Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.     

Large conductance Ca2+-activated K+ channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects

Large conductance calcium activated potassium channels (BK_Ca) are fundamental in the control of cellular excitability. Thus, compounds that activate BK_Ca channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BK_Ca channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BK_Ca channel α-subunit alone or α + β1-subunit complex.Channel activity was determined using a non-radioactive Rb⁺ efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb⁺ efflux both in cells expressing α-subunit alone or α + β1-subunits. Co-expression of the β1-subunit modified the Rb⁺ efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC₄₀ values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BK_Ca channel α + β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC₄₀ values when tested on the BK_Ca α + β1-subunit expressing cells compared to BK_Ca α-subunit expressing cells. Further, the E_max values for BKPr4, BKVV and BKH1 were lower in the BK_Ca channel α + β1-subunit expressing cells.In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BK_Ca channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles.     

Involvement of palmitate/Ca2 +(Sr2 +)-induced pore in the cycling of ions across the mitochondrial membrane

The palmitate/Ca^2 +-induced (Pal/Ca^2 +) pore, which is formed due to the unique feature of long-chain saturated fatty acids to bind Ca^2 + with high affinity, has been shown to play an important role in the physiology of mitochondria. The present study demonstrates that the efflux of Ca^2 + from rat liver mitochondria induced by ruthenium red, an inhibitor of the energy-dependent Ca^2 + influx, seems to be partly due to the opening of Pal/Ca^2 + pores. Exogenous Pal stimulates the efflux. Measurements of pH showed that the Ca^2 +-induced alkalization of the mitochondrial matrix increased in the presence of Pal. The influx of Ca^2 + (Sr^2 +) also induced an outflow of K⁺ followed by the reuptake of the ion by mitochondria. The outflow was not affected by a K⁺/H⁺ exchange blocker, and the reuptake was prevented by an ATP-dependent K⁺ channel inhibitor. It was also shown that the addition of Sr^2 + to mitochondria under hypotonic conditions was accompanied by reversible cyclic changes in the membrane potential, the concentrations of Sr^2 + and K⁺ and the respiratory rate. The cyclic changes were effectively suppressed by the inhibitors of Ca^2 +-dependent phospholipase A₂, and a new Sr^2 + cycle could only be initiated after the previous cycle was finished, indicating a refractory period in the mitochondrial sensitivity to Sr^2 +. All of the Ca^2 +- and Sr^2 +-induced effects were observed in the presence of cyclosporin A. This paper discusses a possible role of Pal/Ca^2 + pores in the maintenance of cell ion homeostasis.     

The use of transgenic mouse models to reveal the functions of Ca2 + buffer proteins in excitable cells

BackgroundCytosolic Ca^2 + buffers are members of the large family of Ca^2 +-binding proteins and are essential components of the Ca^2 + signaling toolkit implicated in the precise regulation of intracellular Ca^2 + signals. Their physiological role in excitable cells has been investigated in vivo by analyzing the phenotype of mice either lacking one of the Ca^2 + buffers or mice with ectopic expression.Scope of ReviewIn this review, results obtained with knockout mice for the three most prominent Ca^2 + buffers, parvalbumin, calbindin-D28k and calretinin are summarized.Major ConclusionsThe absence of Ca^2 + buffers in specific neuron subpopulations, and for parvalbumin additionally in fast-twitch muscles, leads to Ca^2 + buffer-specific changes in intracellular Ca^2 + signals. This affects the excitation–contraction cycle in parvalbumin-deficient muscles, and in Ca^2 + buffer-deficient neurons, properties associated with synaptic transmission (e.g. short-term modulation), excitability and network oscillations are altered. These findings have not only resulted in a better understanding of the physiological function of Ca^2 + buffers, but have revealed that the absence of Ca^2 + signaling toolkit components leads to protein-and neuron-specific adaptive/homeostatic changes that also include changes in neuron morphology (e.g. altered spine morphology, changes in mitochondria content) and network properties.General SignificanceThe complex phenotype of Ca^2 + buffer knockout mice arises from the direct effect of these proteins on Ca^2 + signaling and moreover from the homeostatic mechanisms induced in these mice. For a better mechanistic understanding of neurological diseases linked to disturbed/altered Ca^2 + signaling, a global view on Ca^2 + signaling is expected to lead to new avenues for specific therapies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.     

Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2 + to the second metal binding site

BackgroundFluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase–DNA covalent complex as a topoisomerase–fluoroquinolone–DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.MethodsWe conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg^2 +-, Mn^2 +-, or Ca^2 +-supported DNA cleavage activity of Escherichia coli Topo IV.ResultsIn the absence of any drug, 20–30 mM Mg^2 + was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn^2 + or Ca^2 + was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg^2 + concentrations where Topo IV alone could not efficiently cleave DNA.Conclusions and general significanceAt low Mg^2 + concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg^2 + binding to metal binding site B through the structural distortion in DNA. As Mg^2 + concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg^2 + at site B or inhibition the binding of Mg^2 + to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg^2 + binding.     

Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca2 +-activated K+ channel causing membrane hyperpolarization

TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca^2 + reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP₂, protons, and divalent ions Ca^2 + and Mg^2 +. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 min. Fluid flow stimulated TRPV5 and 6-mediated Ca^2 + entry and increased intracellular Ca^2 + concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La^3 +. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K⁺ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca^2 + signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca^2 + entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20 ng/ml, 48 h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10 μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca^2 + (EGTA; 1 mM) or intracellular Ca^2 + (BAPTA; 5 μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca^2 + influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca^2 + and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.     

Modulation of the conductance-voltage relationship of the BK Ca channel by mutations at the putative flexible interface between two RCK domains

Calcium-dependent gating of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K⁺ conductance (RCK) and the high-affinity Ca²⁺-binding site (the Ca²⁺-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic “assembly interface”. In this study, we tested the structural model of the other interface, the “flexible interface”, by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca²⁺ affinity and voltage sensitivity. Various mutations at Gly-803 shifted the channel's conductance-voltage curve either left or right over a 145-mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.     

Thapsigargin decreases the Na+- Ca2 + exchanger mediated Ca2 + entry in pig coronary artery smooth muscle

Na⁺- Ca^2 + exchanger (NCX) has been proposed to play a role in refilling the sarco/endoplasmic reticulum (SER) Ca^2 + pool along with the SER Ca^2 + pump (SERCA). Here, SERCA inhibitor thapsigargin was used to determine the effects of SER Ca^2 + depletion on NCX–SERCA interactions in smooth muscle cells cultured from pig coronary artery. The cells were Na⁺-loaded and then placed in either a Na⁺-containing or in a Na⁺-substituted solution. Subsequently, the difference in Ca^2 + entry between the two groups was examined and defined as the NCX mediated Ca^2 + entry. The NCX mediated Ca^2 + entry in the smooth muscle cells was monitored using two methods: Ca^2 +sensitive fluorescence dye Fluo-4 and radioactive Ca^2 +. Ca^2 +-entry was greater in the Na⁺-substituted cells than in the Na⁺-containing cells when measured by either method. This difference was established to be NCX-mediated as it was sensitive to the NCX inhibitors. Thapsigargin diminished the NCX mediated Ca^2 + entry as determined by either method. Immunofluorescence confocal microscopy was used to determine the co-localization of NCX1 and subsarcolemmal SERCA2 in the cells incubated in the Na⁺-substituted solution with or without thapsigargin. SER Ca^2 + depletion with thapsigargin increased the co-localization between NCX1 and the subsarcolemmal SERCA2. Thus, inhibition of SERCA2 leads to blockade of constant Ca^2 + entry through NCX1 and also increases proximity between NCX1 and SERCA2. This blockade of Ca^2 + entry may protect the cells against Ca^2 +-overload during ischemia–reperfusion when SERCA2 is known to be damaged.     

CaV1.3 L-type channels,maxiK Ca2 +-dependent K+ channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca^2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Ca_v1.3 L-type Ca²⁺ channels (Ca1.3^−/−) and maxiK Ca²⁺-dependent K⁺ channels (BK^−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca²⁺ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Ca_v1.3, the predominant L-type Ca^2 + channel in RPE cells, varied at different times of day. Ca_V1.3^−/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK^−/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK^−/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca^2 + channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and Ca_V1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.     

The polybasic lysine-rich domain of plasma membrane-resident STIM1 is essential for the modulation of store-operated divalent cation entry by extracellular calcium

