Nature Methods
2, 12 - 14 (2005)
doi:10.1038/nmeth0105-12
Illuminating gap junctionsDavid C SprayDavid C. Spray is in the Departments of Neuroscience and Medicine (Cardiology) at the Albert Einstein College of Medicine, Bronx, New York 10461, USA. spray@accom.yu.edu An imaging method termed LAMP, based on a newly described photo-activatable fluorophore, promises to illuminate how gap junctions mediate intercellular coupling.Gap junction−mediated intercellular coupling is required both for rapid signaling between electrically excitable cells and for slower spread of intercellular second messenger signals between other cell types. It is thus critical to identify factors that determine the strength of such coupling and the impact of pathological conditions on coupling strength. Li et al.1 present a new method, termed local activation of a molecular fluorescent probe (LAMP), by which intercellular diffusion of a gap junction−permeant dye may be quantified noninvasively. This could fulfill a gap junctionologist's dream, potentially enabling measurement of coupling strength and gap junction distribution in vivo in response to physiological and pathological stimuli and perhaps also providing a high-throughput method by which new gap junction−altering drugs could be more rapidly discovered and evaluated.
Since the gap junction was discovered 45 years ago, numerous methods have been developed to determine the effects of physiological and pathological stimuli on diffusion of ions and small molecules through gap junction channels. Electrophysiology has evolved from microelectrode measurements of voltage produced by current injection to whole-cell voltage clamp recordings allowing single-channel analysis. Measurements of the extent and the patterns of dye coupling were advanced by the synthesis of the gap junction−permeant, brightly fluorescent and aldehyde-fixable dye Lucifer yellow2 and by techniques that allowed uptake of dye into cells (through 'scrape-loading'3 to introduce membrane-impermeant dye at a wound edge or into tissue4, and with esterified fluorophores that are membrane-permeant and are trapped inside the cell after esterase action). An additional advance is the use of fluorescence recovery after photobleaching (FRAP), in which fluorophores in a uniformly labeled population are locally photobleached and recovery is quantified as unbleached dye equilibrates through gap junctions3. Calcium-sensitive dyes now permit observation of second messenger transfer between cells in real time, after focal stimulation of one cell or localized photorelease of caged inositol 1,4,5-trisphosphate (IP3; ref. 5), which is gap junction−permeant and liberates calcium to report its diffusion. Finally, the spread of death signals through gap junctions ('bystander cell killing') that has been extended to beneficial tasks such as tumor killing and transfer of essential metabolites (the 'kiss of life' or 'good Samaritan' methods) may ultimately provide a means to rescue cells otherwise destined to die6.
LAMP capitalizes on the best features of several of these methods. An esterified, nonfluorescent coumarin derivative diffuses into cells, where it remains nonfluorescent but is trapped by esterase cleavage. Brief illumination of a single cell or cell region efficiently photoconverts the trapped molecule into a fluorescent, gap junction−permeant species, and fluorescence is followed as a function of time in the coupled neighbor. The high efficiency of photoconversion underlies the noninvasiveness of the method, as it requires relatively low illumination. Because the short excitation and emission wavelengths of the fluorophore do not overlap with those of calcium indicators such as Fluo3, spectral separation allows imaging of dye spread as well as of calcium changes.
Li et al.1 have applied this method to one of the most longstanding questions in the gap junction field: whether intracellular calcium concentration can be high enough to cause gap junction channels to close (for citations and divergent views, see refs. 7 and 8). The reason for the persistent debate is that most studies demonstrating gap junction closure have shown that coupling is lost (and structure is altered) only when very high calcium levels (>>10  M) are reached. Further, the discovery that both calcium and IP3 could diffuse through gap junction channels (thereby conducting calcium waves) seemed to indicate that these second messengers could communicate information between coupled cells, rather than blocking such communication. Finally, there are recent reports that calcium- and calmodulin-dependent kinase activation due to calcium elevation may actually lead to sustained increase in coupling strength in neurons9 and in astrocytes10.
This study reports that elevation of intracellular calcium above 1 M through ionophore treatment or agonist activation is not sufficient to cause gap junction channel closure. However, exposure of cells to thapsigargin, an agent that opens and depletes intracellular calcium stores and thereby activates store-operated calcium channels (SOCs), does cause gap junction channel closure, even though global cell calcium rises even less than with the other treatments. The authors conclude that such SOCs may generate very high levels of calcium in close proximity to the gap junction, thereby achieving gap junction channel closure. Of course, it remains to be proven that the gap junction blocker in these experiments is calcium itself, as any messenger activated by calcium entry could mediate the effect.
At a time when identifying the proteins that bind to gap junctions has become a focus of many laboratories, this paper raises the question of whether there is a high local density of such calcium entry sites near gap junction channels, perhaps forming part of a signaling complex (Fig. 1) involving gap junctions. For example, it is noteworthy that Trpc4, a candidate SOC in astrocytes, colocalizes and is coprecipitated with connexin43, a protein subunit of gap junctions, possibly tethered at connexin43−containing gap junctions through linkage via the scaffolding protein zonula occudins 1 (ref. 11).
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LAMP as demonstrated is limited to examination of changes in coupling in individual cell pairs, although it is likely also to be useful in multiwell plate assays with confluent cell monolayers. Moreover, combined use of patch clamp would make it possible to correlate junctional conductance with permeability in cell pairs expressing different connexins, although cell volumes would still need to be taken into account. Finally, if longer wavelength probes are generated that are better suited for excitation and emission of photoactivatable dye within tissues, there is hope that coupling patterns and strength variations may be visualizable within organs in vivo.
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