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Puertollano, R., and K. Kiselyov (2009) TRPMLs: In sickness and in health. Am. J. Physiol. Renal Physiol. 0:In Press TRPML1, TRPML2 and TRPML3 belong to the mucolipin family of the TRP superfamily of ion channels. The founding member of this family, TRPML1 was cloned during the search for the genetic determinants of the lysosomal storage disease mucolipidosis type IV (MLIV). Mucolipins are predominantly expressed within the endocytic pathway where they appear to regulate membrane traffic and/or degradation. The physiology of mucolipins raises some of the most interesting questions of the modern cell biology. Their traffic and localization is a multi-step process involving a system of adaptor proteins, while their ion channel activity possibly exemplifies the rare cases of regulation of endocytic traffic and hydrolysis by ion channels. Finally, dysregulation of mucolipins results in cell death leading to neurodegenerative phenotypes of MLIV and of the varitint-waddler mouse model of familial deafness. The present review discusses the current knowledge and questions regarding this novel family of disease-relevant ion channels with a specific focus on mucolipin regulation and their role in membrane traffic and cell death. Since mucolipins are ubiquitously expressed, this review may be useful for a wide audience of basic biologists and clinicians. Key words: mucolipin, endocytosis, ion channel, membrane traffic, lysosome. Klionsky, D.J., and . et alia (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151-175 Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response. Kiselyov, K., and S. Muallem (2008) Mitochondrial Ca2+ homeostasis in lysosomal storage diseases. Cell Calcium 44:103-111 Lysosomal storage diseases (LSDs) are a class of genetic disorders in which proteins responsible for digestion or absorption of endocytosed material do not function or do not localize properly. The resulting cellular "indigestion" causes buildup of intracellular storage inclusions that contain unprocessed lipids and proteins that form macromolecular complexes. The buildup of storage material is associated with degenerative processes that are observed in all LSDs, albeit the correlation between the amount of storage inclusions and the severity of the degenerative processes is not always evident. The latter suggests that a specific mechanism set in motion by aberrant lysosomal function drives the degenerative processes in LSDs. It is becoming increasingly clear that in addition to their function in degrading endocytosed material, lysosomes are essential housekeeping organelles responsible for maintaining healthy population of intracellular organelles, in particular mitochondria. The present review surveys the current knowledge on the lysosomal-mitochondrial axis and its possible role as a contributing factor to mitochondrial Ca(2+) homeostasis and to cell death in LSDs. Kim, H.J., Q. Li, S. Tjon-Kon-Sang, I. So, K. Kiselyov, A.A. Soyombo, and S. Muallem (2008) A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype. EMBO J 27:1197-1205 TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca(2+)-permeable channel with a unique form of regulation by extracytosolic (luminal) H(+) (H(+)(e-cyto)). Regulation by H(+)(e-cyto) is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H(+)(e-cyto) to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H(+)(e-cyto), and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H(+)(e-cyto) are altered by both the alpha-helix-destabilizing A419G and the alpha-helix-favouring A419M and A419K. These findings suggest that regulation of TRPML3 by H(+)(e-cyto) is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication. Miedel, M.T., Y. Rbaibi, C.J. Guerriero, G. Colletti, K.M. Weixel, O.A. Weisz, and K. Kiselyov (2008) Membrane traffic and turnover in TRP-ML1-deficient cells: a revised model for mucolipidosis type IV pathogenesis. J. Exp. Med. 205:1477-1490 The lysosomal storage disorder mucolipidosis type IV (MLIV) is caused by mutations in the transient receptor potential-mucolipin-1 (TRP-ML1) ion channel. The "biogenesis" model for MLIV pathogenesis suggests that TRP-ML1 modulates postendocytic delivery to lysosomes by regulating interactions between late endosomes and lysosomes. This model is based on observed lipid trafficking delays in MLIV patient fibroblasts. Because membrane traffic aberrations may be secondary to lipid buildup in chronically TRP-ML1-deficient cells, we depleted TRP-ML1 in HeLa cells using small interfering RNA and examined the effects on cell morphology and postendocytic traffic. TRP-ML1 knockdown induced gradual accumulation of membranous inclusions and, thus, represents a good model in which to examine the direct effects of acute TRP-ML1 deficiency on membrane traffic. Ratiometric imaging revealed decreased lysosomal pH in TRP-ML1-deficient cells, suggesting a disruption in lysosomal function. Nevertheless, we found no effect of TRP-ML1 knockdown on the kinetics of protein or lipid delivery to lysosomes. In contrast, by comparing degradation kinetics of low density lipoprotein constituents, we confirmed a selective defect in cholesterol but not apolipoprotein B hydrolysis in MLIV fibroblasts. We hypothesize that the effects of TRP-ML1 loss on hydrolytic activity have a cumulative effect on lysosome function, resulting in a lag between TRP-ML1 loss and full manifestation of MLIV. van Rossum, DB, D. Oberdick, Y. Rbaibi, G. Bhardwaj, R.K. Barrow, S.H. Snyder, K. Kiselyov, and R.L. Patterson (2008) TRP_2: A lipid-binding/trafficking domain that mediates diacylglycerol-induced vesicle fusion. J. Biol. Chem. 28:11778-11784 We recently modeled Transient Receptor Potential Channels (TRP) channels using the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool(GDDA-BLAST) which derives structural,functional, and evolutionary information from primary amino acid sequence using phylogenetic profiles (1;2). