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We turnover billions of apoptotic cells daily, and these are removed by professional and non-professional phagocytes via efferocytosis 1 . Characterizing the transcriptional program of phagocytes, we discovered a novel solute carrier family (SLC) gene signature (involving 33 SLC members) that is specifically modified during efferocytosis, but not antibody-mediated phagocytosis. Assessing the functional relevance of these SLCs, we noted a robust induction of an aerobic glycolysis program in efferocytic phagocytes, initiated by SLC2A1-mediated glucose uptake, with concurrent suppression of oxidative phosphorylation program. Interestingly, the different steps of phagocytosis 2 , i.e. smell (‘find-me’ signals/ sensing factors released by apoptotic cells), taste (phagocyte-apoptotic cell contact), and ingestion (corpse internalization), activated different SLCs and other molecules to promote glycolysis. Further, lactate, a natural by-product of aerobic glycolysis 3 , was released via another SLC (SLC16A1) that was upregulated after corpse uptake. While glycolysis within phagocytes contributed to actin polymerization and the continued uptake of corpses, the lactate released via SLC16A1 influenced the establishment of an anti-inflammatory tissue environment. Collectively, these data reveal a novel SLC program activated during efferocytosis, identify a previously unknown reliance on aerobic glycolysis during apoptotic cell uptake, and that glycolytic byproducts of efferocytosis can also influence other cells in the microenvironment.

Pannexin 1 (PANX1) subunits form oligomeric plasma membrane channels that mediate nucleotide release for purinergic signalling, which is involved in diverse physiological processes such as apoptosis, inflammation, blood pressure regulation, and cancer progression and metastasis. Here we explore the mechanistic basis for PANX1 activation by using wild type and engineered concatemeric channels. We find that PANX1 activation involves sequential stepwise sojourns through multiple discrete open states, each with unique channel gating and conductance properties that reflect contributions of the individual subunits of the hexamer. Progressive PANX1 channel opening is directly linked to permeation of ions and large molecules (ATP and fluorescent dyes) and occurs during both irreversible (caspase cleavage-mediated) and reversible (α1 adrenoceptor-mediated) forms of channel activation. This unique, quantized activation process enables fine tuning of PANX1 channel activity and may be a generalized regulatory mechanism for other related multimeric channels.

The turnover and clearance of cells is an essential process that is part of many physiological and pathological processes. Improper or deficient clearance of apoptotic cells can lead to excessive inflammation and autoimmune disease. The steps involved in cell clearance include: migration of the phagocyte toward the proximity of the dying cells, specific recognition and internalization of the dying cell, and degradation of the corpse. The ability of phagocytes to recognize and react to dying cells to perform efficient and immunologically silent engulfment has been well-characterized in vitro and in vivo. However, how apoptotic cells themselves initiate the corpse removal and also influence the cells within the neighboring environment during clearance was less understood. Recent exciting observations suggest that apoptotic cells can attract phagocytes through the regulated release of 'find-me' signals. More recent studies also suggest that these find-me signals can have additional roles outside of phagocyte attraction to help orchestrate engulfment. This review will discuss our current understanding of the different find-me signals released by apoptotic cells, how they may be relevant in vivo, and their additional roles in facilitating engulfment.

P anx1 (pannexin 1) channels have emerged as a major means for release of the nucleotides that mediate purinergic signaling, 1 which is involved in multiple physiological and pathophysiological processes such as apoptosis, 2 pyroptosis, 3 tumor cell metastasis, 4 vasoconstriction, 5 leukocyte migration, 6 insulin release from adipocytes, 7 N-methyl-D-aspartate receptor activation, 8 and neuronal survival. 9 Of particular relevance to this current work, Panx1 channels are activated by both caspase-dependent cleavage of the carboxyl terminus and cleavage-independent, receptor-mediated activation mechanisms. During apoptosis, caspase 3/7 cleaves the carboxyl terminus of Panx1, resulting in an irreversibly activated channel that can release ATP as a find-me signal to surrounding phagocytes.2 Alternatively, Panx1 channels, which are also expressed in smooth muscle cells (SMCs), are reversibly activated in a receptor-dependent mechanism. In vascular SMCs, we and others have demonstrated that sympathetic-mediated vasoconstriction and blood pressure are Panx1 dependent based on genetic and pharmacological perturbation.5,10-13 Specifically, activation of α1-adrenergic receptors results in opening of Panx1 channels that activate downstream purinergic receptors through release of ATP. Small ions and large molecules can permeate through open Panx1 channels, which allows for Panx1 channel activities to be assayed in several ways: by entry into cells of dyes, such as TO-PRO-3 or ethidium bromide; by release of nucleotides, such as ATP and UTP (uridine triphosphate); and by plasma membrane Panx1 channel currents (reviewed in [14][15][16] ). Given the paucity of selective compounds capable of targeting Panx1, we used different assays to identify and validate new Panx1 blockers that would be useful in these various pathophysiological contexts, such as treatmentresistant hypertension. Editorial, see p 543 In This Issue, see p 533 Meet the First Author, see p 534Original received November 12, 2017; revision received December 6, 2017; accepted December 12, 2017. In November 2017, the average time from submission to first decision for all original research papers submitted to Circulation Research was 11.99 days. Rationale: Resistant hypertension is a major health concern with unknown cause. Spironolactone is an effective antihypertensive drug, especially for patients with resistant hypertension, and is considered by the World Health Organization as an essential medication. Although spironolactone can act at the mineralocorticoid receptor (MR; NR3C2), there is increasing evidence of MR-independent effects of spironolactone.Objective: Here, we detail the unexpected discovery that Panx1 (pannexin 1) channels could be a relevant in vivo target of spironolactone. Methods and Results:First, we identified spironolactone as a potent inhibitor of Panx1 in an unbiased small molecule screen, which was confirmed by electrophysiological analysis. Next, spironolactone inhibited α-adrenergic vasoconstriction in arterioles from mice and hyperte...

Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.

Apoptotic cell clearance (efferocytosis) elicits an anti-inflammatory response by phagocytes, but the mechanisms underlying this response are still being defined. Here, we uncover a chloridesensing signaling pathway that controls both the phagocyte appetite and its anti-inflammatory response. Efferocytosis transcriptionally altered the genes coding for solute carrier (SLC) proteins SLC12A2 and SLC12A4. Interfering with SLC12A2 expression or function led to significantly Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Background: Activation of GRK2 requires interaction with agonist-occupied GPCRs.Results: Residues on the GRK2 N terminus and kinase domain extension collaborate to create a GPCR docking site.Conclusion: Three GRK subfamilies use similar determinants to create the putative docking site, but subtle differences may dictate selectivity.Significance: Mapping the GRK-GPCR interface is required to understand the mechanism and specificity of GRK activation, and, therefore, the regulation of GPCRs.