Cx37-S319 mutants localize to appositional membrane and form functional gap junction channels. (A) Maximal expression levels for Cx37 in iRin37-WT, iRin37-S319A and iRin37-S319D cells were not different (mean±s.e.m., n=4 samples in triplicate; P=0.44). Data are from slot blot analysis. (B) Pseudo-DIC and immunofluorescence images of iRin37-S319A (3D8) and iRin37-S319D (2C1) induced (bottom) or not (top) to express Cx37. Cells were stained (Hoechst) to reveal nuclei (blue) of all cells in the field of view, and with antibody against Cx37 protein (green). Punctate labeling at appositional membranes (arrowheads), suggestive of gap junction plaques, was evident in the dox+ but not dox− setting. Scale bars: 100 μm. (C) The conductance of junctions formed by each clone (and isoform) were comparable (P=0.6834). Results are mean±s.e.m. [WT, 3.4±0.69 nS (n=30); iRin37-S319A 3D8 and 3E5, 3.9±0.92 nS (n=17) and 4.4±0.73 nS (n=14); iRin37-S319D 2B7 and 2C1 cells, 5.2±0.94 nS (n=9) and 4.2±0.92 nS (n=11)].

Cx37-S319A alleviates whereas -S319D enhances Cx37 mediated growth arrest. (A) Proliferation of expressing (dox+) and non-expressing (dox−) iRin37-S319A clones and iRin37-WT (n=3 for each) did not differ over 21 days (P=0.1582); only for Cx37-WT do the dox+ and dox− curves differ (P<0.0001), indicating lack of growth suppression by the -S319A mutant. The semi-log plot of these data (inset) shows that non-expressing iRin37-WT and both expressing and non-expressing iRin37-S319A mutant cells proliferate at comparable rates, whereas WT-expressing cells do not proliferate. *P<0.0001 significantly different from non-expressing (dox−) cells; ns, not significantly different. (B) The proliferation of iRin cells expressing Cx37-S319D or Cx37-WT is comparable and significantly (*P<0.0001) reduced compared to non-expressing cells of the same clone (n=3 for each). Inset, semi-log plot of proliferation over 6 days (n=3) showing that cell number increases, but does not double, in iRin37-S319D cells. ^†P=0.0024 from WT; ns, not significantly different. (C) 21-day proliferation curves in which Cx37-WT or -S319D expression was induced with dox on day 12 (n=3 for each clone) show suppressed proliferation at higher cell density (than in B). Inset, semi-log plot and regression analysis showing slowed proliferation after versus before induced expression of both Cx37-WT and Cx37-S319D isoforms. *P<0.05 from dox− growth period (2C1 P=0.0026; 2B7 P=0.0229; WT P=0.0024); ns, not significantly different. Double headed arrows in C indicate the period of dox exposure. All error bars denote s.e.m.; statistical differences were assessed using linear regression analysis.

iRin37-S319D cells are growth arrested and accumulate in G₀/G₁. (A) In the presence of serum, Cx37-WT and Cx37-S319D suppressed the proliferation of Rin cells; Cx37-S319A had no growth-suppressive effect. Error bars denote s.e.m.; *P<0.05 (one-way ANOVA with Tukey's post-hoc test). (B–D) In the presence of serum, the percentage of Cx37-S319D cells in G₀ or G₁ (labeled G₁) increased as the percentage in G₂ decreased, suggesting that cells in G₂, when CX37-S319D expression was initiated, were able to complete the cell cycle and were subsequently arrested in G₀ or G₁. (E) Serum deprivation arrested proliferation in an isoform independent manner. (F–H) All phases of the cell cycle were extended in Cx37-WT expressing cells, with no significant increase in any phase over the 5-day period. In contrast, the percentage of S319D cells in G₀ or G₁ increased while simultaneously the percentage in both S and G₂ decreased significantly, suggesting that S and G₂ cells are able to complete the cell cycle and arrest in G₀ or G₁. The Cx37-S319A cells also accumulate in G₀, but for this isoform the percentage in S decreases substantially while G₂ percentage is constant. All error bars denote s.e.m.; *P<0.05 between day 1 vs 5, P values stated in text.

Gap junction channels formed by growth-arresting isoforms of Cx37 prefer the closed state. (A–C) Representative single-channel traces from the indicated genotypes suggesting that Cx37-S319D and -WT prefer the closed state whereas Cx37-S319A prefers a 60–120 pS substate. For the Cx37-WT and -S319A traces, only one channel is active and its closed state corresponds to the 0 pS line indicated by long dashes. For the Cx37-S319D trace, three channels are open at the start of the pulse (652 pS); their successive stable closures result in the peaks at 440 pS (2 channels open) and 192 pS (1 channel open). The size of observed transitions is shown on the left of each trace; all-points histograms, on the right, indicate the frequency of each conductance state for the illustrated trace. ↓ and ↑ indicate polarity and onset/end of pulse in the partner cell. (D) Relative mean±s.e.m. transition frequencies for all three Cx37 isoforms showing transitions between multiple open states and a closed state, including fully open to closed. Together the traces and histogram suggest Cx37-WT channels frequently transition between the ∼90 pS substate and the closed state [traces and previously published studies (Jacobsen et al., 2017) suggest the closed state is preferred]. The Cx37-S319D channels close frequently from multiple open states, but most commonly from the ∼200 pS conductance state (traces suggest closed state is preferred over the many open states). Cx37-WT: n=17, event no.=1917; Cx37-S319A: n=12, event no.=1280; Cx37-S319D: n=20, event no.=4493.

Voltage-dependent gating of Cx37-S319D is diminished compared to Cx37-S319A. (A) Voltage protocol and current traces derived from Cx37-S319D and -S319A mutants, red trace shows response to ±110 mV pulses. (B) Boltzmann fits for Cx37-S319D (n=4) and -S319A (n=9). Fits for positive and negative voltage ranges did not differ for either mutant (or WT). Results are mean±s.e.m. (C) Boltzmann fit parameters for mutants and WT pulsed negative versus positive and compared using ANOVA analysis (negative range, P=0.0008; positive range, P=0.0002). Differences between mutants and between mutants and WT were tested (least squares); where significant differences were found they are indicated in the row labeled Fit.

Behavior of hemichannels formed by Cx37-S319D and -S319A differ. (A) Representative traces show that Cx37-S319A HChs spend more time in open states than either Cx37-S319D or Cx37-WT HChs. The size of transitions is shown on the left of each recording; all-points histograms, on the right, show the frequency of each conductance state for the illustrated trace. (B) Transition frequency (mean±s.e.m. for each conductance bin) and open probability (P_o) for HChs formed by Cx37-S319D and Cx37-S319A. Transitions of multiple amplitudes between fully open (∼750–800 pS) and closed were observed for both isoforms, but their open probabilities differed. Cx37-S319D preferred the closed state and a 60–90 pS subconductance state, whereas Cx37-S319A HChs preferred open states. Cx37-S319A (n=32, event no.=240) and Cx37-S319D (n=35, event no.=1521).