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CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. \"Fast\" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of \"slow\" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate \"slow\" inactivation of sodium channels, but little is known about if/how second messengers control \"slow\" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from \"slow\" inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating \"slow\" inactivation. We postulate that the kinetics of recovery from \"slow\" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca)) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca). We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

The dorsal root ganglia (DRG) house the primary afferent neurons responsible for somatosensation, including pain. We previously identified Jedi-1 (PEAR1/MEGF12) as a phagocytic receptor expressed by satellite glia in the DRG involved in clearing apoptotic neurons during development. Here, we further investigated the function of this receptor in vivo using Jedi-1 null mice. In addition to satellite glia, we found Jedi-1 expression in perineurial glia and endothelial cells, but not in sensory neurons. We did not detect any morphological or functional changes in the glial cells or vasculature of Jedi-1 knockout mice. Surprisingly, we did observe changes in DRG neuron activity. In neurons from Jedi-1 knockout (KO) mice, there was an increase in the fraction of capsaicin-sensitive cells relative to wild type (WT) controls. Patch-clamp electrophysiology revealed an increase in excitability, with a shift from phasic to tonic action potential firing patterns in KO neurons. We also found alterations in the properties of voltage-gated sodium channel currents in Jedi-1 null neurons. These results provide new insight into the expression pattern of Jedi-1 in the peripheral nervous system and indicate that loss of Jedi-1 alters DRG neuron activity indirectly through an intercellular interaction between non-neuronal cells and sensory neurons.

N- and P/Q-type Ca2+channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca2+channels are multisubunit proteins, consisting of a pore-forming α1and accessory β and α2δ subunits each encoded by multiple genes and splice variants. β subunits alter current amplitude and kinetics. The β2asubunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in β2a. In the current manuscript, we studied steady state inactivation properties of native N-and P/Q-type Ca2+channels and recombinant N-type Ca2+channels. When bovine α1Band β2aand human α2δ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P/Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known β subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in β subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca2+channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca2+channels containing β2abut not β2bor β2asubunits mutated to be palmitoylation deficient. Our results suggest that Ca2+channels containing β2asubunits may be regulated by dynamic palmitoylation.

Calpains Play a Role in Insulin Secretion and Action Seamus K. Sreenan 1 , Yun-Ping Zhou 1 , Kenichi Otani 1 8 , Polly A. Hansen 6 , Kevin P.M. Currie 2 , Chien-Yuan Pan 2 , Jean-Pyo Lee 1 , Diane M. Ostrega 1 , William Pugh 1 , Yukio Horikawa 3 5 , Nancy J. Cox 1 4 , Craig L. Hanis 7 , Charles F. Burant 6 , Aaron P. Fox 2 , Graeme I. Bell 1 3 4 5 and Kenneth S. Polonsky 8 1 Medicine 2 Neurobiology, Pharmacology and Physiology 3 Biochemistry and Molecular Biology, and 4 Human Genetics, and the 5 Howard Hughes Medical Institute, the University of Chicago, Chicago, Illinois 6 Department of Cell Biology, Parke-Davis Pharmaceutical Research Division/Warner-Lambert Company, Ann Arbor, Michigan 7 Human Genetics Center, the University of Texas Health Science Center at Houston, Houston, Texas 8 Department of Medicine, Washington University, St. Louis, Missouri Abstract Studies of the genetic basis of type 2 diabetes suggest that variation in the calpain-10 gene affects susceptibility to this common disorder, raising the possibility that calpain-sensitive pathways may play a role in regulating insulin secretion and/or action. Calpains are ubiquitously expressed cysteine proteases that are thought to regulate a variety of normal cellular functions. Here, we report that short-term (4-h) exposure to the cell-permeable calpain inhibitors calpain inhibitor II and E-64-d increases the insulin secretory response to glucose in mouse pancreatic islets. This dose-dependent effect is observed at glucose concentrations above 8 mmol/l. This effect was also seen with other calpain inhibitors with different mechanisms of action but not with cathepsin inhibitors or other protease inhibitors. Enhancement of insulin secretion with short-term exposure to calpain inhibitors is not mediated by increased responses in intracellular Ca 2+ or increased glucose metabolism in islets but by accelerated exocytosis of insulin granules. In muscle strips and adipocytes, exposure to both calpain inhibitor II and E-64-d reduced insulin-mediated glucose transport. Incorporation of glucose into glycogen in muscle also was reduced. These results are consistent with a role for calpains in the regulation of insulin secretion and insulin action. Footnotes Address correspondence and reprint requests to Kenneth S. Polonsky, Department of Medicine, Washington University, 660 S. Euclid, Campus Box 8066, St. Louis, MO 63110. E-mail: polonsky{at}im.wustl.edu . Received for publication 21 June 2000 and accepted in revised form 24 May 2001. S.K.S. is currently affiliated with the Department of Endocrinology, Royal College of Surgeons, James Connolly Memorial Hospital, Blanchardstown, Dublin, Ireland. Y.H. is currently affiliated with the Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan. ANOVA, analysis of varience; AUC, area under the curve; 2-DOG, 2-deoxyglucose; KHB, Krebs-Henseleit buffer; KRB, Krebs-Ringer’s buffer; 3-MG, 3- O -[ 3 H]methyl- d -glucose; TEA, tetraethylammonium.

