Robert Weingart

Conductances and Selective Permeability of Connexin43 Gap Junction Channels Examined in Neonatal Rat Heart Cells

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

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, γj: γj(main state) increases with increasing positivity at the cytoplasmic aspect of the Cx26 connexon and decreases with increasing negativity (slope: 32 pS/100 mV; γj γj(main state) reaches 48 pS as V j approaches 0 mV); γj(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.

Gap junction channels and cardiac impulse propagation.

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.

Electrical Propagation in Synthetic Ventricular Myocyte Strands From Germline Connexin43 Knockout Mice

To characterize the role of connexin43 (Cx43) as a determinant of cardiac propagation, we synthesized strands and pairs of ventricular myocytes from germline Cx43−/− mice. The amount of Cx43, Cx45, and Cx40 in gap junctions was analyzed by immunohistochemistry and confocal microscopy. Intercellular electrical conductance, gj, was measured by the dual-voltage clamp technique (DVC), and electrical propagation was assessed by multisite optical mapping of transmembrane potential using a voltage-sensitive dye. Compared with wild-type (Cx43+/+) strands, immunoreactive signal for Cx43 was reduced by 46% in Cx43+/− strands and was absent in Cx43−/− strands. Cx45 signal was reduced by 46% in Cx43+/− strands and to the limit of detection in Cx43−/− strands, but total Cx45 protein levels measured in immunoblots of whole cell homogenates were equivalent in all genotypes. Cx40 was detected in ≈ 2% of myocytes. Intercellular conductance, gj, was reduced by 32% in Cx43+/− cell pairs and by 96% in Cx43−/− cell pairs. The symmetrical dependence of gj on transjunctional voltage and properties of single-channel recordings indicated that Cx45 was the only remaining connexin in Cx43−/− cells. Propagation in Cx43−/− strands was very slow (2.1 cm/s versus 52 cm/s in Cx43+/+) and highly discontinuous, with simultaneous excitation within and long conduction delays (2 to 3 ms) between individual cells. Propagation was abolished by 1 mmol/L heptanol, indicating residual junctional coupling. In summary, knockout of Cx43 in ventricular myocytes leads to very slow conduction dependent on the presence of Cx45. Electrical field effect transmission does not contribute to propagation in synthetic strands.

Free magnesium in sheep, ferret and frog striated muscle at rest measured with ion‐selective micro‐electrodes

1. Neutral carrier‐based liquid membrane micro‐electrodes were constructed which are suitable for continuous measurements of [Mg2+]i in cardiac and skeletal muscle preparations.

Ungulate cardiac Purkinje fibres: the influence of intracellular pH on the electrical cell‐to‐cell coupling

1. Passive electrical properties of sheep cardiac Purkinje fibres were assessed by performing linear cable analysis. In a separate set of experiments pHi was monitored using recessed‐tip pH‐sensitive micro‐electrodes.

Electrical properties of gap junction channels in guinea-pig ventricular cell pairs revealed by exposure to heptanol.

Cell pairs were isolated from adult guinea pig ventricles to study the electrical properties of gap junction channels. The experiments involved a double voltage-clamp approach and whole-cell, tight-seal recording. Heptanol decreased the intracellular current, In, in a dose-dependent fashion. Before complete uncoupling, In showed fluctuations suggesting the operation of gated channels. In the presence of 3 mM heptanol, In showed quantal steps arising from spontaneous opening and closing of single channels. The IV-relationship of the channels was linear (range: ±95 mV). Analysis of current records revealed the following singlechannel conductances, γn: Mean value = 37 pS; median value = 33 pS. γn was insensitive to the non-junctional membrane potential (range: −90 to +10 mV). 3 mM ATP4− in the pipette solution had no effects on γn, 6 mM ATP4− produced a small decrease, and 6 mM ATP+0.1 mM cAMP− an increase in γn. Channel transitions from closed to open state were variable (range of apparent time constants: 2.5–32 ms; mean: 11 ms).

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.

Intracellular Domains of Mouse Connexin26 and -30 Affect Diffusional and Electrical Properties of Gap Junction Channels

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

Action potential transfer in cell pairs isolated from adult rat and guinea pig ventricles.

An enzymatic procedure was used to obtain ventricular cells from adult rat and guinea pig hearts. Isolated pairs of cells were selected to study the action potential transfer from cell to cell and determine the resistance of the nexal membrane, rn. For this purpose, each cell of a cell pair was connected to a patch pipette so as to enable whole-cell, tight-seal recording. Normal impulse transmission was observed when rn ranged from 5-265 M omega. In these cases, the action potential in both cells occurred virtually simultaneously. An occasional failure in action potential transfer was seen in cell pairs whose rn had increased to 155-375 M omega. In these cases, the impulse transfer across the nexal membrane occurred with considerable delay. Impulse transfer was completely blocked once rn was larger than 780 M omega. Assuming a single connexon conductance of 100 pS, this would mean that more than 13 connexons are necessary to allow impulse transfer from cell to cell. Two single myocytes, gently pushed together, neither showed electrotonic interaction nor impulse transfer, thus rendering unlikely the possibility of an ephaptic signal transmission.