Structural determinants and proliferative consequences of connexin 37 hemichannel function in insulinoma cells

Abstract

Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.

Keywords: Arterial Endothelium; Cell Cycle Control; Cell-Cell Interaction; Connexin; Gap Junction; Hemichannel; Intercellular Communication; Proliferation; Protein Targeting.

FIGURE 1.

Expression and localization of Cx37 are comparable in Cx37-WT, -C₆A, -N55I, and -Q58L iRin cells. A, Cx37 was detected in both the whole cell and Triton X-insoluble protein fractions of iRin cells expressing Cx37-WT, -C₆A, -N55I, and -Q58L; 50 μg of total protein was loaded for each cell type. The 37-GST (GST-rCx37CT_229–333, where CT indicates C terminus) lane shows positive control for antibody detection; note that the Cx37-GST fusion protein typically migrates as multiple bands (this lane was contrast-enhanced for better band visibility) (7, 9). Positions of the 30- and 40-kDa molecular mass markers are shown in the mass marker (MM) lane. B, immunocytochemistry revealed localization of WT and mutant Cx37 (green) at appositional and non-appositional membranes (arrowheads). Red staining corresponds to biotinylated proteins on the extracellular surface of the plasma membrane and to ToPro3-labeled nuclei in the C₆A, N55I and Q58L images (some ToPro3-labeled nuclei are evident in unattached cells above the plane of focus). Yellow staining corresponds to Cx37 localized to the plasma membrane. Scale bars: 10 μm.

FIGURE 2.

HC activity displayed by Cx37-WT, -N55I, and -Q58L-expressing, but not -C₆A-expressing, cells is similar. A–J, distinct HC events with amplitudes of ∼500 pS were observed in normal (left column) and low [Ca²⁺]_o (right column) in iRin cells expressing Cx37-WT (A and B), Cx37-N55I (C and D), and Cx37-Q58L (E and F), but not Cx37-C₆A (G–J). Non-distinct activity centered at the ∼200-pS conductance level was evident in WT- and all mutant-expressing cells, but was diminished in low [Ca²⁺]_o only in WT, N55I, and Q58L cells. Calibration bars (vertical/horizontal): 10 pA/1.0 s.

FIGURE 3.

Cx37-WT, -N55I, and -Q58L HCs mediate NBD dye uptake; Cx37-C₆A HCs do not. A, representative differential interference contrast (DIC) and NBD (NBD-M-TMA)-fluorescence images for each cell line induced to express the indicated form of Cx37 or the non-induced Cx37-WT cells. White: NBD uptake. Rhodamine images are not shown as no cells contained this dye. Scale bar, 100 μm, applies to all frames. B, NBD uptake was greater in cells induced (as compared with non-induced) to express Cx37-WT, -N55I, and -Q58L, but not Cx37-C₆A. Cells counted in non-induced and induced conditions (dox⁻/dox⁺): Cx37-WT, 366/323 (n = 4); Cx37-C₆A, 499/537 (n = 7); Cx37-N55I, 678/688 (n = 5); and Cx37-Q58L, 660/949 (n = 7). Error bars: S.E. * indicates significant difference between dox⁺ (Cx37-expressing) and dox⁻ (non-expressing) cells within the same cell line. NS indicates no significant difference between dox⁺ and dox⁻ cells. C, ratio of dye-positive to total cell number in each of the images obtained from induced Cx37-WT-, -N55I-, and -Q58L-expressing cells.

FIGURE 4.

Expression of Cx37-C₆A, -N55I, or -Q58L fails to suppress the proliferation of iRin cells. Increase in cell number over 15 days was evaluated in iRin cells induced (red filled symbols) or not (white filled symbols) to express Cx37-WT, -C₆A, -N55I, or -Q58L. Proliferation was suppressed only in Cx37-WT-expressing cells. n = 3 for all groups, each experiment performed in triplicate. Error bars: S.E.

FIGURE 5.

Connexin functions necessary for suppression of Rin cell proliferation. Growth-suppressive function of Cx37-WT could require functional (black checks) GJCs, HCs, or C terminus (CT), or a combination of these properties. The C terminus could influence growth through regulatory effects on channel function as well as via channel-independent mechanisms, such as through interactions with growth regulatory proteins. Truncated Cx37 (Cx37-Δ273) forms functional GJCs and HCs, but without its C terminus (red X), it fails to suppress the proliferation of Rin cells (10). Cx37-T154A (9), Cx37-C61A,C65A (11), and Cx37-C₆A (present data) all retain their full-length C terminus, but none forms functional GJCs or HCs and all fail to suppress growth, indicating the necessity of channel function for growth suppression. Cx37-N55I and Cx37-Q58L fail to form functional GJCs, but retain functional HCs and their full-length C terminus, but nevertheless fail to suppress proliferation. Cx43 (purple checks) forms functional GJCs and HCs, although the properties of these channels differ from Cx37, and has a full-length C terminus with a different primary amino acid sequence from Cx37, but fails to suppress Rin cell proliferation (7). Rin cell proliferation is not suppressed by the Cx43*37CT chimera (where CT indicates C terminus) despite the presence of the Cx37 C terminus and formation of functional GJCs, albeit with unique properties (gray checks) (10, 26). NA: not available. Together, these results indicate that Cx37 exerts a growth-suppressive effect only when it is capable of all three functions, intercellular, transmembrane, and intracellular signaling, specifically those supported by the Cx37 sequence.