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Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43-based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease., Introduction The coordinated functioning of the heart as a pump heavily relies on communication between more than 3 billion cardiomyocytes via connexin-based gap junctions (GJs). GJs connecting cardiomyocytes and cells [...]

Aim: Inositol 1,4,5‐trisphosphate receptors (IP3Rs) are intracellular Ca2+‐release channels with crucial roles in cell function. Current IP3R inhibitors suffer from off‐target effects and poor selectivity towards the three distinct IP3R subtypes. We developed a novel peptide inhibitor of IP3Rs and determined its effect on connexin‐43 (Cx43) hemichannels, which are co‐activated by IP3R stimulation. Methods: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on‐nucleus patch‐clamp experiments of IP3R2 channels and carbachol‐induced IP3‐mediated Ca2+ responses in IP3R1, 2 or 3 expressing cells, triple IP3R KO cells and astrocytes. Cx43 hemichannels were studied by patch‐clamp and ATP‐release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP3Rs were verified by co‐immunoprecipitation and affinity pull‐down assays. Results: IP3RPEP6 concentration‐dependently reduced the open probability of IP3R2 channels and competitively inhibited IP3Rs in an IC50 order of IP3R2 (~3.9 μM) < IP3R3 (~4.3 μM) < IP3R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co‐immunoprecipitated with IP3R2 but not with IP3R1; interaction with IP3R3 varied between cell types. The IC50 of IP3RPEP6 inhibition of carbachol‐induced Ca2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19‐inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP3‐Ca2+ responses and strongly increased the EC50 of these responses. Finally, we identified palmitoyl‐8G‐IP3RPEP6 as a membrane‐permeable IP3RPEP6 version allowing extracellular application of the IP3R‐inhibiting peptide. Conclusion: IP3RPEP6 inhibits IP3R2/R3 at concentrations that have limited effects on IP3R1. IP3R activation triggers hemichannel opening, which strongly affects the amplitude and concentration‐dependence of IP3‐triggered Ca2+ responses. [ABSTRACT FROM AUTHOR]

Cx43, a major cardiac connexin, forms precursor hemichannels that accrue at the intercalated disc to assemble as gap junctions. While gap junctions are crucial for electrical conduction in the heart, little is known about the potential roles of hemichannels. Recent evidence suggests that inhibiting Cx43 hemichannel opening with Gap19 has antiarrhythmic effects. Here, we used multiple electrophysiology, imaging, and super-resolution techniques to understand and define the conditions underlying Cx43 hemichannel activation in ventricular cardiomyocytes, their contribution to diastolic [Ca.sup.2+] release from the sarcoplasmic reticulum, and their impact on electrical stability. We showed that Cx43 hemichannels were activated during diastolic [Ca.sup.2+] release in single ventricular cardiomyocytes and cardiomyocyte cell pairs from mice and pigs. This activation involved Cx43 hemichannel [Ca.sup.2+] entry and coupling to [Ca.sup.2+] release microdomains at the intercalated disc, resulting in enhanced [Ca.sup.2+] dynamics. Hemichannel opening furthermore contributed to delayed afterdepolarizations and triggered action potentials. In single cardiomyocytes, cardiomyocyte cell pairs, and arterially perfused tissue wedges from failing human hearts, increased hemichannel activity contributed to electrical instability compared with nonfailing rejected donor hearts. We conclude that microdomain coupling between Cx43 hemichannels and [Ca.sup.2+] release is a potentially novel, targetable mechanism of cardiac arrhythmogenesis in heart failure., Introduction The pumping function of the heart is coordinated by electrical waves of excitation propagating through the myocardium and initiating cardiac contraction. Propagation of action potentials between cardiomyocytes is coordinated [...]