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid-binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG) -sensitive and DAGinsensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2 which alter lipid-binding also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane in response to DAG mobilizing agents, without disrupting SNARE interactions. Importantly, these data accord with the known role of DAG in membrane destabilization which facilitates SNARE-dependent synaptic vesicle fusion (3;4). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality which can have in vivo and mechanistic relevance. Cornell, R.A., M. Aarts, D. Bautista, J. Garcia-Anoveros, K. Kiselyov, and E.R. Liman (2008) A double TRPtych: six views of transient receptor potential channels in disease and health. J Neurosci 28:11778-11784 At the 2008 Annual Meeting of the Society for Neuroscience, a Mini-Symposium entitled "Contributions to TRP Channels to Neurological Disease" included talks from six heads of newly established laboratories, each with a unique research focus, model system, and set of experimental tools. Some of the questions addressed in these talks include the following. What is the role of transient receptor potential (TRP) channels in pain perception? How do normally functioning TRP channels contribute to cell death pathways? What are the characteristics of TRPpathies, disease states that result from overactive or underactive TRP channels? How are TRP channels regulated by signal transduction cascades? This review summarizes recent results from those laboratories and provides six perspectives on the subject of TRP channels and disease. van Rossum, D.B., D. Oberdick, Y. Rbaibi, G. Bhardwaj, R.K. Barrow, N. Nikolaidis, S.H. Snyder, K. Kiselyov, and R.L. Patterson (2008) TRP_2, a lipid/trafficking domain that mediates diacylglycerol-induced vesicle fusion. J Biol Chem 283:34384-34392 We recently modeled transient receptor potential (TRP) channels using the Gestalt Domain Detection Algorithm-Basic Local Alignment Tool (GDDA-BLAST), which derives structural, functional, and evolutionary information from primary amino acid sequences using phylogenetic profiles ( Ko, K. D., Hong, Y., Chang, G. S., Bhardwaj, G., van Rossum, D. B., and Patterson, R. L. (2008) Physics Arch. Quant. Methods arXiv: 0806.2394v1 ). Herein we test our functional predictions for the TRP_2 domain of TRPC3; a domain of unknown function that is conserved in all TRPC channels. Our functional models of this domain identify both lipid binding and trafficking activities. In this study, we reveal: (i) a novel structural determinant of ion channel sensitivity to lipids, (ii) a molecular mechanism for the difference between diacylglycerol (DAG)-sensitive and DAG-insensitive TRPC subfamilies, and (iii) evidence that TRPC3 can comprise part of the vesicle fusion machinery. Indeed, the TRPC3 TRP_2 domain mediates channel trafficking to the plasma membrane and binds to plasma membrane lipids. Further, mutations in TRP_2, which alter lipid binding, also disrupt the DAG-mediated fusion of TRPC3-containing vesicles with the plasma membrane without disrupting SNARE interactions. Importantly, these data agree with the known role of DAG in membrane destabilization, which facilitates SNARE-dependent synaptic vesicle fusion ( Villar, A. V., Goni, F. M., and Alonso, A. (2001) FEBS Lett. 494, 117-120 and Goni, F. M., and Alonso, A. (1999) Prog. Lipid Res. 38, 1-48 ). Taken together, functional models generated by GDDA-BLAST provide a computational platform for deriving domain functionality, which can have in vivo and mechanistic relevance. Kim, H.J., Q. Li, S. Tjon-Kon-Sang, I. So, K. Kiselyov, and S. Muallem (2007) Gain-of-function mutation in TRPML3 causes the mouse varitint-waddler phenotype. J. Biol. Chem. 282:36138-36142 TRPML3 is a member of the TRPML subfamily of the TRP cation channels superfamily. The TRPML3(A419P) mutation causes a severe form while the TRPML3(I362T/A419P) mutation results in a mild form of the varitint-waddler phenotype. The channel properties of TRPML3 and how the mutations cause each phenotype are not known. The present study reports the first channel properties of TRPML3 as a strongly inward rectifying cation channel with a novel regulation by extracytosolic Na+. Pre-incubating the extracytosolic face of TRPML3 in Na+-free media is required for channel activation, but then the channel slowly inactivates. The A419P mutation locks the channel in an open state that shows no inactivation. Similar gain-of-function was observed with the A419G mutation that, like A419P, is expected to destabilize the -helical fifth transmembrane domain of TRPML3. The I362T mutation alone results in inactive channel, but, surprisingly, channel properties of TRPML3(I362T/ A419P) are similar to those of TRPML3 (A419P). However surface expression and current density of TRPML3(I362T/A419P) are lower than