Yu-Hsin Chiu

Unexpected link between an antibiotic, pannexin channels and apoptosis.

Plasma membrane pannexin 1 channels (PANX1) release nucleotide find-me signals from apoptotic cells to attract phagocytes. Here we show that the quinolone antibiotic trovafloxacin is a novel PANX1 inhibitor, by using a small-molecule screen. Although quinolones are widely used to treat bacterial infections, some quinolones have unexplained side effects, including deaths among children. PANX1 is a direct target of trovafloxacin at drug concentrations seen in human plasma, and its inhibition led to dysregulated fragmentation of apoptotic cells. Genetic loss of PANX1 phenocopied trovafloxacin effects, revealing a non-redundant role for pannexin channels in regulating cellular disassembly during apoptosis. Increase in drug-resistant bacteria worldwide and the dearth of new antibiotics is a major human health challenge. Comparing different quinolone antibiotics suggests that certain structural features may contribute to PANX1 blockade. These data identify a novel linkage between an antibiotic, pannexin channels and cellular integrity, and suggest that re-engineering certain quinolones might help develop newer antibacterials.

A molecular signature in the pannexin1 intracellular loop confers channel activation by the α1 adrenoreceptor in smooth muscle cells

The ATP-releasing channel Panx1 is specifically involved in blood pressure regulation by adrenergic signaling. Regulating blood pressure with ATP Blood pressure is dynamically regulated to enable rapid responses to changes in position and physical or emotional stress, such as exercise or anger and fear. Many signals that activate G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs) control vascular tone, including norepinephrine (also known as noradrenaline) released by the sympathetic nervous system, which increases blood pressure. Billaud et al. report that the α1 adrenoreceptor (α1AR)—but not the endothelin-1 or serotonin receptor, which are also Gαq-coupled GPCRs and stimulate vasoconstriction—is specifically coupled to activation of the ATP (adenosine 5′-triphosphate)–releasing channel pannexin1 (Panx1). Mice lacking Panx1 in smooth muscle cells were hypotensive, specifically during their active period of the day. Isolated arteries from these mice did not release ATP and contracted less in response to adrenoreceptor stimulation. Thus, ATP release through Panx1 channels specifically contributes to blood pressure regulation by the sympathetic nervous system. Both purinergic signaling through nucleotides such as ATP (adenosine 5′-triphosphate) and noradrenergic signaling through molecules such as norepinephrine regulate vascular tone and blood pressure. Pannexin1 (Panx1), which forms large-pore, ATP-releasing channels, is present in vascular smooth muscle cells in peripheral blood vessels and participates in noradrenergic responses. Using pharmacological approaches and mice conditionally lacking Panx1 in smooth muscle cells, we found that Panx1 contributed to vasoconstriction mediated by the α1 adrenoreceptor (α1AR), whereas vasoconstriction in response to serotonin or endothelin-1 was independent of Panx1. Analysis of the Panx1-deficient mice showed that Panx1 contributed to blood pressure regulation especially during the night cycle when sympathetic nervous activity is highest. Using mimetic peptides and site-directed mutagenesis, we identified a specific amino acid sequence in the Panx1 intracellular loop that is essential for activation by α1AR signaling. Collectively, these data describe a specific link between noradrenergic and purinergic signaling in blood pressure homeostasis.

Intrinsic properties and regulation of Pannexin 1 channel

Pannexin 1 (Panx1) channels are generally represented as non-selective, large-pore channels that release ATP. Emerging roles have been described for Panx1 in mediating purinergic signaling in the normal nervous, cardiovascular, and immune systems, where they may be activated by mechanical stress, ionotropic and metabotropic receptor signaling, and via proteolytic cleavage of the Panx1 C-terminus. Panx1 channels are widely expressed in various cell types, and it is now thought that targeting these channels therapeutically may be beneficial in a number of pathophysiological contexts, such as asthma, atherosclerosis, hypertension, and ischemic-induced seizures. Even as interest in Panx1 channels is burgeoning, some of their basic properties, mechanisms of modulation, and proposed functions remain controversial, with recent reports challenging some long-held views regarding Panx1 channels. In this brief review, we summarize some well-established features of Panx1 channels; we then address some current confounding issues surrounding Panx1 channels, especially with respect to intrinsic channel properties, in order to raise awareness of these unsettled issues for future research.

