Several domain swap studies showed that the single channel conductance of connexin pores is a property that can be transferred between channels by exchange of M1, particularly its second half (Cx46, Cx37, Cx32; [38
]). Other domain swap studies showed that the charge selectivity of connexin pores can be controlled by E1 (Cx46, Cx32; [40
]), suggesting that E1 contributes to the pore wall. Point mutations in the NT produced changes in the single channel current-voltage relations consistent with electrostatic effects on the permeating ions (Cx32; [32
]). Mutations at two positions in the NT of Cx40 showed that they were essential for spermine block of these channels (the block thought to be at the cytoplasmic vestibule; [36
]). Taken together, these data suggest that the NT, the second half of M1 and at least the initial part of E1 are directly involved in defining the conductance properties of connexin pores.
Involvement of the second half of M1 received experimental support from SCAM studies utilizing two types of thiol-reactive reagents, at both the macroscopic and single channel levels of analysis, carried out on single connexons. Studies using the large thiol reagent MBB (maleimidobutyryl biocytin) identified two sites of reaction in the second half of M1 (Cx46, Cx32; [41
]). Similar studies of M3 were inconclusive due to smaller effects.
A set of MTS (methanethiosulfonate) reagents, which are much smaller than MBB, applied to single connexons in excised patches reacted very rapidly at a series of sites in the second half of M1, extending up to the M1/E1 border (Cx46; [42
]). Modification by MTS reagents of different charge altered the single channel current-voltage relations in a manner that suggested direct electrostatic interaction with the current-carrying ions. No evidence of modification was found for sites in the second half of M3.
In contrast to these findings, measurements of macroscopic current with application of MBB to junctional channels in a cut-open paired oocyte preparation implicated M3 (Cx32; [43
]), originally suggested by hydrophobicity analysis of connexin sequences. All four transmembrane domains were tested for accessibility to MBB. A series of reactive sites separated by two to three amino acids were identified in M3. Several sites in M1 were also reactive, but they were viewed as accessible in the closed but not the open state.
The different implications for pore lining segments would be easily resolved if one could attribute them to differences between the specific connexins studied and/or the fact that one set of data is from single connexons and the other from junctional channels. Unfortunately, these simple explanations do not seem to apply. There are two phylogenetic groups of connexins, with Cx26 and Cx32, members of one group and Cx43 a member of the other [44
]. While there must be some structural differences to account for different limiting pore diameters and charge selectivities, it would be truly remarkable if the fundamental organization and packing of the transmembrane helices were different. More to the point, the transmembrane densities derived from cryo-EM of the M34A mutant of Cx26 [9
] are virtually identical to those derived from cryo-EM of Cx43 [7
By the same token, there must be some differences in the pore-lining structures between unpaired connexons and connexons in junctional channels, simply by virtue of the docking interactions at the extracellular end of the connexons. Again, it would be remarkable if this resulted in wholesale differences in transmembrane packing. In fact, a host of data from measurements of unitary conductances, voltage sensitivities, pharmacological sensitivities and other functional properties of single connexons and junctional channels suggest that this does not occur [20
]. Since the differences cannot be readily explained by the considerations above, they may arise from some combination of the differences in the thiol-reactive reagents used, the different physical configurations of the experiments, and the relative reliabilities, sources of artifact and constraints inherent in the two experimental protocols. Simply put, these different experiments may be revealing different kinds of information about the channels.
In most SCAM studies, the thiol-modifying reagent is presumed to have free access to the molecule of interest. Therefore the rate of modification is considered to be a function of the molecular accessibility of the reagent to the specific group modified. Accessibility can be a function of steric impediment (e.g., the residue is buried deep in the protein interior) and/or a function of the structural states occupied by the target molecule during exposure to the reagent (e.g., how much time a channel is in an open versus closed state). Thus, if a channel is open 90% of the time during incubation with an MTS reagent, one would expect the positions most “accessible” (i.e., most rapidly modified) to MTS modification to be those exposed to the pore lumen when the channel is open, as opposed to those uniquely exposed when closed.
These considerations raise two potential concerns about the SCAM data from the paired oocytes. One is the long time (20 minutes) of exposure to MBB. It is unclear how much of this time was required for diffusion of the large MBB reagent to the junctional molecules through residual oocyte cytoplasmic components. If the delay of action can be thus accounted for, it is not a concern. However, if it takes minutes for modification after reaching the junctions, there is concern that the reactive sites are not sufficiently accessible for the results to be specific for exposed (i.e., pore-lining) residues. The other concern is about the relatively small change in macroscopic currents as a result of modification. On a single channel level, one expects that modification of Cx32 with MBB within the pore will substantially decrease unitary conductance (it decreases Cx46 conductance 80%; Pfahnl and Dahl [46
]). However, the effects on the currents in the oocyte system were much smaller (15–20%). This can mean that either the modification is occurring far enough outside the pore that the MBB only slightly occludes it, or that only a small fraction of the channels are being modified, as if a large fraction of the channels are inaccessible to the reagent. While each of these concerns may be satisfactorily explained, at present they remain unresolved, and stand in contrast to the rapid and dramatic effects seen with the MTS reagents at the single channel level of resolution.