be obtained:

(7)

With this approach, we obtained the following values: for V_j= -50 and 50 mV, τ_i= 1.52 and 1.79 s; for V_j= -100 and 100 mV, τ_i= 0.19 and 0.16 s. These values are in good agreement with those extracted from the I_j records (see Fig. 3A).

De novo formation of gap junction channels

In three other cell pairs, we successfully observed the de novo formation of gap junction channels. Figure 4A illustrates a representative example. After establishing the whole-cell recording conditions, a sustained V_j of -40 mV was applied (V₁= -5 mV; V₂= -45 mV; not shown). Initially, there was no I_j, indicating that the cells were not coupled electrically. A few minutes later, a first channel begun to operate as shown by the paired records I₁ and I₂. They exhibited simultaneous transitions similar in amplitude and of opposite polarity and hence reflect gap junction events. Starting from zero current (continuous lines), I_j slowly increased to a plateau corresponding to the main state, I_j,main (upward deflection in I₁; downward deflection in I₂). The transition between the two levels lasted ∼80 ms. Subsequently, I_j exhibited fast transitions (< 2 ms) between I_j,main and I_j,residual (dashed lines). The latter was different from zero, indicating that the channel did not close completely. An analysis of the signals in Fig. 4A yielded the following single channel conductances: γ_j,main= 156 pS, γ_j,residual= 28 pS. Slow transitions associated with first channel openings have been observed previously in insect cells and transfected HeLa cells (Bukauskas & Weingart, 1994; Bukauskas et al. 1995a).

Figure 4. Single channel activity of a cell pair whose gap junction contained a single channel.

A, sister current records documenting the de novo formation of a gap junction channel. V_j was maintained at -40 mV. Simultaneous transitions in I₁ and I₂ reflect gap junction events (channel opening: upward deflections in I₁, downward deflections in I₂). The very first transition was slow and corresponds to the first opening after channel formation. The subsequent transitions were fast and represent channel flickering between the main state and the residual state (dashed lines). Continuous lines indicate the zero coupling current. B, single channel currents from a weakly coupled cell pair elicited by V_j pulses (V₁ and V₂) of 50 mV (top and middle I₁ trace) and 75 mV (bottom I₁ trace). The coupling currents in the presence of one (top and middle I₁ trace) and two channels (bottom I₁ trace) exhibited fast transitions between the main state and the residual state.

Conductances of single channels

To study single channel currents, we selected cell pairs with only one or two operational channels. This condition prevailed in weakly coupled pairs or in normally coupled pairs after advanced spontaneous uncoupling (27 cell pairs). In a few cases (6 cell pairs), 75 μm SKF-525A was administered to a normally coupled pair to impair I_j and hence to resolve single channel events. As previously shown, this intervention does not affect the conductances of single gap junction channels (Valiunas et al. 1997). Figure 4B shows records from a weakly coupled cell pair. A voltage pulse was delivered repetitively to cell 2 (-50 mV, 200 ms, 1 Hz) while the membrane potential of cell 1 was maintained at -20 mV (traces V₂ and V₁). Initially, the cell pair had one operational channel (upper and middle I₁ trace), i.e. the current signal exhibited fast transitions between I_j,main and I_j,residual (dashed line). Later on, two channels were present (lower I₁ trace), i.e. the signal showed multiple transitions involving three discrete levels corresponding to 2 × I_j,main, I_j,main+I_j,residual and 2 × I_j,residual, respectively.

For further analysis we used current records obtained with maintained and pulsed V_j gradients (compare A and B in Fig. 4). V_j was varied between 25 and 100 mV. The conductances of the main and residual states were determined as the ratios I_j,main/V_j and I_j,residual/V_j, respectively. The histogram in Fig. 5 summarises the data collected from seven cell pairs, plotting the number of events versus conductance. It shows two narrow distributions with no overlap. Both data groups exhibited a binomial distribution and hence were fitted with a Gaussian. The right-hand peak revealed a mean value of 163.2 ± 0.8 pS (n = 221; range 130-218 pS) and corresponds to γ_j,main. The left-hand peak yielded a mean value of 26.4 ± 0.5 pS (n = 53; range 20-35 pS) and corresponds to γ_j,residual. The ratio γ_j,residual/γ_j,main was 0.16. Note that the data included in this analysis were gained at 34-35°C.

Figure 5. Histogram of single channel conductances (γ_j) gained from cell pairs with a single gap junction channel.

