Heteromeric connexons formed by the lens connexins, connexin43 and connexin56

Berthoud, Viviana M.; Montegna, Elisabeth A.; Atal, Namita; Aithal, Naga H.; Brink, Peter R.; Beyer, Eric C.

doi:10.1078/0171-9335-00132

Cited by 37 publications

(31 citation statements)

References 41 publications

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“…One protein was expressed stably, another one was introduced by transient transfection or both were expressed transiently. 72 hours following transfection, connexins and connexons were solubilized in 1%Triton X-100 [18]. Samples were centrifuged at 100,000g ave for 30 min, and the supernatant (containing solubilized connexons) was affinity purified using the μMACS HA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) [19].…”

Section: Affinity Purification Of Ha-tagged Proteinsmentioning

confidence: 99%

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

Gemel

Lin

Collins

et al. 2008

Biochemical and Biophysical Research Communications

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Since most cells in the heart co-express multiple connexins, we studied the possible heteromeric interactions between connexin30.2 and connexin40, connexin43 or connexin45 in transfected cells. Double label immunofluorescence microscopy showed that connexin30.2 extensively co-localized with each co-expressed connexin at appositional membranes. When Triton X-100 solubilized connexons were affinity purified from co-expressing cells, connexin30.2 was isolated together with connexin40, connexin43, or connexin45. Co-expression of connexin30.2 with connexin40, connexin43, or connexin45 did not significantly reduce total junctional conductance. Gap junction channels in cells co-expressing connexin30.2 with connexin43 or connexin45 exhibited voltagedependent gating intermediate between that of either connexin alone. In contrast, connexin30.2 dominated the voltage dependence when co-expressed with connexin40. Our data suggest that connexin30.2 can form heteromers with the other cardiac connexins and that mixed channel formation will influence the gating properties of gap junctions in cardiac regions that co-express these connexins. Keywords Gap Junction; Intercellular Communication; ConnexinCurrent passage between cells in the heart is facilitated by intercellular channels clustered in gap junctions. These channels are formed by docking of two hemichannels (connexons) provided by the opposing cells. Each connexon is a hexamer of subunit proteins called connexins (Cx). As demonstrated in cells expressing individual connexins, each connexin can form channels by itself (homomeric/homotypic channels), and different connexins form channels with different conductance, permeability and gating properties [1]. In cells coexpressing different connexins, heteromeric channels (containing different connexins in the same connexon) can potentially be formed [2].Many studies have shown the presence of three well characterized connexin proteins in the heart, Cx40, Cx43, and Cx45. Cx43 is most abundant in atrial and ventricular myocytes and in Purkinje fibers [3,4]. Cx40 is expressed in atrial myocardium, atrioventricular bundle and ✉Address correspondence to: Eric C. Beyer, MD PhD, University of Chicago, Department of Pediatrics MC4060, 5841 S. Maryland Ave, Chicago, IL 60637-1470, TEL: 773-834-1498.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [5,6]. Cx45 is detected in sinoatrial and atrioventricular nodes, atrioventricular bundle and branches [7,8]. NIH Public AccessRecently, another cardiac connexin, mouse Cx30.2 (human Cx31.9) was discovered [9][10][11]. Cx30.2 expression was ...

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Section: Affinity Purification Of Ha-tagged Proteinsmentioning

confidence: 99%

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

Gemel

Lin

Collins

et al. 2008

Biochemical and Biophysical Research Communications

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“…Oligomerization of connexins into connexons was assessed by immunoblotting fractions obtained after centrifuging cell lysates solubilized with 1% Triton X-100 through a continuous sucrose gradient (5-20%) (3,20).…”

Section: Methodsmentioning

confidence: 99%

“…The cellular process of connexin oligomerization is beginning to be elucidated (1,6,8,18). We and others have identified a number of compatible or incompatible partners for the formation of heteromeric connexons (3,4,5,10,11,15). One of the most interesting compatible combinations is Cx43 and…”

Section: Introductionmentioning

confidence: 99%

A Carboxyl Terminal Domain of Connexin43 Is Critical for Gap Junction Plaque Formation but not for Homo- or Hetero-Oligomerization

Martı́nez¹,

Hayrapetyan²,

Moreno³

et al. 2003

GCAC

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We have initiated a series of experiments to analyze the biosynthesis and oligomerization of Cx43 in cells containing other connexins through the expression of site-directed mutants and chimeric connexin polypeptides. Here we report studies concerning a mutant of Cx43 (Cx43tr) that has been truncated after amino acid 251 to remove most of the Cx43 carboxyterminal region. In stably transfected HeLa cells, full length Cx43 localized primarily to appositional membranes while much more Cx43tr was observed in the cytoplasm. Both Cx43 and Cx43tr showed similar oligomerization profiles based on centrifugation through sucrose gradients. HeLaCx43tr cells showed limited transfer of microinjected Lucifer Yellow but did show electrical coupling. Co-expression of Cx43tr with Cx43 or Cx45 led to Cx43tr localization at appositional membranes and co-localization with the other connexins. Moreover, cells co-expressing Cx43tr with Cx43 or Cx45 showed extensive intercellular dye coupling. Thus, Cx43tr was able to oligomerize and form functional channels when expressed alone or with a compatible connexin, but it only formed plaques when co-expressed. These results suggest that the carboxyl tail of Cx43 is not important for oligomerization, but they implicate critical residues in the formation of gap junction plaques.

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“…For immunofluorescence studies, N2A cells seeded on multi-well slides (LAB TEK, Nalge Nunc International, Naperville, IL) were fixed with 50% methanol-50% acetone for 2 min at room temperature, and incubated sequentially in primary (rabbit poly-clonal anti-Cx50) and secondary (Cy3-conjugated goat anti-rabbit IgG) antibodies according to Berthoud et al (2000). The preparations were observed using a Zeiss Axioplan 2 microscope equipped for epifluorescence.…”

Section: Immunochemical Analysismentioning

confidence: 99%

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

Berthoud

Beyer

et al. 2002

Journal of Membrane Biology

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Gap junction channels formed by connexin 50 (Cx50) are critical for maintenance of lens transparency. Because the C-terminus of Cx50 can be cleaved post-translationally, we hypothesized that channels formed by the truncated Cx50 exhibit altered properties or regulation. We used the dual whole-cell patch-clamp technique to investigate the macroscopic and single-channel properties of gap junctional channels formed by wild-type human Cx50 and a truncation mutant (Cx50A294stop) after transfection of N2A cells. Our results show that wild-type Cx50 formed functional gap junctional channels. The macroscopic G jss -V j relationship was well described by a Boltzmann equation with A of 0.10, V 0 of 43.8 mV and G jmin of 0.23. The single-channel conductance was 212 ± 5 pS. Multiple long-lasting substates were observed with conductances ranging between 31 and 80 pS. Wild-type Cx50 gap junctional channels were reversibly blocked when pH i was reduced to 6.3. Truncating the C-terminus at amino acid 294 caused a loss of pH i sensitivity, but there were no significant changes in single-channel current amplitude or G jss -V j relationship. These results suggest that the C-terminus of human Cx50 is involved in pH i sensitivity, but has little influence over singlechannel conductance, voltage dependence, or gating kinetics.

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Heteromeric connexons formed by the lens connexins, connexin43 and connexin56

Cited by 37 publications

References 41 publications

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

A Carboxyl Terminal Domain of Connexin43 Is Critical for Gap Junction Plaque Formation but not for Homo- or Hetero-Oligomerization

Functional Role of the Carboxyl Terminal Domain of Human Connexin 50 in Gap Junctional Channels

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