Probing the Function of Connexin Channels in Primary Tissues

Meda, Paolo

doi:10.1006/meth.1999.0940

Cited by 20 publications

(25 citation statements)

References 68 publications

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“…The classic method of visualizing gap junction connectivity and function involves charging specific cells with a small (<1 kD) molecule such as a fluorophore or biotin derivative that is permeable to gap junctions of various compositions (30). Current techniques to introduce small molecules into cells are invasive (31,32), inefficient (6,33), or limited in the number of cells that can be studied at one time (34). We reasoned the PLE-CM enzyme-substrate system could provide an alternative method to these techniques by enabling noninvasive introduction of a dye in specific cells and real-time visualization of gap junction connectivity in a cellular network.…”

Section: Resultsmentioning

confidence: 99%

Selective esterase–ester pair for targeting small molecules with cellular specificity

Tian

Yang

Wysocki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

165

212

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Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations. cellular imaging | microscopy | enzyme substrates | fluorophores | pharmacological agents C hemical probes are essential tools in biology for measuring and manipulating cellular properties. Optimization of the structural and electronic features of small molecules allows the fine-tuning of molecular recognition specificity for a particular cellular target. Even with high molecular specificity, however, the application of small molecules in complex biological environments is frequently limited by poor cellular specificity. The inability to target small molecules, such as imaging or pharmacological agents, to defined cellular populations can confound the evaluation and control of discrete subsets of cells within a multicellular environment. A general and efficient strategy for cell-specific targeting, combining the molecular specificity of small molecules with the cellular specificity of genetics, would allow intracellular pathways in defined cell types to be selectively probed in complex tissues.An attractive approach for general cell-specific delivery of small molecules employs selective enzyme-substrate pairs. In this strategy, compounds are masked by attachment of a standard, disposable blocking group that is stable to native cellular enzymes, but labile to a specific exogenous enzyme. Expression of such a protein in a genetically defined cell population permits unmasking of the small molecule with cellular specificity. To be useful across experimental paradigms, such a system should utilize an enzyme that unmasks molecules with high efficiency, expresses in different cell types, and exhibits low cellular toxicity. The cognate masking group must be modular, synthetically efficient, and allow molecules to diffuse passively across the cellular membrane, while also exhibiting favorable solubility and stability in aqueous solution.To date only a few enzyme-substrate pairs have been used as targeted delivery systems for small molecules, and none meet all the criteria outlined above. Strategies employing enzymes encoded by common reporter genes (1) have found some success in targeting small molecules (2-4), ...

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Section: Resultsmentioning

confidence: 99%

Selective esterase–ester pair for targeting small molecules with cellular specificity

Tian

Yang

Wysocki

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

165

212

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“…Individual cells were microinjected for 10 min, using glass pipettes pulled to a resistance of 50-60 MΩ, as assessed in 150 mmol/l LiCl. The tip of the pipette contained the tracer under study, whereas the shaft was filled with 3 mol/l LiCl [28]. The following tracers were compared: 4% Lucifer Yellow (LY; net charge −2; mol.…”

Section: Methodsmentioning

confidence: 99%

“…All tracers were from Sigma-Aldrich (St Louis, MO, USA), except CF, which was from Eastman Kodak (Rochester, NY, USA), and were dissolved in 150 mmol/l LiCl buffered to pH 7.2 with 10 mmol/l HEPES. Following cell penetration, each tracer was iontophoretically injected for 10 min, by applying square pulses of 0.1 nA amplitude, 900 ms duration and 0.5 Hz frequency [12,28]. Pulses were positive for injection of EB and PI, and negative for injection of LY and CF.…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, experiments testing the cell exchange of radioactive precursors [11] and exogenous tracers [8,12,18] have suggested that metabolic coupling within pancreatic islets may be restricted to territories comprising only a few beta cells [8,11,15]. This apparent discrepancy may reflect the different sensitivity of the techniques used to evaluate coupling [28,29], the specific characteristics of the Cx36 channels [4,13,30,31] and/or an influence of the islet setting that could modulate these channels [5,10,13,23,32].…”