Data from 7 cell pairs were pooled in consecutive 5 pS bins. The number of observations was plotted versusγ_j. The continuous curves represent the best fit of data to Gaussians. The left-hand distribution revealed a mean value of 26.4 ± 0.5 pS (n = 53) and reflects the conductance of the incompletely closed channel, γ_j,residual. The right-hand distribution yielded a mean value of 163.2 ± 0.8 pS (n = 221) and corresponds to the conductance of the fully open channel, γ_j,main. Note that these experiments were carried out at 34-35 °C.

Influence of temperature on single channel conductances

To explore the effect of temperature on channel conductances, we used cell pairs with a single operational channel. The temperature of the bath solution was altered with a Peltier element and measured with a thermistor positioned close to the preparation, i.e. 0.5-1 mm (Bukauskas & Weingart, 1993). The experimental protocol involved repetitive application of V_j pulses (±50 mV or ±75 mV; 200 ms; 1 Hz) to one of the cells and assessment of I_j,main and I_j,residual. The mean values of γ_j,main and γ_j,residual were calculated and plotted on a logarithmic scale versus temperature. Figure 6 depicts the resulting graph (4 cell pairs). Over the temperature range examined, i.e. 23-34°C, γ_j,main (○) and γ_j,residual (•) increased with increasing temperature. The continuous lines were obtained by fitting the data to the equation:

(8)

where T is the temperature in°C, γ_j (23) the conductance at 23°C, and Q₁₀ the temperature coefficient. Q₁₀ for γ_j,main and γ_j,residual turned out to be 1.16 ± 0.004 (n = 427) and 1.27 ± 0.025 (n = 170), respectively. Statistical analysis showed that the values of Q₁₀ are significantly different from each other (Student's t test: P < 0.001). This suggests that γ_j,main and γ_j,residual exhibit a different temperature sensitivity. Hence, the ratio γ_j,residual/γ_j,main is expected to be temperature dependent as well. At T values of 23°C (room temperature) and 37°C (body temperature) the ratios were 0.147 (20.8 pS/141.4 pS) and 0.167 (29.1 pS/174.1 pS), respectively.

Figure 6. Influence of temperature on single channel conductances (γ_j).

Plots of single channel conductances γ_j,main (○) and γ_j,residual (•) on a logarithmic scale versus temperature. Symbols correspond to mean values ± 1 s.e.m. obtained from 426 and 170 measurements for γ_j,main and γ_j,residual, respectively. Data were collected during application of V_j pulses of 50 or 75 mV amplitude. Continuous lines were drawn by fitting the data to single exponentials. The temperature coefficients (Q₁₀) for γ_j,main and γ_j,residual were 1.16 and 1.27, respectively.

For comparison, the Q₁₀ of aqueous KCl solution is 1.2-1.3 (Robinson & Stokes, 1970). This suggests that in the case of the residual state, permeation of small ions may be slightly impaired by spatial and/or electrical constraints, whereas in the case of the main state it is not. A similar study with HeLa cells expressing Cx40 yielded Q₁₀ values of 1.21 and 1.3 for γ_j,main and γ_j,residual, respectively (Bukauskas et al. 1995a).

Voltage dependence of single channel conductances

Next we explored the relationship between the single channel conductances and V_j. For this purpose, the values of γ_j,main (○) and γ_j,residual (•) obtained at different V_j were sampled, averaged and plotted versus V_j. The γ_j data for positive and negative voltages were symmetrical and hence were pooled. The number of observations at a given V_j varied from 16 to 207 (12 cell pairs). The values of γ_j,residual collected at V_j= 25 mV were discarded because of the marginal signal/noise ratio and the small number of observations. Figure 7 shows the resulting graph. Over the voltage range examined, i.e. up to ±100 mV, the values of both γ_j,main and γ_j,residual decreased moderately with increasing V_j. The continuous lines correspond to the best fit of data to our gap junction model (Vogel & Weingart, 1998). In the case of γ_j,residual data, the fitting procedure involved numerical calculations. This was necessary because no analytical solution is available. The procedure revealed the following values: Γ_L= 39 pS, V_L= -127 mV. Γ_L represents the conductance of a hemichannel in the low conductance state at V_j= 0 mV (for interpretation of V_L, see Vogel & Weingart, 1998). In the case of the γ_j,main data, the curve fitting was done with the equation:

(9)

Γ_H corresponds to the conductance of a hemichannel in the high conductance state at V_j= 0 mV (for interpretation of V_H, see Vogel & Weingart, 1998). The analysis yielded the following values: Γ_H= 291 pS; V_H= -253 mV. Since a gap junction channel consists of two hemichannels in series, γ_j,main and γ_j,residual at V_j= 0 mV can be calculated as Γ_H/2 = 146 pS and (Γ_H×Γ_L)/(Γ_H+Γ_L) = 34.4 pS, respectively. Note that the data described in this section were gained at 21-23°C. Taking into account the values of Q₁₀ (see ‘Influence of temperature on single channel conductances’), γ_j,main and γ_j,residual at 37°C would be 179 and 48 pS, respectively.

