We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker heart. hMSCs transfected with cardiac gene, mHCN2, by electroporation expressed high levels Cs + -sensitive current (31.1±3.8 pA/pF at −150 mV) activating in diastolic potential range reversal −37.5±1.0 mV, confirming as I f -like. The responded isoproterenol an 11-mV positive shift activation. Acetylcholine had no direct effect, but presence isoproterenol, shifted activation 15 mV negative....

The purpose of this study was to determine whether oligonucleotides the size siRNA are permeable gap junctions and a specific for DNA polymerase beta (pol beta) can move from one cell another via junctions, thus allowing inhibit gene expression in directly. To test hypothesis, fluorescently labelled (morpholinos) 12, 16 24 nucleotides length were synthesized introduced into pair using patch pipette. These probes moved through composed connexin 43 (Cx43). Moreover, rate transfer declined with...

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be cellular repair or delivery system provided that they communicate target such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The patterns Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited coupling each other, HeLa transfected Cx40, acutely isolated...

After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged neuroscience, allowing for precise perturbations neural circuits by light. However, functionality optogenetic tools has not been fully explored outside nonviral, nonembryogenesis-based strategy optogenetics shown before.We demonstrate utility to cardiac muscle tandem cell unit (TCU) strategy, which nonexcitable cells carry exogenous channels, and, when...

Gap junctions formed between transfected cells expressing connexin (Cx) 40 and Cx43 (Cx43-RIN, Cx40-HeLa, Cx43-HeLa) revealed a relationship, g(j)=f(V(j)), at steady state, that is typified by nonsymmetrical behavior similar to previously reported for other heterotypic channels (gap junction conductance [g(j)]; transjunctional voltage [V(j)]). The unitary of the was sensitive polarity V(j). A main state 61 pS found when cell stepped positively or Cx40 negatively (V(j)=70 mV); reverse...

Several proteins including connexin40 (Cx40) and connexin43 (Cx43) form gap junctions between cells of the heart; they may be found separately or coexpressed. These connexins channels with differing conductance permeability properties. Cx40 Cx43 are each required for normal electrical conduction in different regions heart. We hypothesized that major difference these might their selective intercellular passage small molecules such as second messengers, which can assessed using biologically...

Human HeLa cells transfected with mouse Cx45 and rat RIN chicken were used to study the electrical permeability properties of gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels in both cell lines. Multichannel showed a time-dependent activation or deactivation sensitive voltage, Vm. These did not occur nontransfected cells. The hemichannel inhibited by raising Ca2+ acidification CO2. unitary conductance exhibited Vm...

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined interaction Cx43 and Cx40 in HeLa sequentially stably transfected with these two connexins. Immunoblots verified production comparable amounts both connexins, cross-linking showed that formed oligomers, immunofluorescence extensive...

Abstract Myocytes from neonatal rat hearts were used to assess the conductive properties of gap junction channels by means dual voltage-clamp method. The experiments carried out on three types (groups) preparations: (1) induced cell pairs, (2) preformed pairs with few (1 3 channels), and (3) many (100 200 channels) after treatment uncoupling agents such as SKF-525A (75 μmol/L), heptanol (3 mmol/L), arachidonic acid μmol/L). In group 1, first opening a newly formed channel was slow (20 65 ms)...

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination cyclic nucleotide (cAMP) permeability through gap composed Cx43, Cx40, or Cx26 using simultaneous measurements junctional conductance transfer cAMP. For cAMP detection recipient cells were transfected with a reporter gene, nucleotide-modulated channel from sea urchin sperm (SpIH). was...

Mutations in GJB2 and GJB6, the genes that encode human gap junction proteins connexin26 (Cx26) connexin30 (Cx30), respectively, cause hearing loss. Cx26 Cx30 are both expressed cochlea, leading to potential formation of heteromeric hemichannels heterotypic channels. To investigate their interactions, we individually or together HeLa cells. When they were together, appeared interact directly (by colocalization plaques, by coimmunoprecipitation, fluorescence resonance energy transfer)....

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the homotypic connexin26 (Cx26), Cx40, Cx43, Cx45 gap stably expressed in HeLa cells with size net charge. Channel was determined using simultaneous measurements junctional conductance cell-cell flux fluorescent probe. All four allowed passage both cationic anionic probes, but transfer rates...

Mutations in the GJB2 gene (Cx26) cause deafness humans. Most are loss-of-function mutations and nonsyndromic deafness. Some produce a gain of function syndromic associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification poorly understood. We created an animal model for...

This study investigated whether chondrocytes within the cartilage matrix have capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels.

Many cells contain two (or more) gap junction proteins that are able to oligomerize with each other form heteromeric channels and influence the properties of intercellular communication. Cx26 Cx43 found together in a number cell types, but previous data have suggested they might not connexons. We studied possible interactions these connexins by co-expression three different lines. Analysis N2aCx26/Cx43 pairs double whole-cell patch-clamp methods showed were coupled, contained only small...

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim this study was to investigate if chondrocytes from human cartilage express gap junction proteins called connexins (Cxs). We show that in tissue Cx43, Cx45, Cx32, Cx46. also find primary adults retain capacity form functional voltage-dependent junctions. Immunohistochemistry experiments OA patients revealed significantly elevated levels Cx43 Cx45 superficial zone down...

Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression Cx43 increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) not enhanced HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts control but showed...

1 Human HeLa cells expressing mouse connexin30 (Cx30) were used to study the electrical properties of Cx30 gap junction channels. Experiments performed on cell pairs with dual voltage-clamp method. 2 The conductance (gj) at steady state showed a bell-shaped dependence junctional voltage (Vj; Boltzmann fit: Vj,0= 27 mV, gj,min= 0.15, z= 4). instantaneous gj decreased slightly increasing Vj. 3 currents (Ij) declined time following single exponential. constants Ij inactivation (τi) 4 Single...

Abstract The growth of many types cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed suppress the neighboring transformed Direct physical contact between and was required for suppression in system; communication diffusible factors sufficient. significant gap junctional required, indicating that other intercellular junctions mediated regulatory...

We examined whether coupling of a ventricular myocyte to non‐myocyte cell expressing HCN2 could create two‐cell syncytium capable generating sustained pacing. Three types were transfected with the mHCN2 gene and used as sources mHCN2‐induced currents. They human mesenchymal stem cells HEK293 cells, both which express connexin43 (Cx43), HeLa Cx43. Cell–cell between heterologous pairs increased time in co‐culture, hyperpolarization induced currents, indicating current transfer from...

In glands such as the liver and pancreas, gap junctions containing connexin 26 32 (Cx26 Cx32, respectively) couple secretory cells. Uncoupling these compromises function of glands. Lacrimal also contain extensive arrays consisting Cx26 Cx32. We wanted to determine role in fluid secretion. Cx32-deficient mice, immunocytochemistry showed that, male lacrimal gland, remaining was found evenly distributed membrane whereas there little membranes female Western blot analysis that mice expressed...

Cellular delivery of small interfering RNAs to target cells a tissue has the potential travel by two intercellular pathways. For intimately apposed gap junctions allow transport exclusive extracellular space. not in intimate contact, exocytotic release vesicular contents and subsequent retrieval via endocytosis exosomes other represent an alternative system that utilizes Previous studies have shown siRNA/miRNA transfer from cell junction channels. We hypothesized siRNA can be delivered...