those of TRPML3 (A419P). The A419P mutation also affects channel glycosylation and causes massive cell death. These findings show that the varitint-waddler phenotype is due to a gain-of-function of TRPML3(A419P) that is reduced by the TRPML3(I362T/A419P) mutant to result in a milder phenotype. Vergarajauregui, S., R. Oberdick, K. Kiselyov, and R. Puertollano (2007) Mucolipin-1 channel activity is regulated by protein kinase A mediated phosphorylation. Biochem. J. 410:417-425 Mucolipins constitute a family of cation channels with homology to the transient receptor potential family. Mutations in mucolipin-1 (MCOLN1) have been linked to mucolipidosis type IV, a recessive lysosomal storage disease characterized by severe neurological and ophthalmologic abnormalities. At present, little is known about the mechanisms that regulate MCOLN1 activity. Here, we addressed whether MCOLN1 activity is regulated by phosphorylation. We identified two protein kinase A (PKA) consensus motifs in the C-terminal tail of MCOLN1 containing serine 557 and serine 559. Serine 557 was the principal phosphorylation site, as mutation of this residue to alanine caused greater that a 75% reduction in the total levels of phosphorylated MCOLN1 C-terminal tail. Activation of PKA with forskolin promoted MCOLN1 phosphorylation both in vitro and in vivo. In contrast, addition of the PKA inhibitor H89 abolished MCOLN1 phosphorylation. We also found that PKA-mediated phosphorylation regulates MCOLN1 channel activity. Treatment with forskolin decreased MCOLN1 channel activity, while H89 treatment increased MCOLN1 channel activity. The stimulatory effect of H89 on MCOLN1 function was not observed when serine 557 and serine 559 were mutated to alanine, indicating that these two residues are essential for PKA-mediated negative regulation of MCOLN1. This study constitutes the first example of regulation of a member of the mucolipin family by phosphorylation. Worley, P.F., W. Zeng, G. Huang, J.Y. Kim, D.M. Shin, M.S. Kim, J.P. Yuan, K. Kiselyov, and S. Muallem (2007) Homer proteins in Ca2+ signaling by excitable and non-excitable cells. Cell Calcium 42:363-371 Homers are scaffolding proteins that bind Ca(2+) signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca(2+) signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca(2+) signaling complex in an isoform-specific manner. Homer2 increases the GAP activity of RGS proteins and PLCbeta that accelerate the GTPase activity of Galpha subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP(3)Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca(2+) channels Ca(v)1.2 and Ca(v)1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Ca(v)1.2 to regulate E-C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca(2+) signaling and create a negative bias that can be reversed by the immediate early gene form of Homer1. Hence, the Homers should be viewed as the buffers of Ca(2+) signaling that ensure a high spatial and temporal fidelity of the Ca(2+) signaling and activation of downstream effects. Kiselyov, K., J.J. .J.r. Jennigs, Y. Rbaibi, and C.T. Chu (2007) Autophagy, mitochondria and cell death in lysosomal storage diseases. Autophagy 3:259-262 Lysosomal storage diseases (LSDs) are debilitating genetic conditions that frequently manifest as neurodegenerative disorders. They severely affect eye, motor and cognitive functions and, in most cases, abbreviate the lifespan. Postmitotic cells such as neurons and mononuclear phagocytes rich in lysosomes are most often affected by the accumulation of undegraded material. Cell death is well documented in parts of the brain and in other cells of LSD patients and animal models, although little is known about mechanisms by which death pathways are activated in these diseases, and not all cells exhibiting increased storage material are affected by cell death. Lysosomes are essential for maturation and completion of autophagy-initiated protein and organelle degradation. Moreover, accumulation of effete mitochondria has been documented in postmitotic cells whose lysosomal function is suppressed or in aging cells with lipofuscin accumulation. Based upon observations in the literature and our own data showing s Kiselyov, K., D.M. Shin, J.Y. Kim, J.P. Yuan, and S. Muallem (2007) TRPC channels: interacting proteins. Handb. Exp. Pharmacol. 2007:559-574 TRP channels, in particular the TRPC and TRPV subfamilies, have emerged as important constituents of the receptor-activated Ca2+ influx mechanism triggered by hormones, growth factors, and neurotransmitters through activation ofphospholipase C (PLC). Several TRPC channels are also activated by passive depletion of endoplasmic reticulum (ER) Ca2+. Although in several studies the native TRP channels faithfully reproduce the respective recombinant channels, more often the properties of Ca2+ entry and/or the store-operated current are strikingly different from that of the TRP channels expressed in the same cells. The present review aims to discuss this disparity in the context of interaction of TRPC channels with auxiliary proteins that may alter the permeation and regulation of TRPC channels. Kiselyov, K., A. Soyombo, and S. Muallem (2006) TRPpathies. J. Physiol. 