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First published online May 24, 2006
doi: 10.1242/10.1242/jcs.02953

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N-terminal residues in Cx43 and Cx40 determine physiological properties of gap junction channels, but do not influence heteromeric assembly with each other or with Cx26

¹ Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, University of Chicago, Chicago MC4060, 5841 S. Maryland Ave, Chicago, IL 60637-1470, USA
² Department of Pharmacology, SUNY Upstate Medical University, Syracuse, NY 13210, USA

View larger version (21K): [in a new window]   Fig. 1. Immunoblot detection of Cx40 and Cx43 expression in untransfected and stably transfected HeLa and HEK cells. Whole-cell lysates (10 µg protein for HeLa cells, 20 µg protein for HEK cells in Fig. 1A; 10 µg protein for all samples in Fig. 1B) were resolved by SDS-PAGE, transferred to membranes and blotted with (A) anti-Cx40 or (B) anti-Cx43 antibodies. Migration of molecular weight markers is indicated to the left of the blots.

View larger version (55K): [in a new window]   Fig. 2. Immunofluorescent detection of the distributions of immunoreactive Cx40 (A) in stably transfected HeLa-Cx40E12S,E13G cells and Cx43 (B) in stably transfected HeLa-Cx43D12S,K13G cells. Bar, 20 µm.

View larger version (23K): [in a new window]   Fig. 3. Intercellular transfer of microinjected Lucifer Yellow between stably transfected (A) HeLa-Cx40E12S,E13G cells, (B) HeLa-Cx43D12S,K13G cells, and (C) HeLa-Cx40/Cx43D12S,K13G cells.

View larger version (44K): [in a new window]   Fig. 4. Voltage-dependent gating of wild-type (wt) and mutant connexin channels expressed in N2a (A-E) or in HeLa (F) cells. The relationships between normalized steady-state conductance (Gj) and transjunctional voltage (Vj) are shown for cell pairs expressing (A) wtCx40 (stably)-wtCx40 (stably), (B) wtCx43 (stably)-wtCx43 (stably), (C) Cx40E12S,E13G (transiently)-Cx40E12S,E13G (transiently), (D) Cx43D12S,K13G (transiently)-Cx43D12S,K13G (transiently), (E) the heterotypic combination Cx40E12S,E13G (transiently)-wtCx40 (stably), and (F) the heterotypic combination Cx43D12S,K13G (transiently)-wtCx43 (stably). The mathematical fits to the Gj-Vj relationships are described in Tables 2 and 3. All normalized data represent the mean values from 4-6 experiments.

View larger version (62K): [in a new window]   Fig. 5. Spermine blockade of gap junctional currents in N2a cells expressing wild-type (wt) or mutant Cx40. 2 mM spermine was applied unilaterally (in one pipet) during recording of a family of junctional currents. (A) Currents between a pair of cells (stably) expressing wtCx40 were blocked when Vj was positive (center). (B) Currents between a pair of cells (transiently) expressing Cx40E12S,E13G were not affected by 2 mM spermine. (C) Currents between a pair of cells co-expressing wtCx40 (stably) and Cx40E12S,E13G (transiently) were not affected by 2 mM spermine. (D) Currents between a pair of cells co-expressing wtCx40 (stably) and Cx43D12S,K13G (transiently) were slightly affected by 2 mM spermine.

View larger version (40K): [in a new window]   Fig. 6. Single gap junction channel events detected in whole-cell two-current recordings obtained from N2a cells expressing different connexins (during a 30-second Vj pulse of -40 mV): wild-type Cx40 (stably, top trace), wild-type Cx43 (stably, second trace), Cx40E12S,E13G (transiently, third trace), wild-type Cx43 (stably) co-expressed with wild-type Cx40 (transiently, fourth trace), and wild-type Cx43 (stably) co-expressed with Cx40E12S,E13G (transiently, fifth trace). Panels A and B show channel events on different time scales. Panel B shows a 2-seconds excerpt (indicated with a bar above each trace in panel A) displayed at an expanded time scale to illustrate single channel events more clearly.

