NAADP is a potent second messenger that mobilizes Ca 2؉from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca 2؉ release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca 2؉ -permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1 ( ؊/؊ cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP. Ca2ϩ plays a major role in the function of intracellular organelles including biosynthesis and membrane trafficking (1, 2). Although the mechanism controlling Ca 2ϩ in the endoplasmic reticulum has been studied extensively, very little is known about Ca 2ϩ homeostasis by other organelles. Furthermore, although accumulating evidence indicates that acidic organelles such as endosomes and lysosomes are dynamic Ca 2ϩ stores, the molecular basis for Ca 2ϩ release from these so-called "acidic Ca 2ϩ stores" (3) is at present defined poorly. By far, the best characterized route for mobilization of acidic Ca 2ϩ stores is through the production of the potent Ca 2ϩ -releasing second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) 5 (4). The Ca 2ϩ -mobilizing properties of NAADP were discovered in sea urchin eggs (5), in which NAADP was shown to mobilize Ca 2ϩ not from the endoplasmic reticulum but instead from lysosome-related organelles (6). Its effects have subsequently been extended to a variety of cell types, including pancreatic acinar and ␤ cells, smooth muscle cells, neurons, and breast cancer cells (4). NAADP is produced by several extracellular stimuli in an agonist-specific manner and implicated in a number of physiological responses, including fertilization, glucose sensing, exocytosis, and neuronal growth (4). Deregulated lysosomal Ca 2ϩ signaling may also result in disease (2,7,8). Despite the physiological and potential pathophysiological importance of NAADP signaling, the molecular identity of the NAADP receptor is not entirely clear (9). Recently, members of the transient receptor potential mucolipin (TRPML) (10, 11) and two-pore channel (TPC) (12-16) families, which are all present in the endo/lysosomal system, have been proposed as candidates.TRPMLs form a subfamily of the superfamily of the TRP channels. The founding member is TRPML1, which was ide...

Key points• The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K + transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells.• The EP depends upon the diffusion potential elicited by a large K + gradient across the apical surface of the syncytium.• We examined by electrophysiological approaches an involvement of Na + ,K + -ATPase, which occurs at the syncytium's basolateral surface comprising the fibrocytes' membranes and would mediate K + transport across the lateral wall, in maintenance of the EP.• We show that the Na + ,K + -ATPase sustains the syncytium's high [K + ] that is crucial for the K + gradient across the apical surface of the syncytium.• The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus.Abstract The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K + transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K + concentration ([K + ]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K + gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K + transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K + while perfusing into the perilymph of the scala tympani blockers of Na + ,K + -ATPase, the K + pump thought to be essential to the system. Inhibiting Na

Lysosomal storage diseases (LSDs) are caused by inability of cells to process the material captured during endocytosis. While they are essentially diseases of cellular "indigestion", LSDs affect large number of cellular activities and, as such, they teach us about the integrative function of the cell, as well as about the gaps in our knowledge of the endocytic pathway and membrane transport. The present review summarizes recent findings on Ca 2+ handling in LSDs and attempts to identify the key questions on alterations inCa 2+ signaling and membrane transport in this group of diseases, answers to which may lie in delineating the cellular pathogeneses of LSDs. Lysosomal storage diseases as a model for the integrative function of the cellLysosomal storage diseases (LSDs) area diverse set of conditions that impair the uptake, sorting, or digestion of the material captured by cells during endocytosis or claimed by autophagy [1,2]. Degradation of endocytosed extracellular matter and plasma membrane components is a complex process that involves selective membrane fusion, protein and lipid sorting and degradation, and absorption of the products of digestion [3,4]. LSDs occur due to mutations in genes that code for components of the cellular endocytic machinery, or they can be caused by environmental influences such as toxic metals or drugs [5][6][7]. LSDs caused by gene mutations result in improperly delivered, structurally dysfunctional, or acutely inhibited lysosomal digestive enzymes; in some cases LSDs-causing mutations affect absorption of the products of digestion. Additionally, recently accumulated data suggests impaired membrane flow in the endocytic pathway, whether directly or indirectly induced by the genetic mutations, as a contributing factor in LSDs pathogenesis [8].Although they are rarely discussed in the context of LSDs, chemically-induced dysregulated lysosomal function due to acute poisoning or chronic buildup of inhibitors (such as in irondependent lipofuscin buildup in aging cells [9,10], and, perhaps, in Mucolipidosis type IV [11]), share elements of causality as well as cellular and clinical manifestations with the "genetically-induced" LSDs.The inability of cells affected by LSDs to properly handle endocytosed material results in buildup of storage bodies, which are malformed endocytic organelles filled with undigested or © 2009 Elsevier Ltd. All rights reserved.Address correspondence to: Kirill Kiselyov, Department of Biological Sciences, University of Pittsburgh, 519 Langley Hall, 4249 Fifth Ave, Pittsburgh, PA, 15260, USA. Kiselyov@pitt.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal d...

Hydrogen sulfide (H 2 S) is a gaseous neuromodulator produced from L-cysteine. H 2 S is generated by three distinct enzymatic pathways mediated by cystathionine c-lyase (CSE), cystathionine b-synthase (CBS), and mercaptopyruvate sul-furtransferase (MPST) coupled with cysteine aminotransfer-ase (CAT). This study investigated the relative contributions of these three pathways to H 2 S production in PC12 cells (rat pheochromocytoma-derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H 2 S were produced from L-cysteine in the presence of a-ketoglutarate, together with dithiothreitol. The production of H 2 S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L-aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocyto-chemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H 2 S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H 2 S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H 2 S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H 2 S generation. growth factor; PAG, DL-propargylglycine; PGP 9.5, protein gene product 9.5; PLP, pyridoxal 5 0-phosphate; SAMe, S-adenocyl-L-methionine; TRPA1, transient receptor potential channel ankyrin 1; a-KG, a-ketoglutarate.

Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits (alpha-, beta-, and gamma-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant (Ki) value of 0.20 microM. RT-PCR analysis confirmed the presence of transcripts of alpha-, beta-, and gamma-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a Ki value of 0.12 microM at a membrane potential of -64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8) > Na+ (1) >> K+ ( approximately 0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of alpha-, beta-, and gamma-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.