Figure 7. Relationships between single channel conductances (γ_j) and transjunctional voltage (V_j).

Plots of the single channel conductances γ_j,main (○) and γ_j,residual (•) versus V_j. The symbols represent mean values obtained from 313 and 136 measurements, respectively; the bars for ± 1 s.e.m. lay within the size of the symbols. Data derived from negative and positive V_j were pooled. The continuous curves correspond to the best fit of data to equations derived from a mathematical model (for details, see text). Extrapolation to V_j= 0 mV led to γ_j,main and γ_j,residual values of 146 and 34 pS, respectively.

Fast versus slow current transitions

Current transitions associated with single channel activity were usually fast. They were complete within the response time of the experimental set-up, i.e. < 2 ms (see Fig. 4B). This was true for the transitions between channel open states (i.e. the main state and the residual state). However, in conjunction with chemical uncoupling (see ‘Conductances of single channels’), we also observed slow transitions. I_j exhibited slow transitions between a channel open state and the closed state late during wash-in and early during wash-out of 75 μm SKF-525A, i.e. immediately before complete uncoupling and early during recovery from uncoupling. Figure 8 illustrates this finding. It shows the first re-opening of a channel during wash-out. The signals I₁ and I₂ were recorded at a maintained V_j of 50 mV (V₁= -45 mV, V₂= 5 mV). The associated sequence of transitions involved a slow event (∼11 ms) between the closed state (continuous lines) and the main state, a fast event (< 2 ms) between the main state and the residual state (dashed lines), and a slow event (∼36 ms) between the residual state and the closed state. The time course of the first two events becomes evident on the expanded time scale (see inset). At the beginning and end of the current traces, superimposed on the maintained V_j, a depolarising test pulse (200 mV, 75 mV) was administered repetitively to cell 1 giving rise to short current deflections in I₁ but not in I₂. This indicates that the baseline current level (continuous lines) reflects the channel closed state.

Figure 8. Comparison of fast and slow channel transitions.

Sister current records I₁ and I₂ illustrating the re-opening of a gap junction channel previously closed by exposure to 75 μm SKF-525A. V_j was maintained at 50 mV (not shown). The first transition (closed state → main state) was slow (see inset at expanded time scale), the second transition (main state → residual state) was fast (see inset), and the third transition (residual state → closed state) was slow. The values of γ_j,main and γ_j,residual were 140 pS and 25 pS, respectively. The short transitions in I₁ resulted from depolarising test pulses applied to cell 1. Continuous lines indicate zero current, dashed lines indicate residual current.

The analysis of slow events in six cell pairs yielded a mean transition time of 12 ± 1 ms (n = 28). This implies that the channels possess two gating mechanisms, a biophysical one associated with fast transitions and a chemical one accompanied by slow transitions (Bukauskas & Weingart, 1994; Weingart & Bukauskas, 1997).

Determination of main-state probability

Cell pairs with a single gap junction channel were also used to study channel kinetics. At steady state, I_j mainly flickered between two discrete levels, i.e. I_j,main and I_j,residual. To determine the open channel probability, P_o, we used a protocol with V_j pulses of variable amplitude (up to 65 mV) and duration (20-80 s). The initial segment of each I_j record was discarded to avoid non-steady-state data. Figure 9 illustrates segments of such current traces recorded at V_j values of 25, 35, 40 and 55 mV (from top to bottom). The signals indicate that the dwell time in the main state was inversely related to V_j. At a small V_j, i.e. 25 mV, flickering was rare and the channel was preferentially in the main state. At a large V_j, i.e. 55 mV, flickering was also rare but the channel stayed mainly in the residual state (dashed line). At an intermediate V_j, i.e. 35 mV and 40 mV, the channel gradually altered from being in the main state to being in the residual state. The transitions between I_j,main and I_j,residual were always fast (i.e. < 2 ms).

Figure 9. Effects of transjunctional voltage (V_j) on single channel activity.

Long-lasting segments of current records obtained from a cell pair with a single operational gap junction channel. The signals were recorded during steady-state conditions. Upward deflections correspond to channel openings. When V_j was increased from 25 to 35, 40 and 55 mV (from top to bottom), the channel spent progressively less time in the main state and more time in the residual state (dashed lines). Continuous lines refer to the zero coupling current.

