Gudaitis, Lukas

Connexin-36 (Cx36) forms gap junction (GJ) channels that constitute the majority of electrical synapses in mammalian CNS and enable direct signaling between pancreatic beta cells. GJ channels are formed by the docking of two hexameric Cx hemichannels, each gating in response to the transjunctional voltage, Vj. Two distinct Vj gating mechanisms, attributed to the N-terminal domain (NT) and the first extracellular loop, are operative in each hemichannel and can modulate coupling. Uniquely among the 21 human Cx isoforms, intracellular Mg2+ robustly modulates Cx36 GJs, affecting the magnitude of coupling as well as sensitivity to Vj. Previously, we showed that charge substitutions E3Q, E8Q, A13K, and H18K in NT of Cx36 modified sensitivity to Mg2+. Here, we show that these same charge substitutions also alter Vj dependence. Mathematical modeling indicates that Mg2+ effects alone cannot account for the data, implicating modification of intrinsic Vj gating properties. The NT domain forms the cytoplasmic vestibule of a GJ channel and a number of residues function in sensing Vj and stabilizing open/closed configurations. Molecular dynamics simulations show that each of the NT charge substitutions altered the electrostatic profile of the channel pore and produced widespread alterations in interactions between residues in NT and the transmembrane domains that can affect the stability of the putative open conformation. Using heterotypic pairings of WT Cx36 and variants, we established a positive gating polarity for Cx36 and demonstrated polarity reversal for the E3Q substitution, properties indicative that NT-mediated gating plays a predominant role in Vj-dependence of Cx36 GJs.

Unapposed connexin (Cx) hemichannels serve as precursors to gap junction channels but also function independently, playing crucial roles in various physiological processes. Hemichannel gating is influenced by factors such as plasma membrane voltage and extracellular divalent ion concentrations. Excessive hemichannel opening can lead to significant leakage of ions and molecules, and mutations in genes encoding Cxs often result in aberrant gating, contributing to various pathologies. Therefore, evaluating and quantifying Cx hemichannel gating behaviours is important. To address this, we developed a mathematical/computational model describing the voltage-gating properties of Cx hemichannels. The proposed model incorporates two distinct gating mechanisms - fast and loop gating - known to regulate hemichannel closure. These gating transitions are represented within a four-state kinetic scheme, which also accounts for redistribution of voltage upon the closure of either mechanism. Using a sensitivity function matrix approach, we selected voltage protocols that provide sufficient information to constrain the proposed model. The model was then fitted to electrophysiological data recorded from Cx26 and Cx45 hemichannels. Fits to both training datasets and independent validation data indicate that the model can adequately describe the basic characteristics of Cx hemichannel currents. Further analysis using the proposed kinetic scheme provides insights into hemichannel gating behaviour, including the observed delay in current activation upon depolarization and potential discrepancies between gating kinetics of unapposed hemichannels and gap junction channels. Thus, the proposed model can serve as a valuable tool for comparing voltage-gating properties across Cx isoforms and mutants and offers insights into Cx hemichannel gating behaviours. KEY POINTS: Gating of unapposed connexin (Cx) hemichannels plays a crucial role in various physiological processes, whereas mutations in Cx genes that cause aberrant gating are linked to various pathologies. To quantify the voltage-gating properties of Cx hemichannels, we present a novel mathematical/computational model that comprises two established gating mechanisms: fast and loop gating. The validity of the proposed model is demonstrated through fits to electrophysiological data from cells expressing different Cx isoforms, Cx26 and Cx45. The proposed model can serve as a useful tool for comparing the voltage-gating properties across Cx isoforms and mutants, and offers insights into the physiologically relevant mechanistic behaviours of Cx hemichannels.

Gap junction (GJ) channels, formed of connexin (Cx) protein, enable direct intercellular communication in most vertebrate tissues. One of the key biophysical characteristics of these channels is their unitary conductance, which can be affected by mutations in Cx genes and various biochemical factors, such as posttranslational modifications. Due to the unique intercellular configuration of GJ channels, recording single-channel currents is challenging, and precise data on unitary conductances of some Cx isoforms remain limited. In this study, we applied stationary noise analysis, a method successfully used for ion channels with very low unitary conductances, to GJ channels. We modified this technique to account for the residual conductance of GJ channels and present three strategies for estimating unitary conductance, including model-based evaluation of open-state probability and subtraction of residual conductance. To assess the validity, advantages, and limitations of these approaches, we performed mathematical analysis and simulation experiments. We also addressed practical issues such as the underestimation of sample variance in autocorrelated recordings and channel rundown, proposing solutions to these issues. Finally, we applied these strategies to electrophysiological data recorded from cells expressing Cx45. Our findings showed that noise-based estimates of Cx45 unitary conductance from macroscopic currents align well with those obtained from single-channel recordings.

