Connexin Specifi city of Second Messenger Permeation : Real Numbers At Last Andrew


Correspondence to a h a r r i s @ u m d n j . e d u The discovery many years ago that ions and cytoplasmic molecules could diffuse between cells via gap junction channels inspired excited speculation about the roles such intercellular communication could play in development, physiology, and pathology. This view was reinforced when it later emerged that there are many types of gap junction channels, with 20 functionally distinct vertebrate isoforms of the component protein (connexin), which can combine in homomeric and heteromeric channel structures that have distinct conductance, dye permeability, and gating properties. The unitary conductances range from 10 pS to 300 pS; their cation/ anion permeability ratios ( P K+ / P Cl – ) range from 8.0 to 0.8 and their permeabilities to fl uorescent tracers are highly disparate. None of these parameters correlate with each other ( Harris, 2001 ). What is all this variability good for? It seems unlikely to be all about electrical coupling; it makes more sense to think that the distinct pore/permeability properties are important and perhaps designed for the ability to defi ne, mediate, and dynamically modulate the vocabulary of intercellular molecular signals. In fact, evidence for the importance of gap junction as conduits of intercellular molecular signals is growing. In recent years it has been demonstrated, in mostly qualitative ways, that different types of connexin channels have different effective permeabilities to specifi c cytoplasmic molecules, and that the permeability differences among connexin channels cannot be readily inferred from other known functional features. This work has increased speculation that (a) the pores of connexin channels are fi ne tuned for selectivity among specifi c cytoplasmic molecules, and (b) the connexin channels expressed at a particular cellular or tissue location are functionally selected for the ability to mediate highly specifi c intercellular signaling (i.e., for permeability to a specifi c second messenger, or for a specifi c spectrum of permeabilities among a set of second messengers). Several studies have inferred or determined perchannel permeabilities to second messengers, but quantitative data directly comparing the permeabilities of different junctional connexin channels to a second messenger have been lacking. The paper by Kanaporis et al. in this issue (see p. 2 9 3 ) provides such information; it reports the relative and absolute per-channel permeabilities to cAMP of junctional channels formed by three different connexins (Cx26, Cx40, and Cx43). In addition, they provide, for each connexin studied, a quantitative relation between junctional conductance and cAMP permeability and between permeability to the dye Lucifer yellow (LY) and permeability to cAMP. This information permits junctional conductance or LY permeability to be used to quantitatively estimate the corresponding junctional permeability to cAMP (which of course is far more diffi cult to measure). In addition, the cAMP/K + and LY/K + permeability ratios are used to estimate the relative limiting diameters of the three types of channels, with results that tend to confi rm that there is real permeant-pore specifi city when it comes to permeation by second messengers. The experiments use a cyclic nucleotide – modulated channel (SpIH) as a sensor of cytoplasmic cAMP, which is used to report junctional cAMP fl ux from a donor cell that is whole-cell patched with a pipette containing a high concentration of cAMP. Junctional conductance is measured continuously during transfer, and the degradation and synthesis of cAMP are inhibited. The cAMP fl ux into the recipient cell is reported by the increasing magnitude of the SpIH tail currents. The linear portion of the rate of increase is expressed as a function of junctional conductance and as a function of the number of junctional channels. The results show that the per-channel permeability of Cx43 junctional channels to cAMP is 3.2 times higher than Cx26 channels and 5.2 times higher than Cx40 channels. These numbers are further analyzed to yield actual cAMP fl ux rates per channel and fl ux rates normalized to cation fl ux. These results are then compared with analogous data for LY fl ux, which show the same qualitative trend, but lower per-channel permeabilities than to cAMP. Measurements like this are diffi cult and prone to all sorts of confounding factors; both ends of the pore are inside of cells, so the health of the cells must be maintained while altering the levels of a signaling molecule that can be degraded (and synthesized) during the experiment, the junctional conductance must be Connexin Specifi city of Second Messenger Permeation: Real Numbers At Last

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@inproceedings{Harris2008ConnexinSC, title={Connexin Specifi city of Second Messenger Permeation : Real Numbers At Last Andrew}, author={L J Harris}, year={2008} }