Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells

Abstract

Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.

Figure 1

Cxs 40 and 43 coimmunoprecipitated from connexon-enriched fractions. Crude membranes isolated from A7r5 cells after [³⁵S]methionine labeling were solubilized in Triton X-100 and were fractionated on a 5–20% linear sucrose gradient (see text). Individual gradient fractions were immunoprecipitated and analyzed by SDS/PAGE and fluorography. In A, each fraction was immunoprecipitated with affinity-purified anti-Cx43 antibody. A second band with the same mobility as Cx40 is observed in fractions enriched for connexons (e.g., fraction 12). In B, fractions 5 and 12 were immunoprecipitated with preimmune serum or Cx40 antibody. No bands were detected with preimmune serum. A second band with the same mobility as Cx43 is observed in the connexon-enriched fraction 12 but not in the Cx-enriched fraction 5.

Figure 2

Multichannel records obtained from A7r5 (A1 and A2), Rat-1 (B), or N2A-Cx40 (C) cells. The A7r5 cells display event amplitudes not observed in the Rat-1 or N2A-Cx40 cells. In the all-points histograms for the Rat-1 and N2A cells, peaks are narrow, they begin and end at baseline, and their separations correspond to actual event amplitudes. In the all-points histograms for the A7r5 cells, peaks are broad, they do not begin and end at baseline, and peak separations do not correspond to actual event amplitudes. Transjunctional voltage was 40 mV throughout the A7r5 and N2A-Cx40 records except at the asterisks (in A1 and C), where it was 0 mV. Transjunctional voltage was 30 mV throughout the Rat-1 record.

Figure 3

Voltage-dependent gating of gap junction channels in A7r5 cells (A), Rat-1 cells (B), and N2A-Cx40 cells (C). Normalized junctional conductance [steady state relative to instantaneous (G_ss/G_inst)] is plotted as a function of transjunctional voltage for 10 A7r5, 7 Rat-1, and 6 N2A-Cx40 cell pairs. Note the uniformity of responses to voltage in the Rat-1 and N2A cells compared with the diversity observed in the A7r5 cells.