Figure 1
- Model of connexin (Cx32) topology. The molecule is
believed to span the bilayer four times (M1-M4) and to have both
N- and C-termini (NT, CT) at the cytoplasmic side of the
membrane, forming two extracellular loops (E1, E2) and one inner
loop (IL). Two connexin regions are conserved: one spans
approximately the first 100 residues, comprising NT, E1, M1, M2
and the beginning of IL; the other contains M3, M4, E2 and the
beginning of CT. The two remaining regions, most of IL and CT,
vary in sequence and length.
Figure 2
- Model of staggered (one-to-two) interaction between
opposite connexins (12). Based on our present understanding that
M3 lines the channel and both E1 and E2 interact with homologous
domains across the gap, this model places E1 and E2 radially
arranged around the channel with their axes at ~30o
angle from each other. In this model, opposite connexins do not
bind one-to-one but are staggered with each other, such that each
connexin of one membrane interacts with two connexins of the
adjoined membrane. There are two possible configurations of the
staggered model: in one (shown here), both E's would have the
same N-to-C sequence orientation, centrifugal with respect to the
channel, and in the other (see Ref. 12), only E2 would have this
orientation.
Figure 3
- Time course of changes in normalized pCai (A), pHi (B) and junctional conductance (Gj, A and B) in Xenopus oocyte pairs exposed to 100% CO2 for 3 min. pCai and pHi were measured at the oocyte periphery with fura-C18 (a membrane-associated Ca2+ indicator) and BCECF, respectively. Gj was measured by double voltage clamp electrophysiology. Before CO2 exposure, the oocytes had a pCai of 6.66 ± 0.17 (mean ± SD; N = 25) and pHi of 7.63 ± 0.115 (N = 18). With CO2, pCai dropped to 6.37 ± 0.263 (N = 25) at a maximum rate of ~23%/min (A). pCai minima were reached within 8-10 min and pCai recovered to normal or slightly higher than normal values within ~15 min. In contrast, pHi dropped to 6.54 ± 0.113 (N = 18) at a maximum rate of ~34%/min (B). pHi minima were reached within ~4 min and pHi recovered to normal or slightly higher than normal values within ~10 min. The time course of pHi contrasted sharply with that of Gj, which dropped at a maximum rate of ~25%/min and was lowest 8-10 min from the beginning of the CO2 treatment (A and B), whereas the time course of Gj was very close to that of pCai during uncoupling. pCai minima preceded only slightly Gj minima, but pCai recovered at a faster rate (A). From Ref. 28, with permission.
Figure 4
- Effect of arachidonic acid (AA) on electrical coupling studied in Novikoff cell pairs by double whole-cell clamp electrophysiology. The cytosol was buffered for Ca2+ through the pipette solution with either BAPTA or EGTA. The uncoupling effect of AA (20 µM, 20 s) depends on [Ca2+]i buffering. EGTA at concentrations as high as 2 mM was totally ineffective in inhibiting uncoupling by AA (A). In contrast, BAPTA caused a 20% inhibition at concentrations as low as 0.1 mM and completely eliminated the uncoupling effects of AA at 1-2 mM concentrations (B). Indeed, BAPTA is known to be a faster and more efficient intracellular Ca2+ buffer than EGTA. EGTA inhibited uncoupling by ~40% and ~80% at 5 and 10 mM concentrations, respectively. From Ref. 25, with permission.
Figure 5
- Junctional sensitivity to CO2, expressed as normalized junctional conductance (Gj/Gjmax; 100% = control, pretreatment value), in oocyte pairs expressing Cx32, Cx38 or Cx32/38 chimeras (74,75). With Cx38, a 3-min exposure to CO2 decreased Gj to nearly 0%, whereas with Cx32, even a 15-min CO2 treatment decreased Gj by only ~55%. Two chimeras, Cx32/38I (inner loop of Cx32 replaced by that of Cx38) and Cx32/38I2 (second half of inner loop, IL2, of Cx32 replaced by that of Cx38), reproduced the uncoupling efficiency of Cx38. This indicates that IL2 plays an important role in pH gating sensitivity. The N-terminal domain does not appear to be relevant because the chimera Cx32/38N (Cx32 with NT of Cx38) behaved similarly to Cx32 (see Ref. 74).