View larger version (115K): [in a new window]   Fig. 7. Double-label immunolocalization of mutant and wild-type connexins. All panels show stably transfected cells, except M-O, where Cx32 was introduced transiently to make HeLa-Cx43D12S,K13G/Cx32 cells. Individual immunoreactive connexins are detected in the first two columns and appear green or red. Merged images are shown in the right panels where overlap appears yellow. Arrows indicate examples of non-overlapping staining for only a single connexin (long, thin arrows: green only; heavy arrows: red only). (A-C) Fluorescence micrographs of HeLa-Cx40/Cx43D12S,K13G cells showing immunoreactive Cx40 in green (A), Cx43 in red (B) and the overlap of both immunoreactivities (C). Wild-type Cx40 and Cx43D12S,K13G perfectly co-localize. (D-F) Fluorescence micrographs of HEK-Cx40E12S,E13G cells showing immunoreactive Cx40 in green (D), Cx43 in red (E) and the overlap of both immunoreactivities (F). Wild-type Cx43 and Cx40E12S,E13G perfectly co-localize. (H-I) Fluorescence micrographs of HeLa-Cx26/Cx43D12S,K13G cells showing immunoreactive Cx43 in green (G), Cx26 in red (H) and the merged image of both immunoreactivities (I). Wild-type Cx26 and Cx43D12S,K13G do not co-localize. (J-L) Immunofluorescence images of HEK-Cx32 cells showing immunoreactive Cx32 in green (J), Cx43 in red (K) and the merged image of both immunoreactivities (L). Wild-type Cx32 and Cx43 rarely co-localize. (M-O) Immunofluorescence images of HeLa-Cx43D12S,K13G/Cx32 cells showing immunoreactive Cx32 in green (M), Cx43 in red (N) and the merged image of both immunoreactivities (O). Wild-type Cx32 and Cx43D12S,K13G extensively co-localize. Bar, 8 µm for A-C, 6 µm for G-I, 5 µm for D-F and M-O, 4 µm for J-L.

View larger version (40K): [in a new window]   Fig. 8. Affinity purification of HA-tagged connexin and potentially associated proteins. The presence and abundance of Cx40, Cx43 or Cx26 were detected in the cell extract, washes and eluted material by immunoblotting using anti-connexin antibodies. Gels were loaded with 25 µg of protein (corresponding to 1/10 of the amount incubated with MicroBeads) in the lane labeled `Before column'; 1/10 of the fraction collected as a `Flow through', 1/5 of each of the `Washes'; and 1/10 of the `Eluate' (corresponding to 25 µg of protein applied to the purification column). (A) Purification of proteins from HeLa-Cx43 cells transfected with Cx40HA. (B) Purification of proteins from HeLa-Cx43D12S,K13G [Cx43 (mut)] cells transfected with Cx40HA. (C) Purification of proteins from HeLa-Cx26HA cells transfected with Cx43. (D) Purification of proteins from HeLa-Cx26HA cells transfected with Cx43D12S,K13G. (E) Purification of proteins from HeLa-Cx40 cells transfected with Cx26HA. (F) Purification of proteins from HeLa-Cx40E12S,E13G [Cx40 (mut)] cells transfected with Cx26HA.

View larger version (41K): [in a new window]   Fig. 9. Steady-state Gj-Vj relationships in pairs of cells co-expressing a wild-type and a mutant connexin. (A) N2a-Cx40 cells transfected with Cx40E12S,E13G. (B) N2a-Cx40 cells transfected with Cx43D12S,K13G. (C) N2a-Cx43 cells transfected with Cx40E12S,E13G. (D) N2a-Cx43 cells transfected with Cx43D12S,K13G. In all experiments, the first connexin was stably transfected, and the second was introduced transiently. The mathematical fits to the Gj-Vj relationships in panels A-C are provided in Tables 2 and 3.