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1.
《FEBS letters》2014,588(8):1430-1438
Intracellular Ca2+ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca2+]i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca2+-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies. 相似文献
2.
Chen Y Zhou Y Lin X Wong HC Xu Q Jiang J Wang S Lurtz MM Louis CF Veenstra RD Yang JJ 《The Biochemical journal》2011,435(3):711-722
Cx50 (connexin50), a member of the α-family of gap junction proteins expressed in the lens of the eye, has been shown to be essential for normal lens development. In the present study, we identified a CaMBD [CaM (calmodulin)-binding domain] (residues 141-166) in the intracellular loop of Cx50. Elevations in intracellular Ca2+ concentration effected a 95% decline in gj (junctional conductance) of Cx50 in N2a cells that is likely to be mediated by CaM, because inclusion of the CaM inhibitor calmidazolium prevented this Ca2+-dependent decrease in gj. The direct involvement of the Cx50 CaMBD in this Ca2+/CaM-dependent regulation was demonstrated further by the inclusion of a synthetic peptide encompassing the CaMBD in both whole-cell patch pipettes, which effectively prevented the intracellular Ca2+-dependent decline in gj. Biophysical studies using NMR and fluorescence spectroscopy reveal further that the peptide stoichiometrically binds to Ca2+/CaM with an affinity of ~5 nM. The binding of the peptide expanded the Ca2+-sensing range of CaM by increasing the Ca2+ affinity of the C-lobe of CaM, while decreasing the Ca2+ affinity of the N-lobe of CaM. Overall, these results demonstrate that the binding of Ca2+/CaM to the intracellular loop of Cx50 is critical for mediating the Ca2+-dependent inhibition of Cx50 gap junctions in the lens of the eye. 相似文献
3.
Gap junction channels formed by connexin50 (Cx50) are critical for maintenance of eye lens transparency. Cleavage of the carboxyl terminus (CT) of Cx50 to produce truncated Cx50 (Cx50trunc) occurred naturally during maturation of lens fiber cells. The mechanism of its altered properties is under confirmation. It has been suggested that calmodulin (CaM) participates in gating some kinds of gap junction. Here, we performed confocal colocalization and co-immunoprecipitation experiments to study the relationships between Cx50 and CaM. Results exhibited that the CaM could colocalize Ca2+ dependently with CT in the linear area of cell-to-cell contact formed by Cx50trunc, while it could not localize in the linear area without expression of CT. Further study indicated that the CT could interact Ca2+ independently with the cytoplasmic loop (CL) of Cx50. These data put forward the importance of Ca2+-independent intramolecular interaction between CT and CL of Cx50, which mediate the Ca2+-dependent binding of CaM to Cx50. These intra- and intermolecular interactions may further improve our understanding of biological significance of the Cx50 in the eye lens. 相似文献
4.
Identification of the calmodulin binding domain of connexin 43 总被引:2,自引:0,他引:2
Zhou Y Yang W Lurtz MM Ye Y Huang Y Lee HW Chen Y Louis CF Yang JJ 《The Journal of biological chemistry》2007,282(48):35005-35017
Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions. 相似文献
5.
The mechanisms regulating the permeability of lens epithelial cell gap junctions in response to calcium ionophore or ATP agonist-mediated increases in cytosolic Ca2+ (Cai2+) have been investigated using inhibitors of calmodulin (CaM) and PKC. Cell-to-cell transfer of the fluorescent dye AlexaFluor594 decreased after the rapid and sustained increase in Cai2+ (to micromolar concentrations) observed after the addition of ionophore plus Ca2+ but was prevented by pretreatment with inhibitors of CaM but not PKC. In contrast, the delayed, transient decrease in cell-to-cell coupling observed after the addition of ATP that we have reported previously (Churchill G, Lurtz MM, and Louis CF. Am J Physiol Cell Physiol 281: C972-C981, 2001) could be prevented by either the direct or indirect inhibition of PKC but not by inhibition of CaM. Surprisingly, there was no change in the relative proportion of the different phosphorylated forms of lens connexin43 after this ATP-dependent transient decrease in cell-to-cell coupling. Although BAPTA-loaded cells did not display the ATP-dependent transient increase in Cai2+, the delayed, transient decrease in cell-to-cell dye transfer was still observed, indicating it was Cai2+ independent. Thus CaM-mediated inhibition of lens gap junctions is associated with sustained, micromolar Cai2+ concentrations, whereas PKC-mediated inhibition of lens gap junctions is associated with agonist activation of second messenger pathways that are independent of changes in Cai2+. calcium; connexin43; lens gap junctions 相似文献
6.
