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1. 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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2. Using Simulation to Understand the Role of Titration on the Stability of a Peptide–Lipid Bilayer Complex

   https://doi.org/10.1021/acs.langmuir.0c02038  

   Burns, Violetta ; Mertz, Blake ( September 2020 , Langmuir)

   The pH-low insertion peptide (pHLIP) is an anionic membrane-active peptide with promising potential for applications in imaging of cancer tumors and targeted delivery of chemotherapeutics. The key advantage of pHLIP lies in its acid sensitivity: in acidic cellular environments, pHLIP can insert unidirectionally into the plasma membrane. Partitioning–folding coupling is triggered by titration of the acidic residues in pHLIP, transforming pHLIP from a hydrophilic to a hydrophobic peptide. Despite this knowledge, the reverse pathway that leads to exit of the peptide from the plasma membrane is poorly understood. Our hypothesis is that sequential deprotonation of pHLIP is a prerequisite for exit of the peptide from the plasma membrane. We carried out molecular dynamics (MD) simulations to characterize the effect that deprotonation of the acidic residues of pHLIP has on the stability of the peptide when inserted into a model lipid bilayer of 1-palmitoyl-2-oleoyl-sn-3-phosphocholine (POPC). Initiation of the exit mechanism is facilitated by a complex relationship between the peptide, bulk solvent, and the membrane environment. As the N-terminal acidic residues of pHLIP are deprotonated, localized loss of helicity drives unfolding of the peptide and more pronounced interactions with the bilayer at the lipid–water interface. Deprotonation of the C-terminal acidic residues (D25, D31, D33, and E34) leads to further loss of secondary structure distal from the C-terminus, as well as formation of a water channel that stabilizes the orientation of pHLIP parallel to the membrane normal. Together, these results help explain how stabilization of intermediates between the surface-bound and inserted states of pHLIP occur and provide insights into rational design of pHLIP variants with modified abilities of insertion. 

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3. Screening Technologies for Inward Rectifier Potassium Channels: Discovery of New Blockers and Activators

   https://doi.org/10.1177/2472555220905558  

   Walsh, Kenneth B. ( April 2020 , SLAS DISCOVERY: Advancing the Science of Drug Discovery)

   K⁺channels play a critical role in maintaining the normal electrical activity of excitable cells by setting the cell resting membrane potential and by determining the shape and duration of the action potential. In nonexcitable cells, K⁺channels establish electrochemical gradients necessary for maintaining salt and volume homeostasis of body fluids. Inward rectifier K⁺(Kir) channels typically conduct larger inward currents than outward currents, resulting in an inwardly rectifying current versus voltage relationship. This property of inward rectification results from the voltage-dependent block of the channels by intracellular polyvalent cations and makes these channels uniquely designed for maintaining the resting potential near the K⁺equilibrium potential (E_K). The Kir family of channels consist of seven subfamilies of channels (Kir1.x through Kir7.x) that include the classic inward rectifier (Kir2.x) channel, the G-protein-gated inward rectifier K⁺(GIRK) (Kir3.x), and the adenosine triphosphate (ATP)-sensitive (K_ATP) (Kir 6.x) channels as well as the renal Kir1.1 (ROMK), Kir4.1, and Kir7.1 channels. These channels not only function to regulate electrical/electrolyte transport activity, but also serve as effector molecules for G-protein-coupled receptors (GPCRs) and as molecular sensors for cell metabolism. Of significance, Kir channels represent promising pharmacological targets for treating a number of clinical conditions, including cardiac arrhythmias, anxiety, chronic pain, and hypertension. This review provides a brief background on the structure, function, and pharmacology of Kir channels and then focuses on describing and evaluating current high-throughput screening (HTS) technologies, such as membrane potential-sensitive fluorescent dye assays, ion flux measurements, and automated patch clamp systems used for Kir channel drug discovery.

    

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4. Heterozygous Deletion of Epilepsy Gene KCNQ2 Has Negligible Effects on Learning and Memory

   https://doi.org/10.3389/fnbeh.2022.930216  

   Tracy, Gregory C. ; Wilton, Angelina R. ; Rhodes, Justin S. ; Chung, Hee Jung ( July 2022 , Frontiers in Behavioral Neuroscience)

   Neuronal K_v7/Potassium Voltage-Gated Channel Subfamily Q (KCNQ) potassium channels underlie M-current that potently suppresses repetitive and burst firing of action potentials (APs). They are mostly heterotetramers of K_v7.2 and K_v7.3 subunits in the hippocampus and cortex, the brain regions important for cognition and behavior. Underscoring their critical roles in inhibiting neuronal excitability, autosomal dominantly inherited mutations in Potassium Voltage-Gated Channel Subfamily Q Member 2 (KCNQ2) and Potassium Voltage-Gated Channel Subfamily Q Member 3 (KCNQ3) genes are associated with benign familial neonatal epilepsy (BFNE) in which most seizures spontaneously remit within months without cognitive deficits.De novomutations inKCNQ2also cause epileptic encephalopathy (EE), which is characterized by persistent seizures that are often drug refractory, neurodevelopmental delay, and intellectual disability. Heterozygous expression of EE variants ofKCNQ2is recently shown to induce spontaneous seizures and cognitive deficit in mice, although it is unclear whether this cognitive deficit is caused directly by K_v7 disruption or by persistent seizures in the developing brain as a consequence of K_v7 disruption. In this study, we examined the role of K_v7 channels in learning and memory by behavioral phenotyping of theKCNQ2^+/−mice, which lack a single copy ofKCNQ2but dos not display spontaneous seizures. We found that bothKCNQ2^+/−and wild-type (WT) mice showed comparable nociception in the tail-flick assay and fear-induced learning and memory during a passive inhibitory avoidance (IA) test and contextual fear conditioning (CFC). Both genotypes displayed similar object location and recognition memory. These findings together provide evidence that heterozygous loss ofKCNQ2has minimal effects on learning or memory in mice in the absence of spontaneous seizures.

    

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5. Arabidopsis thaliana CYCLIC NUCLEOTIDE‐GATED CHANNEL2 mediates extracellular ATP signal transduction in root epidermis

   https://doi.org/10.1111/nph.17987  

   Wang, Limin ; Ning, Youzheng ; Sun, Jian ; Wilkins, Katie A. ; Matthus, Elsa ; McNelly, Rose E. ; Dark, Adeeba ; Rubio, Lourdes ; Moeder, Wolfgang ; Yoshioka, Keiko ; et al ( February 2022 , New Phytologist)

   Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca²⁺]_cyt) increase as a second messenger. The downstream PM Ca²⁺channels remain enigmatic. Here, theArabidopsis thalianaCa²⁺channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca²⁺]_cytsignalling in roots.

   Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca²⁺]_cytwere measured with aequorin, and root transcriptional changes were determined by quantitative real‐time PCR. Twocngc2loss‐of‐function mutants were used:cngc2‐3anddefence not death1(which expresses cytosolic aequorin).

   Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca²⁺dependent, requiring CNGC2 but not CNGC4 (its channel co‐subunit in immunity signalling). Activation of PM Ca²⁺influx currents also required CNGC2. The eATP‐induced [Ca²⁺]_cytincrease and transcriptional response incngc2roots were significantly impaired.

   CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca²⁺influx, causing depolarization leading to [Ca²⁺]_cytincrease and damage‐related transcriptional response.

    

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