To calculate P_o, the times spent in the main state were determined and divided by the signal duration. The values of P_o obtained from three cell pairs were sampled, averaged and plotted versus V_j. Figure 10 shows the resulting graph. P_o decreased in a sigmoidal fashion between 1 and 0 when V_j was increased from 0 mV to 70 mV. The data points were fitted with the Boltzmann equation:

(10)

Figure 10. Dependence of main-state probability (P_o,main) on transjunctional voltage (V_j).

Values of P_o,main were determined from long lasting current records (20-80 s) during steady-state conditions. The data were collected from cell pairs with a single operational gap junction channel. Data points represent mean values ± 1 s.e.m. from 3 preparations. The continuous curve represents the best fit of data to the Boltzmann equation, with V_j,0= 37.5 mV, P_o,main= 0 and z = 3.

Steady-state kinetics of single channel currents

Currents from preparations with a single channel indicated that Cx30 channels mainly flicker between the main state and the residual state. Occasionally, these states were interrupted by short-lived substates. To study the underlying processes, we examined long segments of I_j records at steady state. The current traces were analysed for dwell times of the channel in the main and the residual states. Because of the rare occurrence and short duration, substates were ignored in this analysis. Figure 11 shows histograms of the open time prevailing at different V_j values, i.e. 10-25 mV (panel A), 30-35 mV (panel B), 40-45 mV (panel C) and 50-55 mV (panel D). The data at V_j values of 10-15 and 20-25 mV were pooled because of the low number of events (3 cell pairs). Over the time domains examined, the distributions of the open times were best approximated by single exponentials, implying that the channels possess a single open state. Curve fitting yielded time constants (τ_o) of 4, 2, 0.67 and 0.4 s, respectively.

Figure 11. Histograms of channel open times.

Current traces of cell pairs with a single gap junction channel were analysed for dwell times in the main state (i.e. channel open times) at different V_j at steady state. The data from 3 cell pairs were sampled and plotted as frequency histograms. The continuous curves correspond to the best fit of data to single exponentials with the following time constants: A, τ_o= 4 s (V_j= 10-25 mV); B, τ_o= 2 s (V_j= 30-35 mV); C, τ_o= 0.67 s (V_j= 40-45 mV); D, τ_o= 0.4 s (V_j= 50-55 mV).

While the determination of the channel open times was straightforward, the assessment of the channel closed times, i.e. the time spent in the residual state, posed problems. These arose from the limited response time of the experimental set-up (1-2 ms). This may have led to the situation that short-lived residual states were interpreted as substates. To eliminate this problem, channel closed times < 50 ms were excluded from the analysis. Another problem was the low number of events, especially at small V_j values. The resulting histograms (data not shown) indicated that the longest closed time increased with increasing V_j, i.e. from 6 s to 11, 42 and 56 s for V_j values of 10-25, 30-35, 40-45 and 50-55 mV, respectively. Furthermore, short closed times were more frequent than long ones at each V_j examined. At V_j values of 40-45 and 50-55 mV, the histograms allowed reliable curve fitting whereas at V_j values of 10-25 and 30-35 mV, the data were too scarce. The distributions in the former cases were best approximated by the sum of two exponentials. The analysis yielded the following time constants for the channel closed times: for V_j= 40-45 mV, τ_c1= 0.3 s and τ_c2= 7.0 s; for V_j= 50-55 mV, τ_c1= 0.23 s and τ_c2= 8.7 s. This suggests that the channels possess two residual states, one with a short lifetime and one with a long lifetime.

The fitted curves in Fig. 11 correspond to the probability density function f(t) =βexp(-βt), where β is the rate constant of channel closing and 1/β represents the mean open time, i.e. the average time a channel spends in the main state. Likewise, the closed time histograms reflect the function f(t) =αexp(-αt), where α is the rate constant of channel opening and 1/α reflects the mean closed time, i.e. the average time a channel spends in the residual state. In Fig. 12A, the data on channel lifetimes obtained in this way were plotted versus V_j, revealing that both channel lifetimes are voltage dependent, i.e. the open times (○) decreased with increasing V_j and the closed times (•: τ_c data; ▴: τ_c1 data; ▪: τ_c2 data) increased. However, the closed times should be regarded with caution because of the limited number of data points and observations.

Figure 12. Steady-state kinetics of gap junction channels.