Xu Q Kopp RF Chen Y Yang JJ Roe MW Veenstra RD 《American journal of physiology. Cell physiology》2012,302(10):C1548-C1556
Calmodulin (CaM) binding sites were recently identified on the cytoplasmic loop (CL) of at least three α-subfamily connexins (Cx43, Cx44, Cx50), while Cx40 does not have this putative CaM binding domain. The purpose of this study was to examine the functional relevance of the putative Cx43 CaM binding site on the Ca(2+)-dependent regulation of gap junction proteins formed by Cx43 and Cx40. Dual whole cell patch-clamp experiments were performed on stable murine Neuro-2a cells expressing Cx43 or Cx40. Addition of ionomycin to increase external Ca(2+) influx reduced Cx43 gap junction conductance (G(j)) by 95%, while increasing cytosolic Ca(2+) concentration threefold. By contrast, Cx40 G(j) declined by <20%. The Ca(2+)-induced decline in Cx43 G(j) was prevented by pretreatment with calmidazolium or reversed by the addition of 10 mM EGTA to Ca(2+)-free extracellular solution, if Ca(2+) chelation was commenced before complete uncoupling, after which g(j) was only 60% recoverable. The Cx43 CL(136-158) mimetic peptide, but not the scrambled control peptide, or Ca(2+)/CaM-dependent kinase II 290-309 inhibitory peptide also prevented the Ca(2+)/CaM-dependent decline of Cx43 G(j). Cx43 gap junction channel open probability decreased to zero without reductions in the current amplitudes during external Ca(2+)/ionomycin perfusion. We conclude that Cx43 gap junctions are gated closed by a Ca(2+)/CaM-dependent mechanism involving the carboxyl-terminal quarter of the connexin CL domain. This study provides the first evidence of intrinsic differences in the Ca(2+) regulatory properties of Cx43 and Cx40. 相似文献
7.
Kurebayashi N Nishizawa H Nakazato Y Kurihara H Matsushita S Daida H Ogawa Y 《American journal of physiology. Cell physiology》2008,294(6):C1419-C1429
To investigate how intercellular coupling can be changed during Ca2+ overloading of ventricular muscle, we studied Ca2+ signals in individual cells and the histochemistry of the major gap junction channel, connexin43 (Cx43), using multicellular preparations. Papillary muscles were obtained from guinea pig ventricles and loaded with rhod-2. Sequential Ca2+ images of surface cells were obtained with a confocal microscope. In intact muscles, all cells showed simultaneous Ca2+ transients in response to field stimulation over a field of view of 0.3 x 0.3 mm2. In severely Ca2+-overloaded muscles, obtained by high-frequency stimulation in nonflowing Krebs solution, cells became less responsive to stimulation. Furthermore, nonsimultaneous but serial onsets of Ca2+ transients were often detected, suggesting a propagation delay of action potentials. The time lag of the onset between two aligned cells was sometimes as long as 100 ms. Similar lags were also observed in muscles with gap junction channels inhibited by heptanol. To investigate whether the phosphorylation state of Cx43 is affected in Ca2+-overloaded muscles, the distributions of phosphorylated and nonphosphorylated Cx43 were determined using specific antibodies. Most of the Cx43 was phosphorylated in the nonoverloaded muscles, whereas nonphosphorylated Cx43 was significantly elevated in severely Ca2+-overloaded muscles. Our results suggest that the propagation delay of action potential within a small area, a few square millimeters, can be a cause of abnormal conduction and a microreentry in Ca2+-overloaded heart. Inactivation of Na+ channels and inhibition of gap junctional communication may underlie the cell-to-cell propagation delay. Ca2+ transient; connexin43; propagation delay; gap junction; arrhythmia 相似文献
8.
Tudor Porumb A. Crivici Perry J. Blackshear M. Ikura 《European biophysics journal : EBJ》1997,25(4):239-247
The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct
family of protein ki-nase C (PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca2+ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling
between Ca2+-CaM signalling and PKC-mediated phosphorylation cascades. We have studied Ca2+ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD)
spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca2+ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified
MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced
with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions
that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled
to the Ca2+ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal
domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca2+ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously.