CaV2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. \"Fast\" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of \"slow\" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate \"slow\" inactivation of sodium channels, but little is known about if/how second messengers control \"slow\" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from \"slow\" inactivation, but an inactive control (4 alpha -PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel beta -subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP- beta -S reduced the effect of PMA suggesting a role for G proteins in modulating \"slow\" inactivation. We postulate that the kinetics of recovery from \"slow\" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

N- and P/Q-type Ca 2+ channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca 2+ channels are multisubunit proteins, consisting of a pore-forming α 1 , and accessory β and α 2 δ subunits each encoded by multiple genes and splice variants. β subunits alter current amplitude and kinetics. The β 2a subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in β 2a . In the current manuscript, we studied steady state inactivation properties of native N- and P/Q-type Ca 2+ channels and recombinant N-type Ca 2+ channels. When bovine α 1B and β 2a and human α 2 δ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P/Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known β subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in β subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca 2+ channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca 2+ channels containing β 2a but not β 2b or β 2a subunits mutated to be palmitoylation deficient. Our results suggest that Ca 2+ channels containing β 2a subunits may be regulated by dynamic palmitoylation.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Effective conservation and management of pond‐breeding amphibians depends on the accurate estimation of population structure, demographic parameters, and the influence of landscape features on breeding‐site connectivity. Population‐level studies of pond‐breeding amphibians typically sample larval life stages because they are easily captured and can be sampled nondestructively. These studies often identify high levels of relatedness between individuals from the same pond, which can be exacerbated by sampling the larval stage. Yet, the effect of these related individuals on population genetic studies using genomic data is not yet fully understood. Here, we assess the effect of within‐pond relatedness on population and landscape genetic analyses by focusing on the barred tiger salamanders (Ambystoma mavortium) from the Nebraska Sandhills. Utilizing genome‐wide SNPs generated using a double‐digest RADseq approach, we conducted standard population and landscape genetic analyses using datasets with and without siblings. We found that reduced sample sizes influenced parameter estimates more than the inclusion of siblings, but that within‐pond relatedness led to the inference of spurious population structure when analyses depended on allele frequencies. Our landscape genetic analyses also supported different models across datasets depending on the spatial resolution analyzed. We recommend that future studies not only test for relatedness among larval samples but also remove siblings before conducting population or landscape genetic analyses. We also recommend alternative sampling strategies to reduce sampling siblings before sequencing takes place. Biases introduced by unknowingly including siblings can have significant implications for population and landscape genetic analyses, and in turn, for species conservation strategies and outcomes. We use SNP data from the pond‐breeding amphibian Ambystoma mavortium generated using a ddRADseq approach to test the effect of within‐pond relatedness on population and landscape genetic analyses. We find that both the inclusion of siblings, but also reduced sample sizes following sibling removal, affected analyses. In particular, we find that analyses dependent on allele frequencies are especially susceptible to sibling‐induced biases and discuss the implications of these findings to future studies.

Burkholderia pseudomallei causes the potentially fatal disease melioidosis. It is generally accepted that B. pseudomallei is a noncommensal bacterium and that any culture-positive clinical specimen denotes disease requiring treatment. Over a 23-year study of melioidosis cases in Darwin, Australia, just one patient from 707 survivors has developed persistent asymptomatic B. pseudomallei carriage. To better understand the mechanisms behind this unique scenario, we performed whole-genome analysis of two strains isolated 139 months apart. During this period, B. pseudomallei underwent several adaptive changes. Of 23 point mutations, 78% were nonsynonymous and 43% were predicted to be deleterious to gene function, demonstrating a strong propensity for positive selection. Notably, a nonsense mutation inactivated the universal stress response sigma factor RpoS, with pleiotropic implications. The genome underwent substantial reduction, with four deletions in chromosome 2 resulting in the loss of 221 genes. The deleted loci included genes involved in secondary metabolism, environmental survival, and pathogenesis. Of 14 indels, 11 occurred in coding regions and 9 resulted in frameshift mutations that dramatically affected predicted gene products. Disproportionately, four indels affected lipopolysaccharide biosynthesis and modification. Finally, we identified a frameshift mutation in both P314 isolates within wcbR , an important component of the capsular polysaccharide I locus, suggesting virulence attenuation early in infection. Our study illustrates a unique clinical case that contrasts a high-consequence infectious agent with a long-term commensal infection and provides further insights into bacterial evolution within the human host. IMPORTANCE Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens. Some bacterial pathogens establish long-term infections that are difficult or impossible to eradicate with current treatments. Rapid advances in genome sequencing technologies provide a powerful tool for understanding bacterial persistence within the human host. Burkholderia pseudomallei is considered a highly pathogenic bacterium because infection is commonly fatal. Here, we document within-host evolution of B. pseudomallei in a unique case of human infection with ongoing chronic carriage. Genomic comparison of isolates obtained 139 months (11.5 years) apart showed a strong signal of adaptation within the human host, including inactivation of virulence and immunogenic factors, and deletion of pathways involved in environmental survival. Two global regulatory genes were mutated in the 139-month isolate, indicating extensive regulatory changes favoring bacterial persistence. Our study provides insights into B. pseudomallei pathogenesis and, more broadly, identifies parallel evolutionary mechanisms that underlie chronic persistence of all bacterial pathogens.