A, values of channel mean open times (i.e. main state; ○) were determined from the probability density function using the rate constant of channel closure, β, and plotted versus V_j. Likewise, values of channel mean closed times (i.e. residual state) were derived from the probability density function, with the rate constant of channel opening, α, and plotted versus V_j. The histograms of the channel closed times were approximated with the sum of two exponentials. This gave rise to two values for the channel mean closed time (•, single exponential with τ_c; ▴, first exponential with τ_c1; ▪, second exponential with τ_c2). B, plots of the rate constants of channel opening, i.e. α (•), α₁ (▴) and α₂ (▪), and channel closing, i.e. β (○), versus V_j (data obtained from Fig. 12A).

Channel lifetimes can also be determined from the dwell times and their frequency of occurrence (see Johnston & Wu, 1995). The channel open times obtained with this procedure were 4.5, 1.99, 0.8 and 0.5 s. These values are close to those deduced from the probability density function (4, 2, 0.67 and 0.4 s) (○, Fig. 12A). The continuous curves were obtained by fitting the open time (○) and closed time data (•) to exponentials with decay constants of 16.6 and 20.2, respectively. In conclusion, these data suggest that the channels stayed in the residual state more often and for longer periods as V_j was increased.

Figure 12B shows plots of the rate constants of channel opening, α (•), α₁ (▴) and α₂ (▪), and channel closing, β (○), versus V_j. The data were obtained from Fig. 12A. The data points for channel open times and closed times were best fitted by exponentials with decay constants of 16.9 and 16.8, respectively. The two curves cross each other at V_j= 32.7 mV.

DISCUSSION

Basic properties of macroscopic gap junction currents

Our data demonstrate that the current flow through Cx30 gap junctions is sensitive to V_j. Hence, these junctions resemble other vertebrate gap junctions (see Bruzzone et al. 1996). On the one hand, the relationship g_j,inst= f(V_j) indicated that I_j,inst is not constant (see Fig. 2B). It decreased slightly with increasing V_j of either polarity. This may reflect (i) a genuine property of the channels, (ii) interference from a V_m-sensitive g_j,inst, or (iii) an artefact. The analysis of I_j records yielded similar values for I_j,inst when a graphical method or a curve fitting procedure was used. Hence, (iii) can be ignored. Explanation (ii) can also be ruled out since there was no indication of a sensitivity of I_j,inst to membrane potential (data not shown). Explanation (i) seems most likely. It is consistent with the single channel data, which indicated that γ_j,main is sensitive to V_j (see ‘Single channel properties’). Computer simulations with our gap junction model (Vogel & Weingart, 1998) showed an excellent fit with the I_j,inst measurements (see Fig. 2B). On the other hand, the relationship g_j,ss/g_j,inst= f(V_j) revealed that I_j,ss decreases with increasing V_j without, however, reaching zero. The data described a bell-shaped relationship and were best approximated with Boltzmann's equation. This is compatible with the observation that channel gating is sensitive to V_j (see ‘Determination of main-state probability’). In comparison with other gap junctions, the Boltzmann parameters for Cx30 channels suggest a strong voltage sensitivity (V_j,0= -28.5 and 26.4 mV and z = 4.1 and 3.9 for negative and positive V_j values). Data previously gained from pairs of injected oocytes revealed a moderately lower V_j sensitivity (V_j,0= -46 mV/38 mV; Dahl et al. 1996).

The single channel measurements offer a plausible explanation for the incomplete decay of g_j,ss at large V_j. They suggest that it reflects partial channel closure, i.e. g_j,min is related to γ_j,residual. This conclusion is consistent with the close match of the values for g_j,min and γ_j,residual/γ_j,main (0.15 and 0.14 for negative and positive V_jversus 0.15). It is also supported by the kinetic studies which demonstrated that the open channel probability, P_o, approaches zero at large V_j (see Fig. 10). This concept has been proposed before for other vertebrate gap junctions, e.g. Cx40 (Bukauskas et al. 1995a), Cx43 (Valiunas et al. 1997) and Cx46 (Sakai et al. 1998).

For comparison, gap junctions whose connexins are structurally closely related to Cx30 exhibit the following properties. Cx26: V_j,0= -93 mV/95 mV; g_j,min= 0.31/0.34; z = 1.2/1.4 (Valiunas et al. 1998). Cx30.3: V_j,0= -62 mV/64 mV; g_j,min= 0.11/0.17; z = 2.4/2.4 (F. F. Bukauskas & R. Weingart, unpublished observation). Cx32: V_j,0= -44 mV/44 mV; g_j,min= 0.22/0.23; z = 2.6/2.5 (Valiunas et al. 1998). Hence, there is no obvious correlation between the amino acid chain length of connexins and the selected electrophysiological parameters.

