Specific amino-acid residues in the N-terminus and TM3 implicated in channel function and oligomerization compatibility of connexin43

Abstract

To identify signals that convey connexin oligomerization compatibility, we have aligned amino-acid sequences of α and β group connexins (Cx)and compared the physico-chemical properties of each homologous amino-acid residue. Four positions were identified that consistently differed betweenα and β-type connexins; two are located in the N-terminal domain(P1 and P2, corresponding to residues 12 and 13 of the Cx43 sequence), and two in the third trans-membrane-spanning domain TM3 (P3 and P4, corresponding to residues 152 and 153 of the Cx43 sequence). Replacement of each of these residues in Cx43 (an α-type connexin) with the corresponding residues of Cx32 (a β-type connexin) resulted in the assembly of all variants into gap junctions; however, only the P4 variant was functional, as indicated by lucifer yellow dye transfer assays. The other three variants exerted a moderate to severe dose-dependent, dominant-negative effect on co-expressed wild-type (wt) Cx43 channel activity. Moreover, a significant dose-dependent,trans-dominant inhibition of channel activity was observed when either one of the N-terminal variants was co-expressed with wt Cx32. Assembly analyses indicated that dominant and trans-dominant inhibitory effects appeared to be based on the oligomerization of wt and variant connexins into mixed connexons. Interestingly, the identified N-terminal amino acids coincide with the position of naturally occurring, disease-causing missense mutations of severalβ-connexin genes (Cx26, Cx30, Cx31, Cx32). Our results demonstrate that three of the identified discriminative amino-acid residues(positions 12, 13 and 152) are crucial for Cx43 channel function and suggest that the N-terminal amino-acid residues at position 12/13 are involved in the oligomerization compatibility of α and β connexins.

Connexin43 with a cytoplasmic loop deletion inhibits the function of several connexins

“…Ad-Cx43del 130-136 had no significant effect upon intercellular communication in cells expressing Cx26. This result was not surprising, since previous observations suggest that Cx43 does not form heteromeric channels with "beta group" connexins such as Cx32 or Cx26 [19,28], and the data presented here showed no overlap between the Cx43del 130-136 and Cx26 immunoreactivities with most of the immunopositive Cx26 at gap junctional plaques.…”

Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC)

“…Having found that TMEM16A assembles into dimeric complexes, we next sought to identify the region responsible for this interaction. Because several other channel proteins (9,11,12) oligomerize via their cytosolic regions, we began by purifying each of the five predicted cytosolic domains (16) from mouse TMEM16A on glutathione-conjugated beads and mixing them with lysate from HEK 293 cells expressing GFP-tagged TMEM16A ( Fig. 2 A and B).…”

“…For example, the NMDA-type glutamate receptor (NR) is a tetramer assembled from two obligate NR1 subunits and a choice of two NR2 subunits ranging from NR2A through NR2D (8), and studies of homologous ionotropic glutamate receptors implicate a cytosolic domain at the amino terminus in tetramerization (9). Similarly, the pentameric cys-loop receptors (10), the tetrameric potassium channels (11), and the gap junctions formed by hexameric hemichannels (12) are all protein complexes composed of subunits whose assembly is driven by channel-specific oligomerization domains.…”

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

“…Charged amino acids within this region influence various physiological properties including transjunctional voltage (V j )-dependent gating (Verselis et al, 1994;Oh et al, 2000;Purnick et al, 2000b;Musa et al, 2004), unitary conductance (Musa et al, 2004;Tong et al, 2004) and sensitivity to regulation by polyamines (Musa et al, 2004). Lagree et al have suggested that amino acids 12 and 13 (numbered according to Cx43) also influence the function and compatibility of connexins (Lagree et al, 2003).…”