NMR analysis of the Ca2+-CaM-MRP peptide complex, as well as CD measurements of Ca2+-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast
to the α-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The
adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues
Lys-75 and Glu-82.
Received: 26 September 1996 / 22 October 1996 相似文献
9.
Kanwardeep S. Kaleka Amber N. Petersen Matthew A. Florence Nashaat Z. Gerges 《Journal of visualized experiments : JoVE》2012,(59)
Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM. 相似文献
10.
Thanutchaporn Kumrungsee Tomomi SaikiSayaka Akiyama Kentaro NakashimaMitsuru Tanaka Yutaro KobayashiToshiro Matsui 《Biochimica et Biophysica Acta (BBA)/General Subjects》2014
Background
Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca2 +-CaM complex formation, a CaMKII activator.Methods
The ability of Trp-His and other peptides to inhibit Ca2 +-CaM complex formation was investigated by a Ca2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.Results
We showed that Trp-His inhibited Ca2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca2 +-binding site of CaM by forming hydrogen bonds with key Ca2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.Conclusions
This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.General significance
The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca2 +-CaM complex formation in the future. 相似文献11.
12.
Martin González-Andrade Rogelio Rodríguez-Sotres Abraham Madariaga-Mazón José Rivera-Chávez Rachel Mata Alejandro Sosa-Peinado 《Journal of biomolecular structure & dynamics》2016,34(1):78-91
In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca2+-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the “open” and “closed” conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM’s inhibitors correlated well with available experimental data as the r2 obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca2+-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca2+-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca2+-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs. 相似文献
13.
Correll RN Pang C Niedowicz DM Satin J Andres DA 《Molecular and cellular biochemistry》2008,310(1-2):103-110
GTPases of the Ras-related RGK family are negative regulators of high voltage-activated (HVA) Ca2+ channel activity. In this study, we examined the role of calmodulin (CaM) association in Rem-mediated Ca2+ channel inhibition. We found that the Rem/CaM interaction is Ca2+-dependent, and that truncation of the Rem C-terminus before position 277 prevents CaM binding. Serial mutagenesis of the
Rem C-terminus between residues 265 and 276 to alanine generated two mutants (RemL271A and RemL274A) that displayed reduced CaM binding, and a subset of these mutants displayed significantly lower cell periphery localization
than RemWT. However, reductions in CaM association or membrane trafficking did not affect function, as all Rem mutants could completely
inhibit Ca2+ channels. The Rem1–275 truncation mutant partially inhibited Ca2+ channel activity despite its inability to bind CaM. Taken together, these studies indicate that CaM association is not essential
for either Rem-mediated Ca2+ channel inhibition or plasma membrane localization.
Jonathan Satin is an established investigator of the American Heart Association. 相似文献
14.
Xu Wang Liang Wen Xiong Amina El Ayadi Darren Boehning John A. Putkey 《The Journal of biological chemistry》2013,288(3):2040-2048
PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca2+ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca2+ binding activity, which may modulate Ca2+ binding to CaM by stabilizing an initial Ca2+-CaM complex or by electrostatically steering Ca2+ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca2+ dynamics, we tested the hypothesis that it influences ligand-dependent Ca2+ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca2+ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca2+ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca2+ binding to CaM greatly inhibit its effect on ATP-induced Ca2+ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca2+ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling. 相似文献
15.
