Yawar J. Qadri

Assessment of the CFTR and ENaC association

Cystic fibrosis (CF) is one of the most common lethal genetic disorders. It results primarily from mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene. These mutations cause inadequate functioning of CFTR, which in turn leads to the severe disruption of transport function in several epithelia across various organs. Affected organs include the sweat glands, the intestine, and the reproductive system, with the most devastating consequences due to the effects of the disease on airways. Despite aggressive treatment, gradual lung failure is the major life limiting factor in patients with CF. Understanding of the exact manner by which defects in the CFTR lead to lung failure is thus critical. In the CF airway, decreased chloride secretion and increased salt absorption is observed. The decreased chloride secretion appears to be a direct consequence of defective CFTR; however, the increased salt absorption is believed to result from the failure of CFTR to restrict salt absorption through a sodium channel named the epithelial Na(+) channel, ENaC. The mechanism by which CFTR modulates the function of ENaC proteins is still obscure and somewhat controversial. In this short review we will focus on recent findings of a possible direct CFTR and ENaC association.

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high PK+/PNa+ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

Heteromeric assembly of acid-sensitive ion channel and epithelial sodium channel subunits.

Amiloride-sensitive ion channels are formed from homo- or heteromeric combinations of subunits from the epithelial Na+ channel (ENaC)/degenerin superfamily, which also includes the acid-sensitive ion channel (ASIC) family. These channel subunits share sequence homology and topology. In this study, we have demonstrated, using confocal fluorescence resonance energy transfer microscopy and co-immunoprecipitation, that ASIC and ENaC subunits are capable of forming cross-clade intermolecular interactions. We have also shown that combinations of ASIC1 with ENaC subunits exhibit novel electrophysiological characteristics compared with ASIC1 alone. The results of this study suggest that heteromeric complexes of ASIC and ENaC subunits may underlie the diversity of amiloride-sensitive cation conductances observed in a wide variety of tissues and cell types where co-expression of ASIC and ENaC subunits has been observed.

Molecular Proximity of Cystic Fibrosis Transmembrane Conductance Regulator and Epithelial Sodium Channel Assessed by Fluorescence Resonance Energy Transfer

We present the evidence for a direct physical association of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC), two major ion channels implicated in the pathophysiology of cystic fibrosis, a devastating inherited disease. We employed fluorescence resonance energy transfer, a distance-dependent imaging technique with capability to detect molecular complexes with near angstrom resolution, to estimate the proximity of CFTR and ENaC, an essential variable for possible physical interaction to occur. Fluorescence resonance energy transfer studies were complemented with a classic biochemical approach: coimmunoprecipitation. Our results place CFTR and ENaC within reach of each other, suggestive of a direct interaction between these two proteins.

ENaCs and ASICs as therapeutic targets

The epithelial Na(+) channel (ENaC) and acid-sensitive ion channel (ASIC) branches of the ENaC/degenerin superfamily of cation channels have drawn increasing attention as potential therapeutic targets in a variety of diseases and conditions. Originally thought to be solely expressed in fluid absorptive epithelia and in neurons, it has become apparent that members of this family exhibit nearly ubiquitous expression. Therapeutic opportunities range from hypertension, due to the role of ENaC in maintaining whole body salt and water homeostasis, to anxiety disorders and pain associated with ASIC activity. As a physiologist intrigued by the fundamental mechanics of salt and water transport, it was natural that Dale Benos, to whom this series of reviews is dedicated, should have been at the forefront of research into the amiloride-sensitive sodium channel. The cloning of ENaC and subsequently the ASIC channels has revealed a far wider role for this channel family than was previously imagined. In this review, we will discuss the known and potential roles of ENaC and ASIC subunits in the wide variety of pathologies in which these channels have been implicated. Some of these, such as the role of ENaC in Liddles syndrome are well established, others less so; however, all are related in that the fundamental defect is due to inappropriate channel activity.

Psalmotoxin-1 docking to human acid sensing ion channel-1

Acid-sensing ion channel-1 (ASIC-1) is a proton-gated ion channel implicated in nociception and neuronal death during ischemia. Recently the first crystal structure of a chicken ASIC was obtained. Expanding upon this work, homology models of the human ASICs were constructed and evaluated. Energy-minimized structures were tested for validity by in silico docking of the models to psalmotoxin-1, which potently inhibits ASIC-1 and not other members of the family. The data are consistent with prior radioligand binding and functional assays while also explaining the selectivity of PcTX-1 for homomeric hASIC-1a. Binding energy calculations suggest that the toxin and channel create a complex that is more stable than the channel alone. The binding is dominated by the coulombic contributions, which account for why the toxin-channel interaction is not observed at low pH. The computational data were experimentally verified with single channel and whole-cell electrophysiological studies. These validated models should allow for the rational design of specific and potent peptidomimetic compounds that may be useful for the treatment of pain or ischemic stroke.

Amiloride Docking to Acid-sensing Ion Channel-1

Amiloride is a small molecule diuretic, which has been used to dissect sodium transport pathways in many different systems. This drug is known to interact with the epithelial sodium channel and acid-sensing ion channel proteins, as well as sodium/hydrogen antiporters and sodium/calcium exchangers. The exact structural basis for these interactions has not been elucidated as crystal structures of these proteins have been challenging to obtain, though some involved residues and domains have been mapped. This work examines the interaction of amiloride with acid-sensing ion channel-1, a protein whose structure is available using computational and experimental techniques. Using molecular docking software, amiloride and related molecules were docked to model structures of homomeric human ASIC-1 to generate potential interaction sites and predict which analogs would be more or less potent than amiloride. The predictions made were experimentally tested using whole-cell patch clamp. Drugs previously classified as NCX or NHE inhibitors are shown to also inhibit hASIC-1. Potential docking sites were re-examined against experimental data to remove spurious interaction sites. The voltage sensitivity of inhibitors was also examined. Using the aggregated data from these computational and experimental experiments, putative interaction sites for amiloride and hASIC-1 have been defined. Future work will experimentally verify these interaction sites, but at present this should allow for virtual screening of drug libraries at these putative interaction sites.