Kinetics of I_j inactivation

Our experiments revealed that the time-dependent decay of I_j is best described by single exponentials with the time constant τ_i, irrespective of the amplitude and polarity of V_j (see Fig. 3A). This is consistent with the presence of a single population of channels whose V_j-sensitive gating is controlled by a two-state process. Over the voltage range examined (i.e. 25-125 mV), the relationship τ_i= f(V_j) obeyed a mono-exponential. The values of τ_i decreased with increasing V_j in a manner symmetrical to V_j= 0 mV. The error bars in Fig. 3A indicate that the data scatter was inversely related to the amplitude of V_j. This means the uncertainty about the course of the function increased with decreasing V_j. Hence, it cannot be excluded that τ_i declines again at very small V_j (see Moreno et al. 1995).

If one considers a two-state process, i.e. open state residual state, the rate constants of channel opening (α) and closing (β) can be deduced from τ_i, g_j,ss, g_j,inst and g_j,min. The ensuing plots for α= f(V_j) and β= f(V_j) indicated that α, the rate constant of channel opening, is considerably less voltage dependent than β, the rate constant of channel closing (see Fig. 3B), i.e. α > β at V_j < V_j,0 and α < β at V_j > V_j,0. Hence, at high V_j the conductance state of the channels is primarily determined by the closing rate constant. The functions α= f(V_j) and β= f(V_j) cross each other at comparable|V_j| values, i.e. at -24.1 and 27.8 mV for negative and positive voltages, respectively. The values of V_j,0 deduced in this way agree with those determined from the relationship g_j,ss/g_j,inst= f(V_j), i.e. -28.5 and 26.4 mV (see Fig. 2B). This close correspondence is somewhat unexpected. Early during V_j pulses (non-stationary condition), channel substates were seen at all voltages, late during V_j pulses (stationary condition), they were virtually absent (Weingart et al. 1996b). Hence, non-stationary data (i.e. α and β) are expected to lead to smaller values of V_j,0 than stationary data (i.e. g_j,ss/g_j,inst). The similarity of V_j,0 values suggests that substates do not contribute significantly to I_j inactivation.

Previous studies with transfected RIN cells expressing Cx43 provided comparable V_j,0 values for non-stationary and stationary data (-60.6 and 60.1 mV and -60.5 and 59.5 mV, respectively; deduced from Banach & Weingart, 1996). Similar results were obtained with SKHep1 cells expressing Cx45 (14 mV and 13.4 mV; Moreno et al. 1995).

Single channel conductances

HeLa cells expressing Cx30 channels exhibited several conductance states, a fully open state, γ_j,main, a residual state, γ_j,residual, several substates, γ_j,substate, and a closed state, γ_j,closed. Hence, they behave like other vertebrate gap junction channels (Weingart et al. 1996a; Valiunas et al. 1998; for an exception, see Valiunas et al. 1998).

One series of experiments was performed at 34-35°C. It yielded an average γ_j,main of 163 pS and a γ_j,residual of 26 pS. If one considers Q₁₀ values of 1.16 and 1.27 for γ_j,main and γ_j,residual, respectively (see Fig. 6), this corresponds to conductances at room and body temperatures (23 and 37°C) of γ_j,main= 141 and 174 pS and γ_j,residual= 21 and 29 pS. Another series of experiments was carried out at 21-23°C, the temperature mainly used in this study. It revealed a distinct V_j dependence for γ_j,main and γ_j,residual; both conductances decreased with increasing V_j (see Fig. 7). Extrapolation to V_j= 0 mV yielded a γ_j,main of 146 pS and a γ_j,residual of 34 pS. If one takes into account the respective Q₁₀ values, this leads to a γ_j,main of 179 pS and a γ_j,residual of 48 pS at 37°C. These values are in good agreement with those obtained from the first series of experiments (see above).

The V_j sensitivity of γ_j,main and γ_j,residual is a novel finding for homotypic gap junction channels of vertebrates. It has been anticipated from investigations on heterotypic gap junction channels (Bukauskas & Weingart, 1995; Weingart et al. 1996a; see also Bukauskas et al. 1995b) and was predicted from our gap junction model (Vogel & Weingart, 1998). This property is based on the assumption that the conductances of hemichannels are voltage dependent (Bukauskas & Weingart, 1995; Weingart et al. 1996a). Recent studies on Cx30 hemichannels expressed in HeLa cells support this prediction (Valiunas & Weingart, 1997; V. Valiunas, R. Vogel & R. Weingart, in preparation). Hence, the V_j sensitivity of γ_j,main and γ_j,residual reflects the sum of a constant conductance of the non-gated hemichannel and a variable conductance of the gated hemichannel. The V_j sensitivity of the hemichannel conductances may arise from a rearrangement of charged amino acid residues of the connexins located inside the channel or at the mouth of the channel. Alternatively, cytoplasmic ions may provoke screening of surface charges during current flow through the channels. For comparison, a V_j-dependent γ_j,residual has also been reported for insect gap junctions (Bukauskas & Weingart, 1994).

