Quantitative Analysis of the Voltage-dependent Gating of Mouse Parotid ClC-2 Chloride Channel

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (Vm) and time dependence of the protopore (Pf) and common (Ps) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (ICl) were measured using whole cell patch clamp. WT channels had ICl that showed inward rectification and biexponential time course. Time constants of fast and slow components were ∼10-fold different at negative Vm and corresponded to Pf and Ps, respectively. Pf and Ps were ∼1 at −200 mV, while at Vm ≥ 0 mV, Pf ∼ 0 and Ps ∼ 0.6. Hence, Pf dominated open kinetics at moderately negative Vm, while at very negative Vm both gates contributed to gating. At Vm ≥ 0 mV, mClC-2 closes by shutting off Pf. Three- and two-state models described the open-to-closed transitions of Pf and Ps, respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter Pf or Ps in other ClC channels. Based on the time and Vm dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to Pf, the dominant regulator of gating, while the C258 residue alters the Vm dependence of Pf, probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue

The inositol 1,4,5-trisphosphate receptor in C. elegans

The soil nematode Caenorhabditis elegans is a genetic model organism whose cellular physiology is closely related to that of mammals, with many signaling cascades and second messengers mirroring those found in higher organisms. Due to the genetic, anatomical, and behavioral simplicity of worms, integrative physiological techniques are relatively straightforward and represent a powerful approach to understand the molecular mechanisms underlying more complex system functions. Studies of the nematode inositol 1,4,5-trisphosphate receptor (InsP 3 R) have led to advances in our understanding of its role in development and behavior

Distinct roles for two Caenorhabditis elegans acid-sensing ion channels in an ultradian clock

Biological clocks are fundamental to an organism’s health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans. An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca2+ wave and forms a core component of the master oscillator that controls the timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca2+ signalling, the generation of an ultradian oscillator, and its metabolic consequences

SLO-2 Is Cytoprotective and Contributes to Mitochondrial Potassium Transport

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance “big” K+ channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K+ transport and APC. The canonical Ca2+-activated BK (or “maxi-K”) channel SLO1 was dispensable for both mitochondrial K+ transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K+ channel SLO2 was required, and that SLO2-dependent mitochondrial K+ transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K+

Integrated calcium and proton signaling during a rhythmic behavior in C. elegans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pharmacology and Physiology, 2014.Biological rhythms are intrinsic to nearly all organisms; yet, they can also be very complex. The genetic model organism C. elegans exhibits several behavioral rhythms that have been studied using integrative physiology. One rhythm in particular, the defecation motor program (DMP), has been shown to be timed by cell-autonomous calcium oscillations. Subsequent trans-epithelial proton fluxes contribute to the DMP’s behavioral output as well as to physiologic events such as development and lifespan. In addition to defining mechanisms that contribute to oscillatory calcium signaling, studies focused on this behavior have emphasized the importance of pH homeostasis and have led to the identification of a novel role for protons in signaling. The work presented here demonstrates that the establishment of an apical intestinal proton gradient, driven by a proton V-ATPase, is necessary for nutrient uptake and development. Proton movement across the basolateral membrane, through the Na+/H+ exchanger NHX-7, is also critical for proper execution of the DMP but not required for pH homeostasis or development. This flux signals posterior body wall muscle contraction and coincides with the underlying IP3R-mediated calcium wave, suggesting calcium-dependent regulation. Structure-function analyses of NHX-7 revealed that its activity is impacted by calcium signaling and feedback from the membrane proton/sodium gradient. Interestingly, the structure-function analysis also indicated that disruption of a consensus binding site for a calcineurin homologous protein, PBO-1, altered the distribution of NHX-7. Genetic analyses further revealed that loss of PBO-1 also affects the localization of the NHX-2 isoform in the apical membrane, which helps to reestablish pH homeostasis following defecation. This ultimately results in a phenotype that resembles a global disruption of intestinal proton transport. The localization deficit appears to be an effect on membrane stability/retention rather than forward protein trafficking. Collectively, these studies support the idea that proton and calcium oscillations synergize to execute the DMP as a means to maintain the intestinal membrane proton motive force. Thus, in addition to contributing to our understanding of oscillatory calcium signaling, pH homeostasis, and proton signaling, our results show that the defecation motor program is an appropriate model to study and potentially target biological rhythms therapeutically

Hypoxia and the Mitochondrial Unfolded Protein Response

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology, 2017.Mitochondria are central to cellular metabolism, generating ATP through oxidative phosphorylation. Additionally, mitochondria are signaling organelles that regulate stress-induced cell death and adaptation. Altered production of small molecule metabolites and/or changes in the localization and distribution of mitochondrial proteins can contribute to signaling cascades that impact mitochondrial and cellular fitness. In particular, perturbations in mitochondrial protein homeostasis trigger a compartment-specific mitochondrial unfolded protein response (UPRmt), which protects against a broad range of stressors including reactive oxygen species (ROS), respiratory chain dysfunction, mitochondrial DNA damage and pathologic bacteria. Furthermore, mitochondrial stress in one tissue can activate the UPRmt in distal tissues suggesting mitochondrial health is monitored remotely for prophylactic adaptive purposes. In the genetic model organism Caenorhabditis elegans, canonical UPRmt signaling requires the transcription factor Activating Transcription Factor associated with Stress 1 (ATFS-1), which contains dual mitochondrial and nuclear localization motifs. Healthy mitochondria import and degrade ATFS-1; whereas dysfunctional mitochondria fail to import ATFS-1 resulting in its nuclear accumulation and binding to promoters of UPRmt target genes. Here, we show that either genetic or pharmacologic stresses that induce UPRmt protect against anoxia-reperfusion (A/R) injury in C. elegans. ATFS-1 is essential for the protective effects of UPRmt activation, and gain-of-function (gf) atfs-1 mutants that exhibit constitutive UPRmt activation are protected against A/R injury. To investigate cell non-autonomous UPRmt signaling we developed a novel genetic system to restrict atfs-1(gf) expression to a single tissue, and using this model we find that atfs-1(gf) alone triggers an autonomous UPRmt in the tissue of expression, but does not activate UPRmt in other tissues. Likewise, cells with atfs-1(gf) showed cell autonomous protection from A/R, but protection is not extended to remote tissues. Our findings contrast with de facto mitochondrial stress, which initiates both local and remote protection. This suggests ATFS-1's centrality is limited to cell autonomous responses to stress and that an alternate pathway underlies remote responses. Preliminary work is also described testing the hypothesis that metabolites accumulated under mitochondrial stress can function as signaling molecules. Specifically, genetic reagents were developed to assess the potential signaling roles of L-2-hydroxyglutarate (L-2-HG), which is produced by lactate dehydrogenase (LDH) under hypoxia, and its enantiomer D-2-HG, which is produced by cancer associated mutations in isocitrate dehydrogenases (IDH1 or IDH2). Both L- and D-2-HG can inhibit epigenetic regulatory enzymes (e.g. HIF prolyl-hydroxylases and JmjC histone demethylases) which utilize α-ketoglutarate as a substrate, providing a potential link between hypoxia and epigenetic modifications that influence UPRmt gene induction. These reagents will be useful to determine the impact of modulating 2-HGs levels under stress, and to investigate their potential signaling roles in the UPRmt. Overall, the results presented herein provide new insights to mitochondrial nuclear-communication and signaling during A/R stress. These insights will help to direct future work in mito-centric and hypoxia-relevant mammalian disease fields including cancer, stem cell biology, and ischemia-reperfusion injuries such as heart attack and stroke, where the UPRmt may contribute to cell survival