Propofol is a widely used general anesthetic, which causes a rapid induction of anesthesia. The most prominent side effect of propofol is the decrease of systemic vascular resistance that leads to hypotension. Moreover, in some cases, propofol has been shown to inhibit cardiac conduction and cause bradycardia, but its pathophysiological mechanism is still not fully understood. On the other hand, propofol was shown to have a protective effect against ventricular arrhythmias during myocardial ischemia. Several studies have shown that propofol may regulate cell coupling through gap junction (GJ) channels formed of Cx43. However, it is unclear how propofol affects all cardiac connexins. It is known that Cx37, Cx40, Cx43 and Cx45 form gap junction channels in cardiovascular system. These connexins are required for coordination of vascular responses and play a key role in ensuring propagation of action potential in cardiac tissue. The aim of this study was to get a better understanding of the capacity of propofol to affect cardiovascular connexins. First, we compared the effect of various propofol concentrations on GJs formed by cardiovascular connexins which were expressed exogenously in human cervix epithelial adenocarcinoma cells (HeLa). The junctional conductance was measured using double whole-cell patch clamp method. Our data show that Cx40 and Cx43 channels exhibit similar sensitivity to propofol (IC50 Cx40 - 17µM; Cx43 – 15µM), while Cx45 channels are sensitive to much higher propofol concentrations (IC50 60µM). Interestingly, the vascular Cx37, which is an important player in dilation of vascular beds, was the most susceptible to propofol (IC50 5µM). Propofol may affect Cx43 GJ channels through activation of protein kinase C (PKC), which in turn phosphorylates Cx43 and reduces coupling through GJs. In our study, the kinase inhibitor GF109203X was used to assess this putative pathway of propofol action on GJs. It is established that low concentrations (40nM) of GF109203X specifically inhibit PKC while higher concentrations (2 µM) inhibit both PKC and protein kinase A (PKA). Our data showed that low concentration of GF109203X significantly reduced inhibition of Cx43 channels by propofol. In contrast, both concentrations of GF109203X had no effect on inhibition of Cx40 and Cx45 channels by propofol. The findings indicate that both low and high kinase inhibitor concentrations considerably lessened propofol’s blocking effect on Cx37.

Propofol is a widely used general anesthetic, which causes a rapid induction of anesthesia and rapid recovery after it. The most prominent side effect of propofol is the decrease of systemic vascular resistance that leads to hypotension [1]. Moreover, in some cases, propofol has been shown to inhibit cardiac conduction and cause bradycardia that can result in cardiac arrest. The propofol effect on the induction of bradycardia has been known for more than 30 years, but its pathophysiological mechanism is still not fully understood. One of the putative mechanisms for the conduction block could be the ability of propofol to change the activity of human atrial muscarinic cholinergic receptors [2]. It is also known that propofol may directly inhibit the sinoatrial (SA) node cells. Furthermore, according to certain research, the effect of propofol on the conduction system varies depending on the concentration of the drug. On the other hand, propofol was shown to have a protective effect against ventricular arrhythmias during myocardial ischemia [3]. Thus, the influence of propofol on the heart rhythm remains controversial. The side effect of propofol may manifest itself through its action on different targets. Indeed, several studies have shown that propofol may regulate cell coupling through gap junction (GJ) channels formed of Cx43. However, it is unclear how propofol affects all cardiac connexins, not just connexin-43 (Cx43). As known Cx43, Cx45, Cx37 and Cx40 form gap junction channels in cardiac and vascular system. These connexins are required for coordination of vascular responses and play a key role in ensuring propagation of action potential in cardiac tissue. The aim of this study was to get a better understanding the capacity of propofol to affect cardiovascular connexins. First, we compared the effect of propofol concentrations on GJs formed by Cx40, Cx43, Cx45 and Cx37 which were expressed exogenously in human cervix epithelial adenocarcinoma cells (HeLa). The junctional conductance was measured using double whole-cell patch clamp method. Our data show that Cx40 and Cx43 channels exhibit similar sensitivity to propofol, while Cx45 channels are only sensitive to much higher propofol concentrations than Cx40 and Cx43 channels. Of all the connexins under research, the most surprising finding showed that Cx37, the vascular system connexin and an important player in coronary heart disease, is also the most susceptible to propofol, meaning it is more sensitive to lower concentrations of the drug. It was suggested that propofol may affect Cx43 GJ channels through activation of protein kinase C (PKC), which in turn phosphorylates Cx43 and reduces coupling through GJs [4]. In our study, the kinase inhibitor GF109203X was used to assess this putative pathway of propofol action on GJs. It is established that low concentrations of GF109203X specifically inhibit PKC while higher concentrations inhibit both PKC and protein kinase A (PKA). Our data showed that low (40 nM) concentration of GF109203X significantly reduced inhibition of Cx43 channels by propofol, indicating that propofol regulates Cx43 channels in PKC-dependent manner, which is in good agreement with already published studies. In contrast, both concentrations of GF109203X had no effect on inhibition of Cx40 channels by propofol, indicating that neither PKC nor PKA is involved in regulation of these channels by the anesthetic. This preliminary data suggest that propofol may have a prominent, connexin specific effect on GJs.