Cx30 gap junction channels also exhibited several substates interposed between the main state and the residual state. These data will be presented in a separate paper (V. Valiunas & R. Weingart, in preparation; see also Weingart et al. 1996b; Valiunas et al. 1996). Closed states of Cx30 channels have been observed in conjunction with chemical interventions, e.g. exposure to lipophilic agents such as SKF-525A (see Fig. 8) or long-chain alkanols such as heptanol (data not shown). Current transitions involving the channel closed state (closed state residual state, closed state substates, closed state main state) were slow (12 ms; see ‘Fast versus slow current transitions’) while those involving open states (main state substates, main state residual state, substate residual state) were fast (< 2 ms). Hence, Cx30 channels resemble other vertebrate gap junction channels such as Cx32 and Cx43 (Valiunas et al. 1997, 1998; Bukauskas & Peracchia, 1997).

For comparison, the closely related connexins Cx26, Cx30.3, Cx31 and Cx32 form channels with a γ_j,main of 102 pS (Valiunas et al. 1998), 73 pS (F. F. Bukauskas & R. Weingart, unpublished observation), 50 pS (Bukauskas & Weingart, 1995) and 31 pS (Valiunas et al. 1998), respectively. Hence, Cx30 channels do not fit into this sequence of values. This suggests that there is no correlation between γ_j,main and the length of the C-terminus of the channel proteins.

Single channel kinetics

Cell pairs with a single gap junction channel have shown that the probability of being in the main state, P_o, decreased with increasing V_j (see Fig. 10). Over the voltage range explored, i.e. V_j= -70 to +70 mV, P_o declined in a sigmoidal manner from 1 to 0. Hence, g_j,min cannot be explained by a partial decrease in P_o (see ‘Basic properties of macroscopic gap junction currents’). It rather corresponds to the product of the number of operational channels, n, and γ_j,residual. The analysis of the P_o data yielded a V_j,0 of 37.5 mV. This value is significantly larger than those gained from the relationship g_j,ss/g_j,inst= f(V_j), i.e. -28.5 and 26.4 mV (see Fig. 2B). Several mechanisms may be responsible for this discrepancy: (i) involvement of substates, (ii) alteration of the driving force across the channels, and (iii) co-operativity between channels.

With regard to (i), the following observations argue against a role of substates. Both data sets, the P_o and g_j,ss/g_j,inst values, were gained at steady state. Since identical experimental conditions are likely to disclose the same channel properties, this should result in similar values of V_j,0. Furthermore, substates were virtually absent at steady state (see ‘Kinetics of I_j inactivation’). With respect to (ii), the two kinds of experiments were performed on cell pairs with single channels and many channels. Hence, V_j,0 may be affected by the number of channels involved. It has been proposed that tightly packed channels increase the channel access resistance and thereby lower the effective voltage gradient across the channels (Hall & Gourdie, 1995). However, this would shift V_j,0 towards larger values, the opposite of what we observed. (iii) Another possibility is that tightly packed channels lead to interactions between individual channels. In transfected HeLa cells the gap junction channels are mostly compacted in membrane plaques, although exceptions have been reported (Hülser et al. 1997). Hence, gating of a channel may affect the operation of adjacent channels. Such a mechanism would be consistent with the observation that I_j inactivation proceeds faster in the presence of many gap junction channels than a single one (V. Valiunas & R. Weingart, in preparation).

For comparison, transfected HeLa cells expressing Cx40 yielded similar V_j,0 values for the P_o and g_j,ss/g_j,inst= f(V_j) data, i.e. 44 mV and -45 and 49 mV, respectively (Bukauskas et al. 1995a). A similar coincidence in V_j,0 has been reported for rat Schwann cells expressing Cx32 (Chanson et al. 1993).