578:641-653 Several members of the TRP family of ion channels have been implicated in human diseases such as mucolipidosis type IV, autosomal dominant polycystic kidney disease, familial focal segmental glomerulosclerosis, hypomagnesemia, and several forms of cancer. While the pathogenesis of some "TRPpathies", such as hypomagnesemia, appears straightforward (a lack of renal and intestinal Mg2+ absorption results in Mg2+ deficiency), in most cases the causal relationship between dysregulation of ion channels and the corresponding diseases remain obscure. The present review focuses on unanswered questions regarding pathogenesis of several TRPpathies for which clear linkage to mutations in TRP channels has been reported in humans. Kiselyov, K., X. Wang, D.M. Shin, W. Zang, and S. Muallem (2006) Calcium signaling complexes in microdomains of polarized secretory cells. Cell Calcium 40:451-459 The highly polarized nature of epithelial cells in exocrine glands necessitates targeting, assembly into complexes and confinement of the molecules comprising the Ca(2+) signaling apparatus, to cellular microdomains. Such high degree of polarized localization has been shown for all Ca(2+) signaling molecules tested, including G protein coupled receptors and their associated proteins, Ca(2+) pumps, Ca(2+) influx channels at the plasma membrane and Ca(2+) release channels in the endoplasmic reticulum. Although the physiological significance of polarized Ca(2+) signaling is clear, little is known about the mechanism of targeting, assembly and retention of Ca(2+) signaling complexes in cellular microdomains. The present review attempts to summarize the evidence in favor of polarized expression of Ca(2+) signaling proteins at the apical pole of secretory cells with emphasis on the role of scaffolding proteins in the assembly and function of the Ca(2+) signaling complexes. The consequence of polarized enrichment of Ca(2+) signaling complexes at the apical pole is generation of an apical to basal pole gradient of cell responsiveness that, at low physiological agonist concentrations, limits Ca(2+) spikes to the apical pole, and when a Ca(2+) wave occurs, it always propagates from the apical to the basal pole. Our understanding of Ca(2+) signaling in microdomains is likely to increase rapidly with the application of techniques to controllably and selectively disrupt components of the complexes and apply high resolution recording techniques, such as TIRF microscopy to this problem. Jennings, J.J., J.r., J.H. Zhu, Y. Rbaibi, X. Luo, C.T. Chu, and K. Kiselyov (2006) Mitochondrial aberrations in mucolipidosis type IV. J. Biol. Chem. 281:39041-39050 Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1 which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in Mucolipidosis type IV is unclear. Here we report that Mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca2+ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolpidoses. Mitochondria accumulate that can not properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca2+ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca2+-mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca2+ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.
Kim, J.Y., W. Zeng, K. Kiselyov, J.P. Yuan, M.H. Dehoff, K. Mikoshiba, P.F. Worely, and S. Muallem (2006) Homer 1 mediates store- and IP3Rs- dependent translocation and retrieval of TRPC3 to the plasma membrane. J. Biol. Chem. 281:32540-32549 Accumulating evidence indicates that members of the TRP channel family are components of store-operated Ca2+ channels (SOCs). Agonist stimulation activates SOCs and TRP channels directly and induces translocation of channels in intracellular vesicles to the plasma membrane (PM). The mechanism of TRP channel translocation in response to store depletion and agonist stimulation is not known. Here we use TRPC3 as a model system to show that IP3 and the scaffold Homer 1 (H1) regulate the rate of translocation and retrieval of TRPC3 from the PM. In resting cells TRPC3 exists in TRPC3-H1b/c-IP3Rs complexes that are located in part at the PM/ER interface and in part in intracellular vesicles. Binding of IP3 to the IP3Rs dissociates the interaction between IP3Rs and H1, but not between H1 and TRPC3. TIRF microscopy and biotinylation assays show robust receptor- and store- dependent translocation of TRPC3 to the PM, and its retrieval upon termination of cell stimulation. The translocation requires store depletion and is prevented by inhibition of the IP3Rs. In HEK293, dissociating H1b/c-IP3Rs with H1a results in TRPC3 translocation to the PM where it is spontaneously active. The TRPC3-H1b/c-IP3Rs complex is reconstituted by infusing recombinant cross linking H1c into these cells. Reconstitution is inhibited by IP3. Deletion of H1 in mice markedly reduces the rates of receptor- and store-stimulated translocation, and antagonist-mediated retrieval of TRPC3 present in intracellular organelles. Conversely, infusion of H1c into H1-/- cells eliminates spontaneous channel activity and increases the rate of channel activation by agonist stimulation. The effects of H1c are inhibited by IP3. These findings together with our earlier studies demonstrating gating of TRPC3 by IP3Rs are used to develop a molecular mechanism in which assembly of the TRPC3-H1b/c-IP3Rs complexes by H1b/c mediate both the translocation of TRPC3 to the PM and gating of TRPC3 by IP3Rs.
Soyombo, A.A., S. Tjon-Kon-Sang, Y. Rbaibi, E. Bashllari, J. Bisceglia, S. Muallem, and K. Kiselyov (2006) TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity. J. Biol. Chem. 281:7294-7301 Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.