Gap junction (GJ) channels, formed by the transmembrane protein connexin-36 (Cx36), facilitate electrical coupling between neurons, retinal and pancreas beta cells. Compared to other connexin (Cx) proteins, Cx36 channels display unique biophysical and biochemical characteristics, including very low unitary conductance, low sensitivity to transjunctional voltage (Vj), and high sensitivity to intracellular free magnesium ion concentration ([Mg2+]). Research has shown that the N-terminus domain of connexin channels plays a key role in determining the Vj gating properties of both Cx hemichannels and GJ channels. In this study, we combined electrophysiological recordings, mathematical/computational modeling, and molecular dynamics simulations to assess the role of specific amino acid residues in the N-terminus on the Vj gating properties of Cx36 channels. Our electrophysiological results revealed that single amino acid substitutions—changing negatively charged glutamic acid at positions 3 and 8 to neutral glutamine (E3Q and E8Q), or neutral alanine and histidine at positions 13 and 18 to positively charged lysine (A13K and H18K)—significantly altered the gating behavior of Cx36 channels. To adequately explain the obtained electrophysiological data, we developed a mathematical model by combining our previously published models of GJ channel Vj gating [1] and Mg2+ regulation of Cx36 channels [2]. Model fitting data indicated that these changes could not be attributed solely to differences in [Mg2+] sensitivity but involved significant modifications to Vj gating properties. Data obtained from molecular dynamics simulations support this hypothesis and may provide mechanistic explanations for the observed changes in gating properties. Mainly, it demonstrated that single residue substitutions at the N-terminus can alter the electrostatic profile of the channel pore and disrupt critical interactions near gating-associated sites. Data obtained from recordings of heterotypic configurations of variant Cx36 channels, as well as from model fitting experiments, allowed us to infer the Vj gating polarity of Cx36 channels—a property not previously reported in other electrophysiological studies. More precisely, our results suggest that Cx36 hemichannels gate in response to relatively positive Vjs, but this gating polarity was reversed by single-residue substitution at the 3rd position of the NT-domain.

Electrophysiological recording via the patch clamp technique allows for the assessment of the biophysical properties of various types of ion channels. However, electrophysiological recordings of gap junction (GJ) channels poses challenges due to their unique intercellular configuration and natural clustering into plaques, making it difficult to obtain reliable data at a single-channel level. Additionally, until recently, no mathematical models adequately explained both the steady-state and kinetic properties of GJ channel gating, which is crucial for model-based evaluation of single-channel level characteristics. Consequently, the methods for accurately correlating data recorded at macroscopic and single-channel levels have been lacking in studies of gap junctional electrophysiology. To address these issues, we combined our previously published four-state model (4SM) of GJ channel gating with probabilistic methods, such as maximum likelihood estimation (MLE)-based analysis of single-channel level currents and stationary noise analysis of macroscopic level electrophysiological recordings. First, we address evaluation of biophysical single-channel-level properties of GJ channels, such as open-state probability and unitary conductance, using data from macroscopic-level recordings. Second, we consider MLE-based methodologies to extract information about gating parameters of GJ channels from electrophysiological recordings with observable unitary events. The validity of the proposed methodologies is first illustrated through stochastic simulations and further extended to real electrophysiological data. Overall, our findings show that these techniques can provide valuable insights into biophysical properties of GJ channels.

Introduction Gap junction (GJ) channels, composed of connexin (Cx) proteins, provide direct metabolic and electrical communication between cells. These specialized channels have been shown to gate robustly in response to transjunctional voltage, Vj. Voltage gating of GJs could play a physiological role, particularly in excitable cells, which can generate large transients in membrane potential during the propagation of action potentials. Mathematical models describing biophysical properties of gap junction channels are valuable, serving as tools for studying GJ channel gating and simulating cell clusters resembling cardiac or nervous tissue. Here, we demonstrate the validation of such a mathematical model and several variants of its applicability

Method & Materials For validation of mathematical model, we used electrophysiological measurements in HeLa cell pairs exogenously expressing Cx45 and in Novikoff cell pairs, which endogenously expresses Cx43. Junctional conductance was measured in selected cell pairs using a dual whole-cell patch-clamp system. Vj was induced by stepping the voltage in one cell while maintaining a constant voltage in the other. Junctional current (Ij) was measured as the change in the current of a neighboring cell, and conductance (gj) was estimated from the relationship gj = -Ij/Vj.