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confidence: 99%

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Beta cells preferentially exchange cationic molecules via connexin 36 gap junction channels

2007

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Aims/hypothesis Pancreatic beta cells are connected by gap junction channels made of connexin 36 (Cx36), which permit intercellular exchanges of current-carrying ions (ionic coupling) and other molecules (metabolic coupling). Previous studies have suggested that ionic coupling may extend to larger regions of pancreatic islets than metabolic coupling. The aim of the present study was to investigate whether this apparent discrepancy reflects a difference in the sensitivity of the techniques used to evaluate beta cell communication or a specific characteristic of the Cx36 channels themselves. Methods We microinjected several gap junction tracers, differing in size and charge, into individual insulin-producing cells and evaluated their intercellular exchange either within intact islets of control, knockout and transgenic mice featuring beta cells with various levels of Cx36, or in cultures of wildtype and Cx36-transfected MIN6 cells. Results We found that (1) Cx36 channels favour the exchange of cations and larger positively charged molecules between beta cells at the expense of anionic molecules; (2) this exchange occurs across sizable portions of pancreatic islets; and (3) during glibenclamide (known as glyburide in the USA and Canada) stimulation beta cell coupling increases to an extent that varies for different gap junction-permeant molecules. Conclusions/interpretationThe data show that beta cells are extensively coupled within pancreatic islets via exchanges of mostly positively charged molecules across Cx36 channels. These exchanges selectively increase during stimulation of insulin secretion. The identification of this permselectivity is expected to facilitate the identification of endogenous permeant molecules and of the mechanism whereby Cx36 signalling significantly contributes to the modulation of insulin secretion.

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“…Because gap junctions are subject to numerous regulatory steps at the posttranslational level, functional electrophysiology studies are needed to determine what open junctional paths exist for various types of ions during different stages of embryogenesis. That gap-junction gating and selectivity properties depend greatly on which connexin family members comprise the junction (Elfgang et al, 1995;Bruzzone et al, 1996b;Meda, 2000) further underscores the necessity for a comprehensive understanding of which connexins are expressed in which embryonic cells during patterning. Overlapping patterns of connexin expression are consistent with the presence of heteromeric or heterotypic gap junctions, which could possess complex gating properties not present in either connexin alone.…”

Section: Expression Of Gap Junction Genesmentioning

confidence: 99%

Early embryonic expression of ion channels and pumps in chick and Xenopus development

Rutenberg

Cheng²,

Levin³

2002

Developmental Dynamics

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An extensive body of literature implicates endogenous ion currents and standing voltage potential differences in the control of events during embryonic morphogenesis. Although the expression of ion channel and pump genes, which are responsible for ion flux, has been investigated in detail in nervous tissues, little data are available on the distribution and function of specific channels and pumps in early embryogenesis. To provide a necessary basis for the molecular understanding of the role of ion flux in development, we surveyed the expression of ion channel and pump mRNAs, as well as other genes that help to regulate membrane potential. Analysis in two species, chick and Xenopus, shows that several ion channel and pump mRNAs are present in specific and dynamic expression patterns in early embryos, well before the appearance of neurons. Examination of the distribution of maternal mRNAs reveals complex spatiotemporal subcellular localization patterns of transcripts in early blastomeres in Xenopus. Taken together, these data are consistent with an important role for ion flux in early embryonic morphogenesis; this survey characterizes candidate genes and provides information on likely embryonic contexts for their function, setting the stage for functional studies of the morphogenetic roles of ion transport.

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Probing the Function of Connexin Channels in Primary Tissues

Cited by 20 publications

References 68 publications

Selective esterase–ester pair for targeting small molecules with cellular specificity

Selective esterase–ester pair for targeting small molecules with cellular specificity

Beta cells preferentially exchange cationic molecules via connexin 36 gap junction channels

Early embryonic expression of ion channels and pumps in chick and Xenopus development

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