A Transient Diffusion Model Yields Unitary Gap Junctional Permeabilities from Images of Cell-to-Cell Fluorescent Dye Transfer Between Xenopus Oocytes

Abstract

As ubiquitous conduits for intercellular transport and communication, gap junctional pores have been the subject of numerous investigations aimed at elucidating the molecular mechanisms underlying permeability and selectivity. Dye transfer studies provide a broadly useful means of detecting coupling and assessing these properties. However, given evidence for selective permeability of gap junctions and some anomalous correlations between junctional electrical conductance and dye permeability by passive diffusion, the need exists to give such studies a more quantitative basis. This article develops a detailed diffusion model describing experiments (reported separately) involving transport of fluorescent dye from a "donor" region to an "acceptor" region within a pair of Xenopus oocytes coupled by gap junctions. Analysis of transport within a single oocyte is used to determine the diffusion and binding characteristics of the cellular cytoplasm. Subsequent double-cell calculations then yield the intercellular junction permeability, which is translated into a single-channel permeability using concomitant measurements of intercellular conductance, and known single-channel conductances of gap junctions made up of specific connexins, to count channels. The preceding strategy, combined with use of a graded size series of Alexa dyes, permits a determination of absolute values of gap junctional permeability as a function of dye size and connexin type. Interpretation of the results in terms of pore theory suggests significant levels of dye-pore affinity consistent with the expected order of magnitude of typical (e.g., van der Waals) intermolecular attractions.

Gap junctions favor normal rat kidney epithelial cell adaptation to chronic hypertonicity

“…Gap junctions could allow the passage, between cells, of small molecules [Ͻ1 kDa, (69)] like ATP or cAMP by passive diffusion (52). Organic osmolytes are good candidates to be transported from cell to cell as they are neutral and small (molecular masses of sorbitol, taurine, myo-inositol, and betaine Ͻ200 Da).…”

Heteromeric, but Not Homomeric, Connexin Channels Are Selectively Permeable to Inositol Phosphates

“…Hemichannels are either homomeric (composed of a single connexin isoform) or heteromeric (more than one isoform). Dramatic and surprising degrees of ionic and molecular permselectivity have been observed for homomeric channels (5)(6)(7)(8)(9)(10)(11)(12)(13). However, most cells express more than one connexin, and heteromeric connexin channels are common in vivo (14 -18).…”

Spatial fluorescence cross‐correlation spectroscopy by means of a spatial light modulator

“…Although our results are rather on a yes or not basis, we showed, for the first time, at a single molecule level, the influence of the permeability upon the sFCCS data, by comparing hydrophobic molecules (R6G) that do quickly pass a lipidic membrane and hydrophilic ones (SRG), that do not. One may think about applying the sFCCS technique to cell membranes, such as the nuclear envelope [38] or the membrane connecting neighbouring cells [39]. However, the average permeability of the membranes, that depends upon the area density of transporters (or pores) spanning the membrane (~50 pores / µm 2 ), the structure of the pores (~10 nm in diameter and 40 nm long) and the size of the molecular permeant, is probably too low (< 100 µm/s) to be assessable by sFCCS [see e.g.…”

“…However, the average permeability of the membranes, that depends upon the area density of transporters (or pores) spanning the membrane (~50 pores / µm 2 ), the structure of the pores (~10 nm in diameter and 40 nm long) and the size of the molecular permeant, is probably too low (< 100 µm/s) to be assessable by sFCCS [see e.g. [38][39][40]. In contrast, the nuclear envelope breakdown occurring during cell division [38] is a situation where the sFCCS technique should be appropriate to measure the density and permeability of disassembled pores.…”