Results From electrophysiological recordings in cell cultures expressing Cx43 or Cx45, the principal isoforms expressed in cardiac tissue, various data sets were fitted simultaneously using global optimization. Model-fitting results showed good correspondence with both kinetic and steady-state data. Our data demonstrate the (4SM) model reproducing a range of experimentally observed GJ behaviors, not achievable through modeling steady-state data alone. Additionally, mathematical analyses showed that the current model can be approximated by a reversible two-state system and solved analytically using a rapid equilibrium assumption (REA), often applied in modeling enzyme kinetics. This model property allows for a substantial reduction in computation time and could be efficiently applied when simulating large clusters of cells.

Conclusion The 4SM model serves as a tool for studying GJ channel gating and its effects on excitation spread in networks of electrically coupled cells.

Propofol is a widely used general anesthetic, which causes a rapid induction of anesthesia and rapid recovery after it. The most prominent side effect of propofol is the decrease of systemic vascular resistance that leads to hypotension [1]. Moreover, in some cases, propofol has been shown to inhibit cardiac conduction and cause bradycardia that can result in cardiac arrest. The propofol effect on the induction of bradycardia has been known for more than 30 years, but its pathophysiological mechanism is still not fully understood. One of the putative mechanisms for the conduction block could be the ability of propofol to change the activity of human atrial muscarinic cholinergic receptors [2]. It is also known that propofol may directly inhibit the sinoatrial (SA) node cells. On the other hand, propofol was shown to have a protective effect against ventricular arrhythmias during myocardial ischemia [3]. Thus, the influence of propofol on the heart rhythm remains controversial. The side effect of propofol may manifest itself through its action on different targets. Indeed, several studies have shown that propofol may regulate cell coupling through gap junction (GJ) channels formed of connexin-43 (Cx43). As known Cx43 and Cx45 form gap junction channels in nervous system and, together with Cx37 and Cx40, are also expressed throughout cardiac and vascular system. These connexins are required for coordination of vascular responses and play a key role in ensuring propagation of action potential in cardiac tissue. The aim of this study was to get a better understanding of the capacity of cardiovascular connexins to participate in side effect caused by propofol. First, we compared the effect of propofol concentrations on GJs formed by Cx40, Cx43 and Cx45, which were expressed exogenously in human cervix epithelial adenocarcinoma cells (HeLa). The junctional conductance was measured using double whole-cell patch clamp method. Our data show that Cx40 and Cx43 channels exhibit similar sensitivity to propofol, while Cx45 channels are only sensitive to much higher propofol concentrations (above 60 µM) than Cx40 and Cx43 channels. It was suggested that propofol may affect Cx43 GJ channels through activation of protein kinase C (PKC), which in turn phosphorylates Cx43 and reduces coupling through GJs [4]. In our study, the kinase inhibitor GF109203X was used to assess this putative pathway of propofol action on GJs. It is established that low concentrations of GF109203X specifically inhibit PKC while higher concentrations inhibit both PKC and protein kinase A (PKA). Our data showed that low (40 nM) concentration of GF109203X significantly reduced inhibition of Cx43 channels conductance by propofol, indicating that propofol regulates Cx43 channels in PKCdependent manner, which is in good agreement with already published studies. In contrast, GF109203X had no effect on inhibition of Cx40 channels by propofol, indicating that neither PKC nor PKA is involved in regulation of these channels by the anesthetic. This preliminary data suggest that propofol can have a prominent, connexin specific effect on GJs.

The advancement of single-channel-level recording via the patch-clamp technique has provided a powerful means of assessing the detailed behaviors of various types of ion channels in native and exogenously expressed cellular environments. However, such recordings of gap junction (GJ) channels are hampered by unique challenges that are related to their unusual intercellular configuration and natural clustering into densely packed plaques. Thus, the methods for reliable cross-correlation of data recorded at macroscopic and single-channel levels are lacking in studies of GJs. To address this issue, we combined our previously published four-state model (4SM) of GJ channel gating by voltage with maximum likelihood estimation (MLE)-based analyses of electrophysiological recordings of GJ channel currents. First, we consider evaluation of single-channel characteristics and the methods for efficient stochastic simulation of single GJ channels from the kinetic scheme described by 4SM using data obtained from macroscopic recordings. We then present an MLE-based methodology for extraction of information about transition rates for GJ channels and, ultimately, gating parameters defined in 4SM from recordings with visible unitary events. The validity of the proposed methodology is illustrated using stochastic simulations of single GJ channels and is extended to electrophysiological data recorded in cells expressing connexin 43 tagged with enhanced green fluorescent protein.