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First published online 29 July 2008
doi: 10.1242/jcs.032482

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An intact connexin N-terminus is required for function but not gap junction formation

¹ Department of Medicine, Section of Cardiology, University of Chicago, Chicago, IL 60637, USA
² Department of Pediatrics, Section of Hematology/Oncology, University of Chicago, Chicago, IL 60637, USA

View larger version (38K): [in this window] [in a new window]   Fig. 1. Diagram showing the topology of the connexin polypeptide relative to the membrane (top) and the amino acid sequences of the N-terminal domains of wild-type CX37 and the deletion or substitution mutants (bottom). NT, amino terminal domain; CL, cytoplasmic loop; CT, C-terminal domain; TM1-TM4, transmembrane domains 1-4; E1 and E2, extracellular loops.

View larger version (42K): [in this window] [in a new window]   Fig. 2. Fluorescence microscopic localization of wild-type CX37 (A) and deletion mutants (B-I) after transient transfection of HeLa cells with GFP-labeled constructs. Wild-type CX37 (A), 5-21 (E), 2-8 (F), 9-21 (G), 2-13 (H) and 14-21 (I) each frequently showed localization to appositional membranes consistent with formation of gap junction plaques. (B) 2-21 was always localized in the cytoplasm, and no gap junction plaques were ever detected. 4-21 most commonly was found in the cytoplasm (C) and rarely between cells, in small gap junction plaques (D). Scale bar: 20 µm for A-D,G,H; 25 µm for E,F,I.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Intercellular transfer of microinjected gap junction permeant tracers in transfected HeLa cells. (A, B) Photomicrographs obtained after microinjection of a solution containing Lucifer yellow (yellow) and neurobiotin (red) into HeLa cells expressing wild-type CX37 (A) or 2-8 (B). Neither cells expressing CX37 nor those expressing 2-8 showed significant intercellular transfer of Lucifer yellow beyond the injected cell (indicated by asterisk). Cells expressing wild-type CX37 displayed extensive transfer of neurobiotin, whereas those expressing 2-8 did not allow significant intercellular passage of this tracer. Scale bar: 30 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Wild-type CX37 produced large hemichannel currents in Xenopus oocytes, but 2-21 did not produce currents nor inhibited wild-type currents. (A-D) Hemichannel current traces from control oocytes (A), or oocytes expressing wild-type CX37 (B), 2-21 (C) or wild-type CX37 and 2-21 (D) elicited in zero divalent OR2 by a series of voltage steps held for 1 second from a holding potential of –40 mV in 10 mV steps. Control currents shown in A were obtained from an oocyte that was injected with 2 ng antisense oligonucleotide to endogenous connexin CX38 only. Although an oocyte injected with 10 ng cRNA encoding wild-type CX37 showed hemichannel currents (B), no currents were observed above background in oocytes injected with 10 ng 2-21 cRNA alone (C). Oocytes injected with 10 ng wild-type CX37 cRNA and 10 ng 2-21 cRNA showed hemichannel currents similar in magnitude to the currents observed in oocytes injected with wild-type CX37 cRNA. (E) Current-voltage relationships obtained from control oocytes (x), or oocytes injected with wild-type CX37 (), 2-21 (), or a 1:1 mixture of 2-21 and CX37 (). Data are shown as mean ± s.e.m.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Mutants 2-8 and 9-21 did not produce conducting hemichannels, but coexpression of either one with wild-type CX37 inhibited currents produced from wild-type CX37. (A) Current-voltage relationships for control oocytes (x), wild-type CX37 (), 2-8 () and differing ratios of 2-8 coexpressed with wild-type CX37 (, 1:1; , 0.2:1; , 0.05:1). (B) Current-voltage relationships for wild-type CX37 (), 9-21 (), and differing ratios of 9-21 coexpressed with wild-type CX37 (, 1:1; , 0.2:1; , 0.1:1). (C,D) Graphs of mole fraction vs fractional current for 2-8 coexpressed with wild-type CX37 (C) and for 9-21 coexpressed with wild-type CX37 (D). Curves represent the theoretical values for inhibition by one (lower) or two (upper) mutant subunits within a hemichannel. Both 2-8 and 9-21 fit closely to the curve for inhibition of hemichannel function by one mutant subunit. Data are shown as mean ± s.e.m.

View larger version (35K): [in this window] [in a new window]   Fig. 6. A mutant containing alanine residues substituted for the amino acids at positions 2-8 formed plaques in transfected HeLa cells, did not form functional hemichannels but inhibited wild-type CX37 function. (A) Fluorescence microscopic localization of 2-8Ala in HeLa cells after transient transfection. Scale bar: 20 µm. (B) Current-voltage relationships for control oocytes (x), wild-type CX37 (), 2-8Ala () and 2-8Ala coexpressed with wild-type CX37 at a 1:1 ratio (). Data are shown as mean ± s.e.m.

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