Kiselyov, K., J. Chen, Y. Rbaibi, D. Oberdick, S. Tjon-Kon-Sang, N. Shcheynikov, S. Muallem, and A. Soyombo (2005) TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage. J. Biol. Chem. 280:43218-43223 Mutations in the gene MCOLN1 coding for the TRP (transient receptor potential) family ion channel TRP-ML1 lead to the lipid storage disorder mucolipidosis type IV (MLIV). The function and role of TRP-ML1 are not well understood. We report here that TRP-ML1 is a lysosomal monovalent cation channel. Both native and recombinant TRP-ML1 are cleaved resulting in two products. Recombinant TRP-ML1 is detected as the full-length form and as short N- and C-terminal forms, whereas in native cells mainly the cleaved N and C termini are detected. The N- and C-terminal fragments of TRP-ML1 were co-immunoprecipitated from cell lysates and co-eluted from a Ni2+ column. TRP-ML1 undergoes proteolytic cleavage that is inhibited by inhibitors of cathepsin B (CatB) and is altered when TRP-ML1 is expressed in CatB-/- cells. N-terminal sequencing of purified C-terminal fragment of TRP-ML1 expressed in Sf9 cells indicates a cleavage site at Arg200 downward arrow Pro201. Consequently, the conserved R200H mutation changed the cleavage pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channel activity. This work provides the first example of inactivation by cleavage of a TRP channel. The significance of the cleavage to the function of TRP-ML1 is under investigation.
Kiselyov, K., J.Y. Kim, W. Zeng, and S. Muallem (2005) Protein-protein interaction and functionTRPC channels. Pflug. Arch. Eur. J Phy. 451:116-124 Since their identification in the concluding years of the last century, the mammalian transient receptor potential (canonical) (TRPC) channels have remained in the limelight as the primary candidates for the Ca(2+) entry pathway activated by the hormones, growth factors, and neurotransmitters that exert their effect through activation of PLC. Although TRPC channels have been shown clearly to mediate, at least in part, receptor-activated Ca(2+) entry in literally all cell types, several of their central characteristics, as recorded in expression systems using recombinant channels, differ from those of the native receptor-dependent Ca(2+) influx channels. The present review attempts to highlight the interaction of TRPC channels with other proteins, which may explain the variability of TRPC channel activation and regulatory mechanisms observed with the native and recombinant channels. These include the homologous and heterotopous interactions of TRPC channel isoforms, the interaction of TRPC channels with calmodulin, PLCgamma, IP(3) receptors, and with scaffolding proteins like InaD, EBP50/NEHRF, caveolin, Janctate and Homers.
van Rossum, D.B., R.L. Patterson, K. Kiselyov, D. Boehning, R.K. Barrow, D.L. Gill, and S.H. Snyder (2004) Agonist-induced Ca2+ entry determined by inositol 1,4,5-trisphosphate recognition. Proc. Natl. Acad. Sci., USA 101:2323-2327 It has been considered that Ca2+ release is the causal trigger for Ca2+ entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (IP3R), the lack of Ca2+ entry in response to receptor activation is attributed to the absence of Ca2+ release. We reveal in this article that IP3R recognition of IP3 determines agonist-induced Ca2+ entry (ACE), independent of its Ca2+ release activity. In DT40 IP3R(-/-) cells, endogenous ACE can be rescued with type 1 IP3R mutants (both a DC-terminal truncation mutant and a D2550A pore mutant), which are defective in Ca2+ release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an IP3R-dependent manner without Ca2+ store release. Conversely, ACE cannot be rescued with mutant IP3Rs lacking IP3 binding (both the D90-110 and R265Q IP3-binding site mutants). We conclude that an IP3-dependent conformational change in the IP3R, not endoplasmic reticulum Ca2+ pool release, triggers ACE.
Kiselyov, K., D.M. Shin, and S. Muallem (2003) Signalling specificity in GPCR-dependent Ca2+ signalling. Cell. Signal. 15:243-253 Cells use signalling networks to translate with high fidelity extracellular signals into specific cellular functions. Signalling networks are often composed of multiple signalling pathways that act in concert to regulate a particular cellular function. In the centre of the networks are the receptors that receive and transduce the signals. A versatile family of receptors that detect a remarkable variety of signals are the G protein-coupled receptors (GPCRs). Virtually all cells express several GPCRs that use the same biochemical machinery to transduce their signals. Considering the specificity and fidelity of signal transduction, a central question in cell signalling is how signalling specificity is achieved, in particular among GPCRs that use the same biochemical machinery. Ca(2+) signalling is particularly suitable to address such questions, since [Ca(2+)](i) can be recorded with excellent spatial and temporal resolutions in living cells and tissues and now in living animals. Ca(2+) is a unique second messenger in that both biochemical and biophysical components form the Ca(2+) signalling complex to regulate its concentration. Both components act in concert to generate repetitive [Ca(2+)](i) oscillations that can be either localized or in the form of global, propagating Ca(2+) waves. Most of the key proteins that form Ca(2+) signalling complexes are known and their activities are reasonably well understood on the biochemical and biophysical levels. We review here the information gained from studying Ca(2+) signalling by GPCRs to gain further understanding of the mechanisms used to generate cellular signalling specificity.