Figure 6
- Decrease in junctional conductance (Gj)
in Xenopus oocyte pairs, expressing wild-type Cx32 or Cx32
deleted of most of the C-terminus, with exposure to 100% CO2 for either 15 min (A and B) or 3 min
(B). Note that deletion of the C-terminus by over 80% (D225,
D222, D219) did not affect CO2
sensitivity. With 3 min CO2, Gj dropped to 82 ± 8%, 91 ± 7% and 90
± 3% (mean ± SEM) with D225, D222 and D219, respectively, and
with 15 min CO2, to 53.5 ± 10%,
65 ± 11% and 53 ± 7% with D225, D222 and D219, respectively.
Figure 7
- Summary of the effects of partial or total
replacement of arginine (R) residue with asparagine (N) or
threonine (T) residues, in the initial domain (C1)
of the C-terminus chain, on normalized Gj
(Gj/Gj max;
100% = control, pretreatment value), following 3-min or 15-min
exposure to CO2. Note that
replacement of all of the 5 R with N or T residues greatly
increased the CO2 sensitivity of
Cx32, whereas partial R/N replacement resulted in intermediate CO2 sensitivities. This
indicates that the R residues differ in their ability to inhibit
the CO2 sensitivity of Cx32.
R215 appears to have greater inhibitory power than R219-220. In
contrast, R223-224 seems to partly counteract the inhibitory
activity of both R215 and R219-220, because 2R/N and 1R/N were
more sensitive to 15-min exposure to CO2
than 4R/N and 3R/N#1, respectively.
Figure 8
- Model of potential electrostatic interactions among
three cytoplasmic domains (IL1,
IL2 and C1)
of Cx32, displayed in alpha-helical conformation. In view of the
fact that 1) IL2 and C1 are positively charged, 2) the
inhibitory action of C1 depends
on its positive charges, and 3) the only cytoplasmic domain with
negative charges is IL1 (not
considering some acidic residues of the C-terminal domain that
can be deleted without gating consequences), we propose that open
and closed channel states depend on charge interactions among IL1, IL2
and C1. In coupled conditions
the negative charges of IL1
would be unavailable for interaction, whereas with CO2 conformational changes would expose
them, enabling IL2 and C1 to competitively interact with IL1. IL1-IL2 interaction would result in closed
channel, whereas IL1-C1 interaction would maintain the
channel open.
Figure 9
- Gap junction channels can be homotypic (made of two
connexons expressing the same connexin) (a) or heterotypic (made
of two connexons each expressing a different connexin) (b).
Similarly, connexons can be homomeric (made of the same connexin)
(a and b) or heteromeric (composed of different connexins) (c and
d). Therefore, cell-cell channels can be homomeric-homotypic (a),
homomeric-heterotypic (b), monoheteromeric (one connexon
heteromeric and the other homomeric) (c), or biheteromeric (both
connexons heteromeric) (d).
Figure 10
- Sensitivity to CO2
presented as normalized junctional conductance (Gj/Gj max; 100% = control, pretreatment
value) in oocyte pairs expressing heteromeric or heterotypic
channels. Pairs in which one oocyte expressed a 50/50 mixture of
Cx32 and 5R/N mutant (mixed) and the other either Cx32 (32)
or 5R/N (R/N) were less sensitive to CO2
than 32-32 and R/N-R/N pairs, respectively. Their
sensitivity is consistent with the idea that in heteromeric
hemichannels (mixed) gating is impaired and suggests that
gating may require connexin cooperativity. In contrast, the
sensitivity of heterotypic channels (32-R/N) was close to
that theoretically predicted, indicating that the two
hemichannels of a cell-cell channel are likely to gate
independently from each other.