STIM1 acts as an endoplasmic reticulum Ca^2 + sensor that communicates the filling state of the intracellular stores to the store-operated channels. In addition, STIM1 is expressed in the plasma membrane, with the Ca^2 + binding EF-hand motif facing the extracellular medium; however, its role sensing extracellular Ca^2 + concentrations in store-operated Ca^2 + entry (SOCE), as well as the underlying mechanism remains unclear. Here we report that divalent cation entry stimulated by thapsigargin (TG) is attenuated by extracellular Ca^2 + in a concentration-dependent manner. Expression of the Ca^2 +-binding defective STIM1(D76A) mutant did not alter the surface expression of STIM1 but abolishes the regulation of divalent cation entry by extracellular Ca^2 +. Orai1 and TRPC1 have been shown to play a major role in SOCE. Expression of the STIM1(D76A) mutant did not alter Orai1 phosphoserine content. TRPC1 silencing significantly attenuated TG-induced Mn^2 + entry. Expression of the STIM1(K684,685E) mutant impaired the association of plasma membrane STIM1 with TRPC1, as well as the regulation of TG-induced divalent cation entry by extracellular Ca^2 +, which suggests that TRPC1 might be involved in the regulation of divalent cation entry by extracellular Ca^2 + mediated by plasma membrane-resident STIM1. Expression of the STIM1(D76A) or STIM1(K684,685E) mutants reduced store-operated divalent cation entry and resulted in loss of dependence on the extracellular Ca^2 + concentration, providing evidence for a functional role of plasma membrane-resident STIM1 in the regulation of store-operated divalent cation entry, which at least involves the EF-hand motif and the C-terminal polybasic lysine-rich domain.     

Alterations in ryanodine receptors and related proteins in heart failure

Sarcoplasmic reticulum (SR) Ca^2 + release plays an essential role in mediating cardiac myocyte contraction. Depolarization of the plasma membrane results in influx of Ca^2 + through l-type Ca^2 + channels (LTCCs) that in turn triggers efflux of Ca^2 + from the SR through ryanodine receptor type-2 channels (RyR2). This process known as Ca^2 +-induced Ca^2 +release (CICR) occurs within the dyadic region, where the adjacent transverse (T)-tubules and SR membranes allow RyR2 clusters to release SR Ca^2 + following Ca^2 + influx through adjacent LTCCs. SR Ca^2 + released during systole binds to troponin-C and initiates actin–myosin cross-bridging, leading to muscle contraction. During diastole, the cytosolic Ca^2 + concentration is restored by the resequestration of Ca^2 + into the SR by SR/ER Ca^2 +-ATPase (SERCA2a) and by the extrusion of Ca^2 + via the Na⁺/Ca^2 +-exchanger (NCX1). This whole process, entitled excitation–contraction (EC) coupling, is highly coordinated and determines the force of contraction, providing a link between the electrical and mechanical activities of cardiac muscle. In response to heart failure (HF), the heart undergoes maladaptive changes that result in depressed intracellular Ca^2 + cycling and decreased SR Ca^2 + concentrations. As a result, the amplitude of CICR is reduced resulting in less force production during EC coupling. In this review, we discuss the specific proteins that alter the regulation of Ca^2 + during HF. In particular, we will focus on defects in RyR2-mediated SR Ca^2 + release. This article is part of a Special Issue entitled: Heart failure pathogenesis and emerging diagnostic and therapeutic interventions.     

TMC8 (EVER2) attenuates intracellular signaling by Zn2 + and Ca2 + and suppresses activation of Cl− currents

Eight paralogue members form the family of transmembrane channel-like (TMC) proteins that share considerable sequence homology to anoctamin 1 (Ano1, TMEM16A). Ano1 is a Ca^2 + activated Cl⁻ channel that is related to head and neck cancer, often caused by human papilloma virus (HPV) infection. Mutations in TMC 6 and 8 (EVER1, EVER2) cause epidermodysplasia verruciformis. This rare skin disease is characterized by abnormal susceptibility to HPV infection and cancer. We found that in contrast to Ano1 the common paralogues TMC4–TMC8 did not produce Ca^2 + activated Cl⁻ currents when expressed in HEK293 cells. On the contrary, TMC8 was found to be localized in the endoplasmic reticulum (ER), where it inhibited receptor mediated Ca^2 + release, activation of Ano1 and volume regulated LRRC8-related Cl⁻ currents. Zn^2 + is co-released from the ER together with Ca^2 + and thereby further augments Ca^2 + store release. Because TMC8 is required to lower cytosolic Zn^2 + concentrations by the Zn^2 + transporter ZnT-1, we hypothesize that HPV infections and cancer caused by mutations in TMC8 are related to upregulated Zn^2 +/Ca^2 + signaling and activation of Ano1.     