Yuan, J.P., K. Kiselyov, D.M. Shin, J. Chen, N. Shcheynikov, S.H. Kang, M.H. Dehoff, M.K. Schwarz, P.H. Seeburg, S. Muallem, and P.F. Worley (2003) Homer binds TRPC family channels and is required for gating of TRPC1 by IP3 receptors. Cell 114:777-789 Receptor signaling at the plasma membrane often releases calcium from intracellular stores. For example, inositol triphosphate (IP3) produced by receptor-coupled phospholipase C activates an intracellular store calcium channel, the IP(3)R. Conversely, stores can induce extracellular calcium to enter the cell through plasma membrane channels, too. How this "reverse" coupling works was unclear, but store IP(3)Rs were proposed to bind and regulate plasma membrane TRP cation channels. Here, we demonstrate that the adaptor protein, termed Homer, facilitates a physical association between TRPC1 and the IP(3)R that is required for the TRP channel to respond to signals. The TRPC1-Homer-IP(3)R complex is dynamic and its disassembly parallels TRPC1 channel activation. Homer's action depends on its ability to crosslink and is blocked by the dominant-negative immediate early gene form, H1a. Since H1a is transcriptionally regulated by cellular activity, this mechanism can affect both short and long-term regulation of TRPC1 function.
Kiselyov, K., D.M. Shin, X. Luo, S.B. Ko, and S. Muallem (2002) Ca2+ signaling in polarized exocrine cells. Adv. Exp. Med. Biol. 506:175-183 Receptor signaling at the plasma membrane often releases calcium from intracellular stores. For example, inositol triphosphate (IP3) produced by receptor-coupled phospholipase C activates an intracellular store calcium channel, the IP(3)R. Conversely, stores can induce extracellular calcium to enter the cell through plasma membrane channels, too. How this "reverse" coupling works was unclear, but store IP(3)Rs were proposed to bind and regulate plasma membrane TRP cation channels. Here, we demonstrate that the adaptor protein, termed Homer, facilitates a physical association between TRPC1 and the IP(3)R that is required for the TRP channel to respond to signals. The TRPC1-Homer-IP(3)R complex is dynamic and its disassembly parallels TRPC1 channel activation. Homer's action depends on its ability to crosslink and is blocked by the dominant-negative immediate early gene form, H1a. Since H1a is transcriptionally regulated by cellular activity, this mechanism can affect both short and long-term regulation of TRPC1 function. Choi, J.Y., D. Muallem, K. Kiselyov, M.G. Lee, P.J. Thomas, and S. Muallem (2001) Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis. Nature 410:94-97 Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl(-)-coupled HCO3- transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO3- and pH affect mucin viscosity and bacterial binding. We have examined Cl(-)-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.
Kiselyov, K., D.M. Shin, N. Shcheynikov, T. Kurosaki, and S. Muallem (2001) Regulation of Ca2+-release-activated Ca2+ current (Icrac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells. Biochem. J. 360:17-22 Persistence of capacitative Ca(2+) influx in inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-deficient DT40 cells (DT40(IP(3)R-/-)) raises the question of whether gating of Ca(2+)-release activated Ca(2+) current (I(crac)) by conformational coupling to Ca(2+)-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of I(crac) Ca(2+) current in wild-type and DT40(IP(3)R-/-) cells. In both cell types passive depletion of internal Ca(2+) stores by infusion of EGTA activated a Ca(2+) current with similar characteristics and time course. The current was highly Ca(2+)-selective and showed strong inward rectification, all typical of I(crac). The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of I(crac), and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited I(crac) activation in DT40(IP(3)R-/-) cells, even after complete depletion of intracellular Ca(2+) stores by ionomycin. Wild-type and DT40(IP(3)R-/-) cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40(IP(3)R-/-) cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP(3)Rs and RyRs can efficiently gate I(crac) in DT40 cells and explain the persistence of I(crac) gating by internal stores in the absence of IP(3)Rs.
Kiselyov, K.I., D.M. Shin, Y. Wang, I.N. Pessah, P.D. Allen, and S. Muallem (2000) Gating of store-operated channels by conformational coupling to ryanodine receptors. Mol. Cell 6:421-431 We report here that RyRs interact with and gate the store-operated hTrp3 and Icrac channels. This gating contributes to activation of hTrp3 and Icrac by agonists. Coupling of hTrp3 to IP3Rs or RyRs in the same cells was found to be mutually exclusive. Biochemical and functional evidence suggest that mutually exclusive coupling reflects clustering and segregation of hTrp3-IP3R and hTrp3-RyR complexes in plasma membrane microdomains. Gating of CCE by RyRs indicates that gating by conformational coupling is not unique to skeletal muscle but is a general mechanism for communication between events in the plasma and endoplasmic reticulum membranes.
Kiselyov, K.I., S.B. Semyonova, A.G. Mamin, and G.N. Mozhayeva (1999) Miniature Ca2+ channels in excised plasma-membrane patches: activation by IP3. Pflug. Arch. Eur. J Phy. 437:305-314 In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.