Aims Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at −70 mV. Methods and results Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy. Conclusion Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation. [ABSTRACT FROM AUTHOR]

The eight members of the calcium channel γ subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of γ2, γ3, γ4 and γ8 (the TARPs) modulate AMPA receptor localization and function, the γ1 and γ6 subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the γ6 subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of γ6 subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of γ6 is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix–helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of γ6 with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of γ6 reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the γ6 calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density. [ABSTRACT FROM AUTHOR]

Aim: Inositol 1,4,5-trisphosphate receptors (IP 3 Rs) are intracellular Ca 2+ -release channels with crucial roles in cell function. Current IP 3 R inhibitors suffer from off-target effects and poor selectivity towards the three distinct IP 3 R subtypes. We developed a novel peptide inhibitor of IP 3 Rs and determined its effect on connexin-43 (Cx43) hemichannels, which are co-activated by IP 3 R stimulation., Methods: IP3RPEP6 was developed by in silico molecular docking studies and characterized by on-nucleus patch-clamp experiments of IP 3 R2 channels and carbachol-induced IP 3 -mediated Ca 2+ responses in IP 3 R1, 2 or 3 expressing cells, triple IP 3 R KO cells and astrocytes. Cx43 hemichannels were studied by patch-clamp and ATP-release approaches, and by inhibition with Gap19 peptide. IP3RPEP6 interactions with IP 3 Rs were verified by co-immunoprecipitation and affinity pull-down assays., Results: IP3RPEP6 concentration-dependently reduced the open probability of IP 3 R2 channels and competitively inhibited IP 3 Rs in an IC 50 order of IP 3 R2 (~3.9 μM) < IP 3 R3 (~4.3 μM) < IP 3 R1 (~9.0 μM), without affecting Cx43 hemichannels or ryanodine receptors. IP3RPEP6 co-immunoprecipitated with IP 3 R2 but not with IP 3 R1; interaction with IP 3 R3 varied between cell types. The IC 50 of IP3RPEP6 inhibition of carbachol-induced Ca 2+ responses decreased with increasing cellular Cx43 expression. Moreover, Gap19-inhibition of Cx43 hemichannels significantly reduced the amplitude of the IP 3 -Ca 2+ responses and strongly increased the EC 50 of these responses. Finally, we identified palmitoyl-8G-IP3RPEP6 as a membrane-permeable IP3RPEP6 version allowing extracellular application of the IP 3 R-inhibiting peptide., Conclusion: IP3RPEP6 inhibits IP 3 R2/R3 at concentrations that have limited effects on IP 3 R1. IP 3 R activation triggers hemichannel opening, which strongly affects the amplitude and concentration-dependence of IP 3 -triggered Ca 2+ responses., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Aims: Connexin-based gap junctions are crucial for electrical communication in the heart; they are each composed of two docked hemichannels (HCs), supplied as unpaired channels via the sarcolemma. When open, an unpaired HC forms a large pore, high-conductance and Ca2+-permeable membrane shunt pathway that may disturb cardiomyocyte function. HCs composed of connexin 43 (Cx43), a major cardiac connexin, can be opened by electrical stimulation but only by very positive membrane potentials. Here, we investigated the activation of Cx43 HCs in murine ventricular cardiomyocytes voltage-clamped at -70 mV., Methods and Results: Using whole-cell patch-clamp, co-immunoprecipitation, western blot analysis, immunocytochemistry, proximity ligation assays, and protein docking studies, we found that stimulation of ryanodine receptors (RyRs) triggered unitary currents with a single-channel conductance of ∼220 pS, which were strongly reduced by Cx43 knockdown. Recordings under Ca2+-clamp conditions showed that both RyR activation and intracellular Ca2+ elevation were necessary for HC opening. Proximity ligation studies indicated close Cx43-RyR2 apposition (<40 nm), and both proteins co-immunoprecipitated indicating physical interaction. Molecular modelling suggested a strongly conserved RyR-mimicking peptide sequence (RyRHCIp), which inhibited RyR/Ca2+ HC activation but not voltage-triggered activation. The peptide also slowed down action potential repolarization. Interestingly, alterations in the concerned RyR sequence are known to be associated with primary familial hypertrophic cardiomyopathy., Conclusion: Our results demonstrate that Cx43 HCs are intimately linked to RyRs, allowing them to open at negative diastolic membrane potential in response to RyR activation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

The eight members of the calcium channel gamma subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of gamma(2), gamma(3), gamma(4) and gamma(8) (the TARPs) modulate AMPA receptor localization and function, the gamma(1) and gamma(6) subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the gamma(6) subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of gamma(6) subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of gamma(6) is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of gamma(6) with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of gamma(6) reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the gamma(6) calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density.