A quantized mechanism for activation of pannexin channels

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.

Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes

Objective Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. Methods We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. Results Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. Conclusions We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

Hematopoietic pannexin 1 function is critical for neuropathic pain

Neuropathic pain symptoms respond poorly to available therapeutics, with most treated patients reporting unrelieved pain and significant impairment in daily life. Here, we show that Pannexin 1 (Panx1) in hematopoietic cells is required for pain-like responses following nerve injury in mice, and a potential therapeutic target. Panx1 knockout mice (Panx1−/−) were protected from hypersensitivity in two sciatic nerve injury models. Bone marrow transplantation studies show that expression of functional Panx1 in hematopoietic cells is necessary for mechanical hypersensitivity following nerve injury. Reconstitution of irradiated Panx1 knockout mice with hematopoietic Panx1−/− cells engineered to re-express Panx1 was sufficient to recover hypersensitivity after nerve injury; this rescue required expression of a Panx1 variant that can be activated by G protein-coupled receptors (GPCRs). Finally, chemically distinct Panx1 inhibitors blocked development of nerve injury-induced hypersensitivity and partially relieved this hypersensitivity after it was established. These studies indicate that Panx1 expressed in immune cells is critical for pain-like effects following nerve injury in mice, perhaps via a GPCR-mediated activation mechanism, and suggest that inhibition of Panx1 may be useful in treating neuropathic pain.

Revisiting multimodal activation and channel properties of Pannexin 1

Chiu and colleagues review the primary evidence for divergent activation mechanisms and unitary properties of Pannexin 1 channels.

Pannexin 1 Channels as an Unexpected New Target of the Anti-Hypertensive Drug Spironolactone

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 &agr;-adrenergic vasoconstriction in arterioles from mice and hypertensive humans, an effect dependent on smooth muscle Panx1, but independent of the MR NR3C2. Last, spironolactone acutely lowered blood pressure, which was dependent on smooth muscle cell expression of Panx1 and independent of NR3C2. This effect, however, was restricted to steroidal MR antagonists as a nonsteroidal MR antagonist failed to reduced blood pressure. Conclusions: These data suggest new therapeutic modalities for resistant hypertension based on Panx1 inhibition.

Response by Good et al to Letter Regarding Article, “Pannexin-1 Channels as an Unexpected New Target of the Antihypertensive Drug Spironolactone”

In our recent publication, we reported the discovery that spironolactone—an antihypertensive—is also a new Panx1 (pannexin 1) inhibitor. In addition, we found that spironolactone interferes with α1AR (α1 adrenoceptor)-mediated vasoconstriction of resistance vessels and acutely lowers blood pressure in mice; importantly, these effects require Panx1 channel expression in vascular smooth muscle cells but are independent of the MR (mineralocorticoid receptor)—the traditional target of spironolactone. Based on the accumulated evidence, we proposed that Panx1 is a novel target of spironolactone that, in combination with MR-dependent actions, may contribute to the beneficial blood pressure-lowering effects of spironolactone that are especially relevant for treatment of resistant hypertensive patients. We are pleased that Drs Wright and Angus recognize that this novel action adds to the well-accepted MR-dependent antihypertensive effects of spironolactone and that this unveils a potentially important mechanism with translational clinical implications. In addition, however, these authors raise some points of contention with our studies that can be summarized in 2 general arguments: (1) based on perceived differences with their own pharmacological reports, they insist that additional pharmacological studies are necessary to support the fundamental underlying mechanism (ie, that Panx1-mediated ATP release contributes to α1AR-mediated vasoconstriction) and (2) that spironolactone acts less potently at Panx1 than at MR and thus may require concentrations not achieved clinically. Below, we address both of these points. Despite the narrow focus of their argument on work involving a single Panx1 inhibitor (mefloquine)1 and a single P2X1 blocker (NF449),2 there is now substantial pharmacological and, importantly, genetic evidence supporting a role for Panx1-mediated ATP release in α1AR-mediated vasoconstriction. For Panx1, this …