Kiselyov, K., and S. Muallem (1999) Fatty acids, diacylglycerol, Ins(1,4,5)P3 receptors and Ca2+ influx. Trends Neurosci. 22:334-337 I(min) is a plasma membrane-located, Ca(2+)-selective channel that is activated by store depletion and regulated by inositol 1,4, 5-trisphosphate (IP(3)). In the present work we examined the coupling between I(min) and IP(3) receptors in excised plasma membrane patches from A431 cells. I(min) was recorded in cell-attached mode and the patches were excised into medium containing IP(3). In about 50% of experiments excision caused the loss of activation of I(min) by IP(3.) In the remaining patches activation of I(min) by IP(3) was lost upon extensive washes of the patch surface. The ability of IP(3) to activate I(min) was restored by treating the patches with rat cerebellar microsomes reach in IP(3) receptors but not by control forebrain microsomes. The re-activated I(min) had the same kinetic properties as I(min) when it is activated by Ca(2+)-mobilizing agonists in intact cells and by IP(3) in excised plasma membrane patches and it was inhibited by the I(crac) inhibitor SKF95365. We propose that I(min) is a form of I(crac) and is gated by IP(3) receptors.
Zubov, A.I., E.V. Kaznacheeva, A.V. Nikolaev, V.A. Alexeenko, K. Kiselyov, S. Muallem, and G.N. Mozhayeva (1999) Regulation of the miniature plasma membrane Ca2+ channel I(min) by inositol 1,4,5-trisphosphate receptors. J. Biol. Chem. 274:25983-25985 I(min) is a plasma membrane-located, Ca(2+)-selective channel that is activated by store depletion and regulated by inositol 1,4, 5-trisphosphate (IP(3)). In the present work we examined the coupling between I(min) and IP(3) receptors in excised plasma membrane patches from A431 cells. I(min) was recorded in cell-attached mode and the patches were excised into medium containing IP(3). In about 50% of experiments excision caused the loss of activation of I(min) by IP(3.) In the remaining patches activation of I(min) by IP(3) was lost upon extensive washes of the patch surface. The ability of IP(3) to activate I(min) was restored by treating the patches with rat cerebellar microsomes reach in IP(3) receptors but not by control forebrain microsomes. The re-activated I(min) had the same kinetic properties as I(min) when it is activated by Ca(2+)-mobilizing agonists in intact cells and by IP(3) in excised plasma membrane patches and it was inhibited by the I(crac) inhibitor SKF95365. We propose that I(min) is a form of I(crac) and is gated by IP(3) receptors.
Kiselyov, K., G.A. Mignery, M.X. Zhu, and S. Muallem (1999) The N-terminal domain of the IP3 receptor gates store-operated hTrp3 channels. Mol. Cell 4:423-429 In the present work, we studied the interaction and effect of several IP3 receptor (IP3R) constructs on the gating of the store-operated (SOC) hTrp3 channel. Full-length IP3R coupled to silent hTrp3 channels in intact cells but did not activate them until stores were depleted of Ca2+. By contrast, constructs containing the IP3-binding domain activated silent hTrp3 channels in unstimulated cells and restored gating of hTrp3 by IP3 in excised plasma membrane patches. We conclude that the N-terminal domain of the IP3R functions as a gate and is sufficient for activation of SOCs. The sensing and transduction domains of the IP3R are required to maintain SOCs in an inactive state.
Mozhayeva, M.G., and K.I. Kiselyov (1998) Involvement of Ca2+-induced Ca2+ release in the biphasic Ca2+ response evoked by readdition of Ca2+ to the medium after UTP-induced store depletion in A431 cells. Pflug. Arch. Eur. J Phy. 435:859-864 We have recently shown that the Ca2+ response in endothelial cells evoked by readdition of Ca2+ to the medium after store depletion caused by a submaximal concentration of agonist can involve Ca2+ release from Ca2+ stores sensitive to both inositol 1,4, 5-trisphosphate and ryanodine. The present experiments were performed to determine whether this mechanism might also exist in other types of cell. For this purpose, we used the human carcinoma cell line A431, which has a varied resting [Ca2+]i. We found that the amplitude of the Ca2+ response evoked by Ca2+ readdition did not correlate with the amplitude of the preceding UTP-evoked Ca2+ release, but did positively correlate with the initial [Ca2+]i. An inspection of the two patterns of response seen in this study (the large biphasic and small plateau-shaped Ca2+ responses) revealed that there is an accelerating rise in [Ca2+]i during the biphasic response. Application of ryanodine during the plateau-shaped Ca2+ response reversibly transformed it into the biphasic type. Unlike ryanodine, caffeine did not itself evoke Ca2+ release, but it caused a further [Ca2+]i rise when [Ca2+]i had already been elevated by thapsigargin. These data suggest that in A431 cells, as in endothelial cells, the readdition of Ca2+ after agonist-evoked store depletion can evoke Ca2+-induced Ca2+ release. This indicates that Ca2+ entry may be overestimated by this widely used protocol.
Kiselyov, K., X. Xu, G. Mozhayeva, T. Kuo, I. Pessah, G. Mignery, X. Zhu, L. Birnbaumer, and S. Muallem (1998) Functional interaction between InsP3 receptors and store-operated Htrp3 channels. Nature 396:478-482 Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).
