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1. Connexin 46 and connexin 50 gap junction channel properties are shaped by structural and dynamic features of their N-terminal domains

   Yue, Benny ; Haddad, Bassam G. ; Khan, Umair ; Chen, Hongchong ; Atalla, Mena ; Zhang, Ze ; Zuckerman, Daniel M. ; Reichow, Steve L. ; Bai, Donglin ( April 2021 , The journal of physiology)

   null (Ed.)

   Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long-range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino-terminal (NT) domain as a major contributor for isoform-specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46-50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free-energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage-dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra- and inter-subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract-linked connexin variants. 

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2. Connexin-46/50 in a dynamic lipid environment resolved by CryoEM at 1.9 Å

   https://doi.org/10.1038/s41467-020-18120-5  

   Flores, Jonathan A. ; Haddad, Bassam G. ; Dolan, Kimberly A. ; Myers, Janette B. ; Yoshioka, Craig C. ; Copperman, Jeremy ; Zuckerman, Daniel M. ; Reichow, Steve L. ( August 2020 , Nature Communications)

   Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels.

    

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3. A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels

   https://doi.org/10.1085/jgp.202012607  

   Gaete, Pablo S. ; Lillo, Mauricio A. ; López, William ; Liu, Yu ; Jiang, Wenjuan ; Luo, Yun ; Harris, Andrew L. ; Contreras, Jorge E. ( October 2020 , Journal of General Physiology)

   Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease–causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

    

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4. MitoBK Ca channel is functionally associated with its regulatory β1 subunit in cardiac mitochondria

   https://doi.org/10.1113/JP277769  

   Balderas, Enrique ; Torres, Natalia S. ; Rosa‐Garrido, Manuel ; Chaudhuri, Dipayan ; Toro, Ligia ; Stefani, Enrico ; Olcese, Riccardo ( July 2019 , The Journal of Physiology)

   Association of plasma membrane BK_Cachannels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BK_Cachannel (mitoBK_Ca) by BK‐β subunits is not established.

   MitoBK_Ca‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.

   A large fraction of mitoBK_Cadisplay properties similar to that of plasma membrane BK_Cawhen associated with BK‐β1 (left‐shifted voltage dependence of activation,V_1/2 = −55 mV, 12 µmmatrix Ca²⁺).

   In BK‐β1 knockout mice, cardiac mitoBK_Cadisplayed a lowP_oand a depolarizedV_1/2of activation (+47 mV at 12 µmmatrix Ca²⁺)

   Co‐expression of BK_Cawith the BK‐β1 subunit in HeLa cells doubled the density of BK_Cain mitochondria.

   The present study supports the view that the cardiac mitoBK_Cachannel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBK_Cashapes mitochondrial Ca²⁺handling.

   Association of the plasma membrane BK_Cachannel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBK_Ca) with regulatory subunits is unknown. We report that mitoBK_Cafunctionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBK_Cais a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K⁺of 300 pS. Additionally, mitoBK_Cadisplays a high open probability (P_o) and voltage half‐activation (V_1/2 = −55 mV,n = 7) resembling that of plasma membrane BK_Cawhen associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BK_Ca‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBK_Cacurrents (five patches with mitoBK_Caactivity out of 28 total patches fromn = 5 different hearts), displaying a depolarizedV_1/2of activation (+47 mV in 12 µmmatrix Ca²⁺). The reduced activity of mitoBK_Cawas accompanied by a high expression of BK_Catranscript in the BK‐β1 KO, suggesting a lower abundance of mitoBK_Cachannels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BK_Cathat specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca²⁺overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBK_Caassociates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBK_Cachannel that helps to maintain mitochondrial Ca²⁺homeostasis.

    

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5. A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

   https://doi.org/10.1113/JP279917  

   Ferreira, Juan J. ; Pequera, Germán ; Launikonis, Bradley S. ; Ríos, Eduardo ; Brum, Gustavo ( October 2020 , The Journal of Physiology)

    Accumulation of inorganic phosphate (P_i) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P_iinto SR are fully established.

    We showed that P_ipromoted Ca²⁺release at concentrations below 10 mmand decreased it at higher concentrations. This decrease correlated well with that of [Ca²⁺]_SR.

    Pre‐treatment of permeabilized myofibres with 2 mmCl⁻channel blocker 9‐anthracenecarboxylic acid (9AC) inhibited both effects of P_i.

    The biphasic dependence of Ca²⁺release on [P_i] is explained by a direct effect of P_iacting on the SR Ca²⁺release channel, combined with the intra‐SR precipitation of Ca²⁺salts. The effects of 9AC demonstrate that P_ienters the SR via a Cl⁻pathway of an as‐yet‐undefined molecular nature.

   Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P_i). P_idiffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca²⁺salts, thus contributing to the impairment of Ca²⁺release. Information on the P_ientrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca²⁺spark frequency is correlated with the Ca²⁺load of the SR and can be used to monitor this variable. We studied the effects of P_ion Ca²⁺sparks in permeabilized fibres of the frog. Relative event frequency (f/f_ref) rose with increasing [P_i], reaching 2.54 ± 1.6 at 5 mm,and then decreased monotonically, reaching 0.09 ± 0.03 at [P_i] = 80 mm. Measurement of [Ca²⁺]_SRconfirmed a decrease correlated with spark frequency at high [P_i]. A large [Ca²⁺]_SRsurge was observed upon P_iremoval. Anion channels are a putative path for P_iinto the SR. We tested the effect of the chloride channel blocker 9‐anthracenecarboxylic acid (9AC) on P_ientrance. 9AC (400 µm)applied to the cytoplasm produced a non‐significant increase in spark frequency and reduced the P_ieffects on this parameter. Fibre treatment with 2 mm9AC in the presence of high cytoplasmic Mg²⁺suppressed the effects of P_ion [Ca²⁺]_SRand spark frequency up to 55 mm[P_i]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that P_iimpairs Ca²⁺release by accumulating and precipitating with Ca²⁺inside the SR, thus contributing to myogenic fatigue.

    

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