Calmodulin (CaM) is a highly conserved calcium-binding protein consisting of two homologous domains, each of which contains two EF-hands, that is known to bind well over 300 proteins and peptides. In most cases the (Ca2+)4-form of CaM leads to the activation of a key regulatory enzyme or protein in a myriad of biological processes. Using the nitroxide spin-labeling reagent, 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl, bovine brain CaM was modified at 2–3 methionines with retention of activity as judged by the activation of cyclic nucleotide phosphodiesterase. X-band electron paramagnetic resonance (EPR) spectroscopy was used to measure the spectral changes upon addition of Ca2+ to the apo-form of spin-labeled protein. A significant loss of spectral intensity, arising primarily from reductions in the heights of the low, intermediate, and high field peaks, accompanied Ca2+ binding. The midpoint of the Ca2+-mediated transition determined by EPR occurred at a higher Ca2+ concentration than that measured with circular dichroic spectroscopy and enzyme activation. Recent data have indicated that the transition from the apo-state of CaM to the fully saturated form, [(Ca2+)4-CaM], contains a compact intermediate corresponding to [(Ca2+)2-CaM], and the present results suggest that the spin probes are reporting on Ca2+ binding to the last two sites in the N-terminal domain, i.e. for the [(Ca2+)2-CaM] → [(Ca2+)4-CaM] transition in which the compact structure becomes more extended. EPR of CaM, spin-labeled at methionines, offers a different approach for studying Ca2+-mediated conformational changes and may emerge as a useful technique for monitoring interactions with target proteins. 相似文献
16.
Atrial Fibrillation-Linked Germline GJA5/Connexin40 Mutants Showed an Increased Hemichannel Function
Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca2+]o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation. 相似文献
17.
In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis. 相似文献
18.
Caitlin Hyland Michael Mfarej Giorgos Hiotis Sabrina Lancaster Noelle Novak M. Kathryn Iovine Matthias M. Falk 《Molecular biology of the cell》2021,32(20)
Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of Cxs of only 1–5 h, resulting in constant endocytosis and biosynthetic replacement of gap junction channels, has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256–289), a region known to encode key residues regulating gap junction turnover, is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and a zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication. Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques, and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that continuous Cx43 gap junction endocytosis is an essential aspect of gap junction function and, when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function. 相似文献
19.
Gap junction channels formed by connexin50 (Cx50) are critical for the maintenance of eye lens transparency, which is sensitive to pH and external Ca2+ concentration, but the mechanism is still not clear. In this study we performed dye uptake-leakage assays, patch clamping and confocal co-localization experiments to confirm the function of calmodulin (CaM) and Ca2+ in the Cx50 hemichannel. Below pH 7.4, lucifer yellow (LY)-preloaded Cx50-HeLa cells allow dye to leak out when washed with Ca2+-free solution or incubated in solution containing 50 microg/ml W7 (CaM inhibitor) first, then washed in solution containing 2 mM Ca2+, whereas little or no dye leakage was observed when LY-preloaded Cx50-HeLa cells were incubated in solution containing 2 mM Ca2+. Moreover, in the absence of Ca2+, polarizing pulses applied to Cx50-HeLa activated outward transmembrane currents, which were inhibited by 2 mM external Ca2+. When Cx50-HeLa cells were incubated with 2 mM Ca2+ and 50 microg/ml W7, the transmembrane currents were activated again. This indicates that Ca2+ and CaM play a gating role in Cx50 hemichannels. Either the chelation of Ca2+ or the inhibition of CaM increased the permeability of Cx50 hemichannels. The same phenomena were observed below pH 6.5. Furthermore, CaM could be localized in gap junctions formed by Cx50 below pH 6.5. Our results demonstrate that CaM and Ca2+ can cooperate in the gating control of Cx50 hemichannels. 相似文献
20.
Francesco Zonta Fabio Mammano Mauro Torsello Nicola Fortunati Laura Orian Antonino Polimeno 《Biochemical and biophysical research communications》2014
Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca2+ concentration ([Ca2+]e). It is well established that hemichannel activity is inhibited at normal (∼1 mM) [Ca2+]e, whereas lowering [Ca2+]e to micromolar levels fosters hemichannel opening. Atomic force microscopy imaging shows significant and reversible changes of pore diameter at the extracellular mouth of Cx26 hemichannels exposed to different [Ca2+]e, however, the underlying molecular mechanisms are not fully elucidated. Analysis of the crystal structure of connexin 26 (Cx26) gap junction channels, corroborated by molecular dynamics (MD) simulations, suggests that several negatively charged amino acids create a favorable environment for low-affinity Ca2+ binding within the extracellular vestibule of the Cx26 hemichannel. In particular a highly conserved glutammic acid, found in position 47 in most connexins, is thought to undergo post translational gamma carboxylation (γGlu47), and is thus likely to play an important role in Ca2+ coordination. γGlu47 may also form salt bridges with two conserved arginines (Arg75 and Arg184 in Cx26), which are considered important in stabilizing the structure of the extracellular region. 相似文献