Hematopoietic stem cell (HSC) aging has become a concern in chemotherapy of older patients. Humoral and paracrine signals from the bone marrow (BM) hematopoietic microenvironment (HM) control HSC activity during regenerative hematopoiesis. Connexin-43 (Cx43), a connexin constituent of gap junctions (GJs) is expressed in HSCs, down-regulated during differentiation, and postulated to be a self-renewal gene. Our studies, however, reveal that hematopoietic-specific Cx43 deficiency does not result in significant long-term competitive repopulation deficiency. Instead, hematopoietic Cx43 (H-Cx43) deficiency delays hematopoietic recovery after myeloablation with 5-fluorouracil (5-FU). 5-FU-treated H-Cx43-deficient HSC and progenitors (HSC/P) cells display decreased survival and fail to enter the cell cycle to proliferate. Cell cycle quiescence is associated with down-regulation of cyclin D1, up-regulation of the cyclin-dependent kinase inhibitors, p21 ^cip1. and p16 ^INK4a , and Forkhead transcriptional factor 1 (Foxo1), and activation of p38 mitogen-activated protein kinase (MAPK), indicating that H-Cx43-deficient HSCs are prone to senescence. The mechanism of increased senescence in H-Cx43-deficient HSC/P cells depends on their inability to transfer reactive oxygen species (ROS) to the HM, leading to accumulation of ROS within HSCs. In vivo antioxidant administration prevents the defective hematopoietic regeneration, as well as exogenous expression of Cx43 in HSC/P cells. Furthermore, ROS transfer from HSC/P cells to BM stromal cells is also rescued by reexpression of Cx43 in HSC/P. Finally, the deficiency of Cx43 in the HM phenocopies the hematopoietic defect in vivo. These results indicate that Cx43 exerts a protective role and regulates the HSC/P ROS content through ROS transfer to the HM, resulting in HSC protection during stress hematopoietic regeneration.

In addition to forming gap-junction channels, a subset of connexins (Cxs) also form functional hemichannels. Most hemichannels are activated by depolarization, and opening depends critically on the external Ca ²⁺ concentration. Here we describe the mechanisms of action and the structural determinants underlying the Ca ²⁺ regulation of Cx32 hemichannels. At millimolar calcium concentrations, hemichannel voltage gating to the full open state of ≈90 pS is inhibited, and ion conduction at negative voltages of the partially open hemichannels (≈18 pS) is blocked. Thus, divalent cation blockage should be considered as a physiological mechanism to protect the cell from the potentially adverse effects of leaky hemichannels. A ring of 12 Asp residues within the external vestibule of the pore is responsible for the binding of Ca ²⁺ that accounts for both pore occlusion and blockage of gating. The residue Asp-169 of one subunit and the Asp-178 of an adjacent subunit must be arranged precisely to allow interactions with Ca ²⁺ to occur. Interestingly, a naturally occurring mutation (D178Y) that causes an inherited peripheral neuropathy induces a complete Ca ²⁺ deregulation of Cx32 hemichannel activity, suggesting that this dysfunction may be involved in the pathogenesis of the neuropathy.

Neurotransmission through electrical synapses plays an important role in the spike synchrony among neurons and oscillation of neuronal networks. Indeed, electrical transmission has been implicated in the hypersynchronous electrical activity of epilepsy. We have investigated the influence of intracellular pH on the strength of electrical coupling mediated by connexin36 (Cx36), the principal gap junction protein in the electrical synapses of vertebrates. In striking contrast to other connexin isoforms, the activity of Cx36 channels decreases following alkalosis rather than acidosis when it is expressed in Xenopus oocytes and N2A cells. This uncoupling of Cx36 channels upon alkalinization occurred in the vertebrate orthologues analyzed (human, mouse, chicken, perch, and skate). While intracellular acidification caused a mild or moderate increase in the junctional conductance of virtually all these channels, the coupling of the skate Cx35 channel was partially blocked by acidosis. The mutational analysis suggests that the Cx36 channels may contain two gating mechanisms operating with opposing sensitivity to pH. One gate, the dominant mechanism, closes for alkalosis and it probably involves an interaction between the C- and N-terminal domains, while a secondary acid sensing gate only causes minor, albeit saturating, changes in coupling following acidosis and alkalosis. Thus, we conclude that neuronal Cx36 channels undergo unique regulation by pH _i since their activity is inhibited by alkalosis rather than acidosis. These data provide a novel basis to define the relevance and consequences of the pH-dependent modulation of Cx36 synapses under physiological and pathological conditions.

Connexin-43 (Cx43), a gap junction protein involved in control of cell proliferation, differentiation and migration, has been suggested to have a role in hematopoiesis. Cx43 is highly expressed in osteoblasts and osteogenic progenitors (OB/P). To elucidate the biologic function of Cx43 in the hematopoietic microenvironment (HM) and its influence in hematopoietic stem cell (HSC) activity, we studied the hematopoietic function in an in vivo model of constitutive deficiency of Cx43 in OB/P. The deficiency of Cx43 in OB/P cells does not impair the steady state hematopoiesis, but disrupts the directional trafficking of HSC/progenitors (Ps) between the bone marrow (BM) and peripheral blood (PB). OB/P Cx43 is a crucial positive regulator of transstromal migration and homing of both HSCs and progenitors in an irradiated microenvironment. However, OB/P Cx43 deficiency in nonmyeloablated animals does not result in a homing defect but induces increased endosteal lodging and decreased mobilization of HSC/Ps associated with proliferation and expansion of Cxcl12-secreting mesenchymal/osteolineage cells in the BM HM in vivo. Cx43 controls the cellular content of the BM osteogenic microenvironment and is required for homing of HSC/Ps in myeloablated animals.