Single channel time histograms

The analysis of single channel records at steady state enabled us to construct channel time histograms (see Fig. 11). Over the time domain explored, the distributions of the open times were best approximated by a single exponential, suggesting that the channels possess a single open state. Curve fitting yielded open time constants (τ_o) of 4, 2, 0.67 and 0.4 s for V_j values of 17.5, 32.5, 42.5 and 52.5 mV, respectively. In contrast, the channel closed times were best described by the sum of two exponentials, indicating that the channels possess two residual states. Curve fitting yielded the following closed time constants: for V_j= 42.5 mV, τ_c1= 0.3 s and τ_c2= 7.0 s; for V_j= 52.5 mV, τ_c1= 0.23 s and τ_c2= 8.7 s. At a given V_j, short closed times were more abundant than long ones. A comparison of the open and closed times indicates that the former decreased with increasing V_j while the latter increased. On average, the channel open times were shorter than the closed times.

The establishment of lifetime histograms was somewhat hampered by the presence of substates (Weingart et al. 1996b; Valiunas et al. 1996). However, because of their low incidence and short duration they were ignored in the present context. While this had no serious effect on the analysis of channel open times, it influenced the analysis of the channel closed times. The problem was that the short lifetimes of the substates (5-10 ms) made it difficult to distinguish residual states from substates. To circumvent this difficulty, events shorter than 50 ms were excluded, which lowered the number of observations. This rendered it impractical to do curve fitting at V_j= 17.5 mV and 22.5 mV.

Using the channel lifetimes, we made an attempt to determine the rate constants of channel opening (α) and closing (β). The former involved the use of the probability density function (see Fig. 11), the latter the use of the channel dwell times and their frequency of occurrence. This procedure is a compromise for the missing histograms of the channel closed times at smaller voltages. It turned out that α decreases exponentially with increasing V_j while β increases (see Fig. 12B). This suggests that the channels stayed in the residual state more often and for longer periods as V_j was increased. This explains the decrease in P_o with increasing V_j. The functions α= f(V_j) and β= f(V_j) obtained from microscopic currents crossed each other at V_j= 32.7 mV (see Fig. 12B) which corresponds to V_j,0. For comparison, the functions α= f(V_j) and β= f(V_j) gained from macroscopic currents intersected at V_j values of -24.1 and 27.8 mV, respectively (see Fig. 3B). This discrepancy resembles that previously discussed for the P_o data (see ‘Single channel kinetics’). It indicates that single channel analyses lead to larger V_j,0 values than multichannel analyses, a phenomenon which may be related to interactions between channels (V. Valiunas & R. Weingart, in preparation).

Previous investigations on steady-state gating kinetics of gap junction channels relied on multichannel preparations or records. Rat Schwann cells expressing Cx32 yielded preliminary data on channel open times (Chanson et al. 1993). The mean value decreased from 1.62 s to 0.36 s when V_j was altered from 20 mV to 60 mV. The electrical synapse of the zebrafish retina revealed a mean open time of 31 ms at V_j= 50 mV (McMahon & Brown, 1994). This discrepancy may reflect different signal filtering used by these investigators (30-100 Hz versus 500 Hz). The latter laboratory also reported that gap junction channels in horizontal cells of bass retina exhibit two open states (Lu & McMahon, 1996). Smooth muscle cells expressing Cx43 exhibited mean open times of 0.44 and 5.25 s and mean closed times of 0.21 and 1.49 s (V_j= 80-30 mV; Brink et al. 1996). A secondary and extremely slow current decay was attributed to mode shifting.

Biological role of Cx30

In the present paper we report that Cx30 channels have a conductance of 180 pS. This value is rather large when compared with other gap junction channels (range 40-300 pS; see Waltzman & Spray, 1995). Hence, Cx30 channels permit the transfer of small ions as well as molecules of considerable size (D. Manthey et al., in preparation). This may be relevant for tissues whose physiology relies on strong intercellular signalling and/or metabolic co-operation. In this paper we also show that Cx30 channels exhibit a strong V_j sensitivity (V_j,0= 27 mV). This renders Cx30 hemichannels interesting candidates for the formation of rectifying channels. Hemichannels of connexins with a strong V_j dependence (e.g. Cx26, Cx40, Cx43), when docked onto hemichannels with a weak V_j sensitivity, are expected to form heterotypic channels with pronounced rectification (Bukauskas & Weingart, 1995; Bukauskas et al. 1995b). Indeed, experiments have shown that this is the case for Cx30-Cx26 and Cx30-Cx43 channels (Dahl et al. 1996; Weingart et al. 1996b; V. Valiunas & R. Weingart, unpublished observation). Cx30 expression studies suggest that the central nervous system, skin and lung may meet the requirements for forming rectifying channels (Dahl et al. 1996; Kunzelmann et al. 1999). However, precise immunohistochemical localisations are needed to identify such cellular areas.