Dual Voltage-clamp Technique Research Articles

Single channel properties of pannexin-1 and connexin-43 hemichannels and P2X7 receptors in astrocytes cultured from rodent spinal cords.

Astrocytes express surface channels involved in purinergic signaling. Among these channels, pannexin-1 (Px1) and connexin-43 (Cx43) hemichannels (HCs) release ATP that acts directly, or through its derivatives, on neurons and glia via purinergic receptors. Although HCs are functional, that is, open and close under physiological and pathological conditions, single channel properties of Px1 HCs in astrocytes have not been defined. Here, we developed a dual voltage clamp technique in HeLa cells expressing human Px1-YFP, and then applied this system to rodent spinal astrocytes to compare their single channel properties with other surface channels, that is, Cx43 HCs and P2X7 receptors (P2X7Rs). Channels were recorded in cell attached patches and evoked with ramp cycles applied through another pipette in whole cell voltage clamp. The mean unitary conductances of Px1 HCs were comparable in HeLa Px1-YFP cells and spinal astrocytes, ~42 and ~48 pS, respectively. Based on their unitary conductance, voltage-dependence, and unitary activity after pharmacological and gene silencing, Px1 HCs in astrocytes could be distinguished from Cx43 HCs and P2X7Rs. Channel activity of Px1 HCs and P2X7Rs was greater than that of Cx43 HCs in control astrocytes during ramps. Unitary activity of Px1 HCs was decreased and that of Cx43 HCs and P2X7Rs increased in astrocytes treated with fibroblast growth factor 1 (FGF-1). In summary, we resolved single channel properties of three different surface channels involved in purinergic signaling in spinal astrocytes, which were differentially modulated by FGF-1, a growth factor involved in neurodevelopment, inflammation and repair.

Coupling between cardiac cells-An important determinant of electrical impulse propagation and arrhythmogenesis.

Cardiac arrhythmias are an important cause of sudden cardiac death-a devastating manifestation of many underlying causes, such as heart failure and ischemic heart disease leading to ventricular tachyarrhythmias and ventricular fibrillation, and atrial fibrillation causing cerebral embolism. Cardiac electrical propagation is a main factor in the initiation and maintenance of cardiac arrhythmias. In the heart, gap junctions are the basic unit at the cellular level that host intercellular low-resistance channels for the diffusion of ions and small regulatory molecules. The dual voltage clamp technique enabled the direct measurement of electrical conductance between cells and recording of single gap junction channel openings. The rapid turnover of gap junction channels at the intercalated disk implicates a highly dynamic process of trafficking and internalization of gap junction connexons. Recently, non-canonical roles of gap junction proteins have been discovered in mitochondria function, cytoskeletal organization, trafficking, and cardiac rescue. At the tissue level, we explain the concepts of linear propagation and safety factor based on the model of linear cellular structure. Working myocardium is adequately represented as a discontinuous cellular network characterized by cellular anisotropy and connective tissue heterogeneity. Electrical propagation in discontinuous cellular networks reflects an interplay of three main factors: cell-to-cell electrical coupling, flow of electrical charge through the ion channels, and the microscopic tissue structure. This review provides a state-of-the-art update of the cardiac gap junction channels and their role in cardiac electrical impulse propagation and highlights a combined approach of genetics, cell biology, and physics in modern cardiac electrophysiology.

Neonatal Rat Cardiomyocytes Show Characteristics of Nonhomotypic Gap Junction Channels

Neonatal rat cardiomyocytes mainly coexpress the connexins Cx40, Cx43, and to a small amount Cx45, leading to potential formation of mixed (heteromeric/heterotypic) gap junction channels. Using the dual-voltage clamp technique with switching clamp circuits, the authors investigated voltage sensitivity of gap junction channels between cell pairs of Cx40, Cx43, and Cx45 stably transfected HeLa cells and compared those data to data obtained from cell pairs of cultured neonatal rat cardiomyocytes. In accordance to previously published data, the relationship between normalized conductance and transjunctional voltage (g/V(j)) was quasisymmetrical for the transfected HeLa cells, indicating homotypic gap junction channels. Boltzmann curves fitted to data obtained from neonatal rat cardiomyocyte pairs expressing both Cx40 and Cx43 showed an asymmetrical inactivation pattern, which cannot be explained by the presence of pure populations of homotypic gap junction channels of either isoform. In conclusion the authors assume the additional presence of heterotypic and possibly even heteromeric gap junction channels in neonatal rat cardiomyocytes.

Pro-arrhythmogenic potential of immature cardiomyocytes is triggered by low coupling and cluster size