Molecular dynamics simulation of cation–phospholipid clustering in phospholipid bilayers: Possible role in stalk formation during membrane fusion

In this study, we performed all-atom long-timescale molecular dynamics simulations of phospholipid bilayers incorporating three different proportions of negatively charged lipids in the presence of K⁺, Mg^2 +, and Ca^2 + ions to systemically determine how membrane properties are affected by cations and lipid compositions. Our simulations revealed that the binding affinity of Ca^2 + ions with lipids is significantly stronger than that of K⁺ and Mg^2 + ions, regardless of the composition of the lipid bilayer. The binding of Ca^2 + ions to the lipids resulted in bilayers having smaller lateral areas, greater thicknesses, greater order, and slower rotation of their lipid head groups, relative to those of corresponding K⁺- and Mg^2 +-containing systems. The Ca^2 + ions bind preferentially to the phosphate groups of the lipids. The complexes formed between the cations and the lipids further assembled to form various multiple-cation-centered clusters in the presence of anionic lipids and at higher ionic strength—most notably for Ca^2 +. The formation of cation–lipid complexes and clusters dehydrated and neutralized the anionic lipids, creating a more-hydrophobic environment suitable for membrane aggregation. We propose that the formation of Ca^2 +–phospholipid clusters across apposed lipid bilayers can work as a “cation glue” to adhere apposed membranes together, providing an adequate configuration for stalk formation during membrane fusion.     

Mechanism of dopamine D2 receptor-induced Ca2 + release in PC-12 cells

Intracellular Ca^2 + levels are tightly regulated in the neuronal system. The loss of Ca^2 + homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca^2 + signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3 μM ATP caused an early Ca^2 + release followed by a delayed Ca^2 + release. The delayed Ca^2 + release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca^2 + release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca^2 + release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca^2 + release phenomenon. siRNA targeting of calcyon blocked the delayed Ca^2 + release, decreased ATP-evoked IP₃R-mediated Ca^2 + release, and impaired subsequent Ca^2 + oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP₃R-mediated Ca^2 + release in PC-12 cells. In schizophrenia, Ca^2 + dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca^2 + levels as well as dopamine signaling.     

Involvement of store-operated Ca2 + entry in activation of AMP-activated protein kinase and stimulation of glucose uptake by M3 muscarinic acetylcholine receptors in human neuroblastoma cells

G_q/11-coupled muscarinic acetylcholine receptors (mAChRs) belonging to M₁, M₃ and M₅ subtypes have been shown to activate the metabolic sensor AMP-activated protein kinase (AMPK) through Ca^2 +/calmodulin-dependent protein kinase kinase-β (CaMKKβ)-mediated phosphorylation at Thr172. However, the source of Ca^2 + required for this response has not been yet elucidated. Here, we investigated the involvement of store-operated Ca^2 + entry (SOCE) in AMPK activation by pharmacologically defined M₃ mAChRs in human SH-SY5Y neuroblastoma cells. In Ca^2 +-free medium the cholinergic agonist carbachol (CCh) caused a transient increase of phospho-Thr172 AMPK that rapidly ceased within 2 min. Conversely, in the presence of extracellular Ca^2 + CCh-induced AMPK phosphorylation lasted for at least 180 min. The SOCE modulator 2-aminoethoxydiphephenyl borate (2-APB), at a concentration (50 μM) that suppressed CCh-induced intracellular Ca^2 + ([Ca^2 +]_i) plateau, inhibited CCh-induced AMPK phosphorylation. CCh triggered the activation of the endoplasmic reticulum Ca^2 + sensor stromal interaction molecule (STIM) 1, as indicated by redistribution of STIM1 immunofluorescence into puncta, and promoted the association of STIM1 with the SOCE channel component Orai1. Cell depletion of STIM1 by siRNA treatment reduced both CCh-induced [Ca^2 +]_i plateau and AMPK activation. M₃ mAChRs increased glucose uptake and this response required extracellular Ca^2 + and was inhibited by 2-APB, STIM1 knockdown, CaMKKβ and AMPK inhibitors, and adenovirus infection with dominant negative AMPK. Thus, the study provides evidence that SOCE is required for sustained activation of AMPK and stimulation of downstream glucose uptake by M₃ mAChRs and suggests that SOCE is a critical process connecting M₃ mAChRs to the control of neuronal energy metabolism.     

Modeling squid axon K+ channel by a nucleation and growth kinetic mechanism

A kinetic model accounting for all salient features of the K⁺ channel of the squid giant axon, including the rising phase of the ON gating charge and the Cole-Moore effect, is provided. Upon accounting for a significant feature distinguishing K⁺, Na⁺ and Ca^2 + channels from channel-forming peptides modeled in our previous 2016 BBA paper, the nucleation-and-growth kinetic model developed therein is extended to simulate ON ionic and gating currents of the K⁺ channel of the squid giant axon at different depolarization potentials by the use of only two free parameters. K⁺ channel opening is considered to proceed by progressive aggregation of single subunits, while they are moving their gating charge outward under depolarizing conditions within their tetrameric structure; K⁺ channel closing proceeds in the opposite direction, by repolarization-induced disaggregation of subunits, accompanied by inward movement of their gating charge.