Microglial Signaling in Chronic Pain with a Special Focus on Caspase 6, p38 MAP Kinase, and Sex Dependence:

Microglia are the resident immune cells in the spinal cord and brain. Mounting evidence suggests that activation of microglia plays an important role in the pathogenesis of chronic pain, including chronic orofacial pain. In particular, microglia contribute to the transition from acute pain to chronic pain, as inhibition of microglial signaling reduces pathologic pain after inflammation, nerve injury, and cancer but not baseline pain. As compared with inflammation, nerve injury induces much more robust morphologic activation of microglia, termed microgliosis, as shown by increased expression of microglial markers, such as CD11b and IBA1. However, microglial signaling inhibitors effectively reduce inflammatory pain and neuropathic pain, arguing against the importance of morphologic activation of microglia in chronic pain sensitization. Importantly, microglia enhance pain states via secretion of proinflammatory and pronociceptive mediators, such as tumor necrosis factor α, interleukins 1β and 18, and brain-derived growth factor. Mechanistically, these mediators have been shown to enhance excitatory synaptic transmission and suppress inhibitory synaptic transmission in the pain circuits. While early studies suggested a predominant role of microglia in the induction of chronic pain, further studies have supported a role of microglia in the maintenance of chronic pain. Intriguingly, recent studies show male-dominant microglial signaling in some neuropathic pain and inflammatory pain states, although both sexes show identical morphologic activation of microglia after nerve injury. In this critical review, we provide evidence to show that caspase 6—a secreted protease that is expressed in primary afferent axonal terminals surrounding microglia—is a robust activator of microglia and induces profound release of tumor necrosis factor α from microglia via activation of p38 MAP kinase. The authors also show that microglial caspase 6/p38 signaling is male dominant in some inflammatory and neuropathic pain conditions. Finally, the authors discuss the relevance of microglial signaling in chronic trigeminal and orofacial pain.

Two PKC consensus sites on human acid-sensing ion channel 1b differentially regulate its function

Human acid-sensing ion channel 1b (hASIC1b) is a H(+)-gated amiloride-sensitive cation channel. We have previously shown that glioma cells exhibit an amiloride-sensitive cation conductance. Amiloride and the ASIC1 blocker psalmotoxin-1 decrease the migration and proliferation of glioma cells. PKC also abolishes the amiloride-sensitive conductance of glioma cells and inhibits hASIC1b open probability in planar lipid bilayers. In addition, hASIC1bs COOH terminus has been shown to interact with protein interacting with C kinase (PICK)1, which targets PKC to the plasma membrane. Therefore, we tested the hypothesis that PKC regulation of hASIC1b at specific PKC consensus sites inhibits hASIC1b function. We mutated three consensus PKC phosphorylation sites (T26, S40, and S499) in hASIC1b to alanine, to prevent phosphorylation, and to glutamic acid or aspartic acid, to mimic phosphorylation. Our data suggest that S40 and S499 are critical sites mediating the modulation of hASIC1b by PKC. We expressed mutant hASIC1b constructs in Xenopus oocytes and measured acid-activated currents by two-electrode voltage clamp. T26A and T26E did not exhibit acid-activated currents. S40A was indistinguishable from wild type (WT), whereas S40E, S499A, and S499D currents were decreased. The PKC activators PMA and phorbol 12,13-dibutyrate inhibited WT hASIC1b and S499A, and PMA had no effect on S40A or on WT hASIC1b in oocytes pretreated with the PKC inhibitor chelerythrine. Chelerythrine inhibited WT hASIC1b and S40A but had no effect on S499A or S40A/S499A. PKC activators or the inhibitor did not affect the surface expression of WT hASIC1b. These data show that the two PKC consensus sites S40 and S499 differentially regulate hASIC1b and mediate the effects of PKC activation or PKC inhibition on hASIC1b. This will result in a deeper understanding of PKC regulation of this channel in glioma cells, information that may help in designing potentially beneficial therapies in their treatment.

Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain

ABSTRACT Introduction: Currently the treatment of chronic pain is inadequate and compromised by debilitating central nervous system side effects. Here we discuss new therapeutic strategies that target dorsal root ganglia (DRGs) in the peripheral nervous system for a better and safer treatment of chronic pain. Areas covered: The DRGs contain the cell bodies of primary sensory neurons including nociceptive neurons. After painful injuries, primary sensory neurons demonstrate maladaptive molecular changes in DRG cell bodies and in their axons. These changes result in hypersensitivity and hyperexcitability of sensory neurons (peripheral sensitization) and are crucial for the onset and maintenance of chronic pain. We discuss the following new strategies to target DRGs and primary sensory neurons as a means of alleviating chronic pain and minimizing side effects: inhibition of sensory neuron-expressing ion channels such as TRPA1, TRPV1, and Nav1.7, selective blockade of C- and Aβ-afferent fibers, gene therapy, and implantation of bone marrow stem cells. Expert opinion: These peripheral pharmacological treatments, as well as gene and cell therapies, aimed at DRG tissues and primary sensory neurons can offer better and safer treatments for inflammatory, neuropathic, cancer, and other chronic pain states.