Cell transplantation strategies to regenerate compromised myocardium take advance of in vitro generated cardiomyocytes. Common in those immature myocytes is spontaneous impulse formation and a restricted ability to establish proper electrical interaction. Spontaneous impulse formation and impaired cell-to-cell coupling have been shown to be arrhythmogenic. To investigate whether these features harbour a pro-arrhyhmogenic potential for cell transplantation, a co-culture of spontaneously active neonatal rat cardiomyocytes (NRC) and quiescent adult dog cardiomyocytes (ADC) was used. ADCs and NRCs were isolated and cultured on laminin-coated substrates. Connexin43, N-cadherin and alpha-actinin expression was evaluated with immunohistochemistry. Intercellular coupling was measured in cell pairs using the dual voltage clamp technique and fluorescent dye injection. One day after isolation, NRCs were beating spontaneously, while ADCs remained quiescent in monoculture. ADC resting membrane potential was -80.3+/-0.2 mV (mean+/-SEM, N=24) and did not change significantly over time. NRCs had a maximal diastolic potential of -65.0+/-2.8 mV (N=4). After one day of co-culture, pseudopodia-like extensions developed at the former intercalated discs of ADCs, contacting the NRCs. Only ADCs that contacted three or more NRCs started to beat in synchrony. Expression of connexin43 and N-cadherin indicated presence of electrical and mechanical junctions at the interface between the two cell-types. Transfer of Lucifer Yellow demonstrated junctional permeability between ADCs and NRCs. Junctional conductance between ADC-ADC (31.9+/-5.1 nS, N=10) and NRC-NRC (35.0+/-9.6 nS, N=6) pairs was significantly higher compared to ADC-NRC pairs (9.7+/-2.9 nS, N=8). Gap-junctional blockade with halothane reversibly abolished NRC-triggered beating of ADCs. Computer simulations demonstrated that within a delicate 'window' of gap junctional conductance small clusters of spontaneously active cells are able to induce triggered activity in quiescent mature myocytes but also in a two-dimensional sheet of ventricular cells. Spontaneously active immature cardiomyocytes are able to trigger mature cardiomyocytes depending on their level of electrical coupling and the amount of coupled immature myocytes.

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.

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties.

HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e. the dependence of the instantaneous and steady-state conductances (gj,inst, gj,ss) on the transjunctional voltage (Vj) and the kinetics of inactivation of the gap junction current (Ij). Pairs of singly transfected cells showed homogeneous behaviour at both Vj polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions gj,inst=f(Vj) and gj,ss=f(Vj). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions gj,inst=f(Vj) and gj,ss=f(Vj) suggesting that connexons Cx43 and Cx45 gate with positive and negative Vj, respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasi-homogeneous behaviour at one Vj polarity and heterogeneous behaviour at the other polarity. The former yielded Boltzmann parameters intermediate between those of Cx43-Cx43, Cx45-Cx45 and Cx43-Cx45 preparations; the latter could not be explained by homotypic and heterotypic combinations of homomeric connexons. Each pair of doubly transfected cells (Cx43/Cx45) yielded unique functions gj,inst=f(Vj) and gj,ss=f(Vj). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomeric-homotypic, homomeric-heterotypic channels as well as heteromeric-homotypic and heteromeric-heterotypic channels. This has implications for the impulse propagation in specific areas of the heart.

Dual patch clamp.

AbstractThe main feature of gap junctions is the passage of ions and small molecules between adjacent cells. In the heart, for example, junctional currents between adjacent cells are elicited by the membrane potential difference when one of the cells generates an action potential, while the other cell is still at resting membrane potential, thereby allowing propagation of the action potential. Experimentally, such gap junctional currents can be directly evoked and measured by independently controlling the membrane voltage of both cells of a cell pair. This is accomplished by “dual voltage clamping” a cell-pair, a method introduced by Spray et al. (1). Using separate electrodes for voltage control and current injection, this technique allowed accurate determination of kinetics and conductance of gap junctions (1–3). At present, the dual voltage-clamp technique is usually performed with two patch pipets (4), each on one cell of a cell pair, which control voltage and measure current simultaneously (5–8). KeywordsSeries ResistanceMembrane ResistancePatch PipetsCell PairJunctional ConductanceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Molecular Dissection of Transjunctional Voltage Dependence in the Connexin-32 and Connexin-43 Junctions

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

Intramolecular interactions mediate pH regulation of connexin43 channels

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

PH sensitivity of the cardiac gap junction proteins, connexin 45 and 43.

. Intercellular communication through gap junction channels can be regulated by changes in intracellular pH (pHi). This regulation may play an important role in ischemic heart tissue. Using the dual voltage-clamp technique, we compared the pHi sensitivity of gap junction channels composed of connexin 43 (Cx43) and Cx45, two of the gap junction proteins that are expressed in heart. We made use of SKHep1 cells, endogenously expressing low levels of Cx45 and SKHep1 cells stably transfected with rat Cx43. To manipulate the pHi we applied the NH3/NH+4 pH-clamp method. At pHi 6.7 the gj of Cx45 channels was reduced to approximately 20% of control values (pHi 7.0) and at pHi 6.3 all channels closed. The gj of Cx43 channels was approximately 70% of control values at pHi 6.7 and approximately 40% at pHi 6.3. Cx43 channels closed at pHi 5.8. Single channel conductances were 17.8 pS for Cx45 and 40.8 pS for Cx43 at pHi 7.0 and did not change significantly at lower pHi. This suggests that the decrease in macroscopic conductance observed at low pHi results from the decrease in open probability of gap junctional channels rather than from a decrease in single channel conductance. Our results demonstrate that gap junction channels built of Cx45 are far more pH sensitive than channels built of Cx43.

Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43.

We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild-type and mutant rat cardiac Cx43 channels were expressed in antisense-injected Xenopus oocytes. Junctional conductance was measured using the dual voltage-clamp technique, and intracellular acidification was induced by superfusion with a sodium acetate-containing solution balanced at a pH of 6.2. H95 was substituted by other amino acids by use of oligonucleotide-directed site-specific mutagenesis. Replacing H95 for the hydrophobic residues methionine or phenylalanine, for the charged basic residue arginine, or for the noncharged residue glutamine (H95Q) yielded nonfunctional channels. Functional expression of H95Q was rescued by placing a histidine residue in position 93 (H95Q-L93H), 94 (H95Q-A94H), or 97 (H95Q-F97H) but not in position 96. Further experiments showed that replacing H95 with either aspartate (an acidic residue) or tyrosine (a polar uncharged residue) led to the expression of functional channels with a reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)