The Permeability of Gap Junction Channels to Probes of Different Size Is Dependent on Connexin Composition and Permeant-Pore Affinities

Abstract

Gap junctions have traditionally been characterized as nonspecific pores between cells passing molecules up to 1 kDa in molecular mass. Nonetheless, it has become increasingly evident that different members of the connexin (Cx) family mediate quite distinct physiological processes and are often not interchangeable. Consistent with this observation, differences in permeability to natural metabolites have been reported for different connexins, although the physical basis for selectivity has not been established. Comparative studies of different members of the connexin family have provided evidence for ionic charge selectivity, but surprisingly little is known about how connexin composition affects the size of the pore. We have employed a series of Alexa dyes, which share similar structural characteristics but range in size from molecular weight 350 to 760, to probe the permeabilities and size limits of different connexin channels expressed in Xenopus oocytes. Correlated dye transfer and electrical measurements on each cell pair, in conjunction with a three-dimensional mathematical model of dye diffusion in the oocyte system, allowed us to obtain single channel permeabilities for all three dyes in six homotypic and four heterotypic channels. Cx43 and Cx32 channels passed all three dyes with similar efficiency, whereas Cx26, Cx40, and Cx45 channels showed a significant drop-off in permeability with the largest dye. Cx37 channels only showed significant permeability for the smaller two dyes, but at two- to sixfold lower levels than other connexins tested. In the heterotypic cases studied (Cx26/Cx32 and Cx43/Cx37), permeability characteristics were found to resemble the more restrictive parental homotypic channel. The most surprising finding of the study was that the absolute permeabilities calculated for all gap junctional channels in this study are, with one exception, at least 2 orders of magnitude greater than predicted purely on the basis of hindered pore diffusion. Consequently, affinity between the probes and the pore creating an energetically favorable in-pore environment, which would elevate permeant concentration within the pore and hence the flux, is strongly implicated.

Structural Determinants and Proliferative Consequences of Connexin 37 Hemichannel Function in Insulinoma Cells

“…In contrast, like Cx43, Cx37 traffics to the cell surface with and without disulfide bond formation between ECL cysteines; however, unlike Cx43, Cx37 without ECL cysteines is unable to form obviously functional HCs. That Cx37 behaves differently from either Cx26 or Cx43 is perhaps not surprising given the many functional differences between these proteins (conductance, selectivity (20,34), HC function in normal [Ca 2ϩ ] o , potent growth suppression (7)), but certainly, our data and those of others show that structure-function behaviors of Cx37 are not readily predicted by structure-function relationships determined from other Cxs, even another ␣-Cx, Cx43 (33).…”

Structural and Functional Similarities of Calcium Homeostasis Modulator 1 (CALHM1) Ion Channel with Connexins, Pannexins, and Innexins*

“…The pore properties of connexins have been explored by imaging permeation of fluorescent dyes of different sizes (30,31). As an independent approach to evaluate the CALHM1 functional pore diameter, we measured uptake into CALHM1-expressing neuroblastoma N2a cells of different sized fluorescent molecules with the same electrical valence (Alexa488 (A488), Alexa594 (A594), and Alexa633 (A633)) that are structurally related as well as Lucifer Yellow (LY) ( Table 2).…”

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

“…Additional insight into the biologically relevant second messenger propagated via Cx43 in response to FGF2 may be gleaned from the permeability of gap junctions. Depending on the connexin subunits that make up a gap junction channel, gap junctions have varying size and charge permeability characteristics (Goldberg et al, 2004;Weber et al, 2004). Gap junctions formed by Cx43 have among the largest pore diameter, permitting the diffusion of molecules up to 1.2 kDa, with permeability preferences for molecules that are negatively charged (Weber et al, 2004;Harris, 2007).…”

“…Depending on the connexin subunits that make up a gap junction channel, gap junctions have varying size and charge permeability characteristics (Goldberg et al, 2004;Weber et al, 2004). Gap junctions formed by Cx43 have among the largest pore diameter, permitting the diffusion of molecules up to 1.2 kDa, with permeability preferences for molecules that are negatively charged (Weber et al, 2004;Harris, 2007). The second messengers produced in response to FGFR activation, IP3, DAG, calcium and arachidonic acid, are all small enough to theoretically pass through Cx43 channels.…”

“…The second messengers produced in response to FGFR activation, IP3, DAG, calcium and arachidonic acid, are all small enough to theoretically pass through Cx43 channels. Importantly, Cx45 gap junctions form channels with markedly reduced pore size, permitting the diffusion of molecules of up to ϳ0.3 kDa and exhibiting a preference for positively charged molecules (Veenstra et al, 1994(Veenstra et al, , 1995Weber et al, 2004). Previous work has shown that overexpression of chicken Cx45 in Cx43-expressing osteoblasts, results in a dramatic decrease in the chemical and electrical coupling of the resultant gap junctions, indicating that in a mixed Cx43/ Cx45 background the biophysical properties of Cx45 prevail (Koval et al, 1995;Li et al, 1999;Martinez et al, 2002).…”