Kiselyov, K.I., A.G. Mamin, S.B. Semyonova, and G.N. Mozhayeva (1997) Low-conductance high selective inositol (1,4,5)-trisphosphate activated Ca2+ channels in plasma membrane of A431 carcinoma cells. FEBS Lett. 407:309-312 In many cells, activation of receptors coupled to PIP2 turnover results in Ca2+ release from the intracellular stores accompanied by Ca2+ influx across the PM. It is not well established yet whether Ca2+ influx is activated by IP3 or by an unknown signal generated upon Ca2+ store depletion. We report here a single-channel study of low-conductance IP3-activated channels of very high selectivity for Ca2+ in the PM of A431 carcinoma cells. The channels are strongly potential dependent and sensitive to [Ca2+]i within the physiological range. The data obtained argues for IP3 acting directly on plasma membrane Ca2+ channels.
Mamin, A.G., K.I. Kiselyov, and G.N. Mozhayeva (1996) Effect of intracellular calcium on ATP-activated, GTP-dependent calcium channels in rat macrophages. J. Physiol. 491:697-705 1. In order to study the effect of intracellular free Ca2+ concentration ([Ca2+]i) on the activity of ATP-activated, GTP-dependent Ca2+ channels in rat macrophages, experiments were performed using the inside-out configuration of the patch-clamp technique. 2. Channel activity was observed in the cell-attached mode when 100 microM ATP was added to the pipette solution containing 105 mM Ba2+, but it disappeared rapidly after patch excision. The activity could be restored by the application of 100 microM GTP or GTP gamma S onto the internal surface of the plasma membrane. 3. The properties of the GTP gamma S-evoked channels are identical to those of channels activated by extracellular application of ATP. The channels exhibited four current sublevels with conductances of about 3.5, 7, 10 and 15 pS when 105 mM Ba2+ was the only permeant cation. The extrapolated reversal potentials were similar for all the sublevels and averaged about +40 mV. 4. Elevation of [Ca2+]i within the range 0.01-1 microM resulted in a decrease in mean inward current. The half-maximal value of the mean current was about 0.08 microM. 5. This decreases in mean current resulted from a redistribution of sublevel occupancies: the 1st sublevel tended to be come more abundant with elevation of [Ca2+]i, while the relative weights of the high-conductance 3rd and 4th sublevels decreased. 6. The open-channel current fell with an increase in [Ca2+]i as quickly as the mean current did, indicating that the sublevel redistribution alone is sufficient to produce the revealed decrease in net inward current. 7. It is concluded that [Ca2+]i elevation does not fix the channel in a closed state but rather decreases the ability of the channel to operate in high-conductance states.
Naumov, A.P., E.V. Kaznacheyeva, K.I. Kiselyov, Y.A. Kuryshev, A.G. Mamin, and G.N. Mozhayeva (1995) ATP-activated inward current and calcium-permeable channels in rat macrophage plasma membranes. J. Physiol. 486:323-337 1. To study mechanisms of receptor-operated Ca2+ influx in non-excitable cells, membrane currents of rat peritoneal macrophages were recorded using whole-cell cell-attached and outside-out configurations of the patch clamp technique. Under whole-cell recording conditions, ATP applied in micromolar concentrations elicited an inward current response when the bath solution contained Ba2+, Ca2+ or Na+ as the only permeant cations. 2. Increasing the Mg2+ concentration had an inhibitory effect on the ATP-induced inward current indicating that the active form of ATP responsible for the cation entry is ATP4-. The nucleotide potency order was ATP > ATP gamma S > ADP. UTP was completely ineffective (n = 19). The data obtained are consistent with the ATP receptor being of the P2Z type. 3. The macrophage plasma membrane was impermeable to Tris+ during the ATP-induced current at ATP4- concentrations varying from 0.07 to 500 microM. At higher concentrations, ATP produced a large inward steady-state current, which could be attributed to membrane permeabilization. 4. Activity of single channels was recorded when ATP was applied to the external surface of the patch membrane both in cell-attached and outside-out experiments. A specific property of the channels appeared to be the existence of at least four conductance sublevels. With 105 mM Ba2+ as the permeant cation, the conductance sublevels were 3.5, 7, 10 and 15 pS. With 10 mM Ca2+ the sublevel conductances were equal to 4, 9, 13 and 17 pS. 5. The unitary conductance estimated from the whole-cell current noise analysis (3.5-4.5 pS for 105 mM Ba2+) was significantly lower than that obtained from single channel measurements at the main (3rd) current level, but it was very close to the conductance of the minimum (1st) level. 6. Extrapolated reversal potential values estimated from current-voltage curves for predominant conductance levels were equal to +40 and +26 mV for 105 mM Ba2+ and 10 mM Ca2+, respectively. The permeability ratios fell in the sequence: PCa:PBa:PK = 71.:29:1. Thus, ATP-activated channels in the macrophage membrane are rather selective for divalent vs. monovalent cations, with the predominant permeability being for Ca2+.
Naumov, A.P., K.I. Kiselyov, A.G. Mamin, E.V. Kaznacheyeva, Y.A. Kuryshev, and G.N. Mozhayeva (1995) ATP-operated calcium-permeable channels activated via a guanine nucleotide-dependent mechanism in rat macrophages. J. Physiol. 486:339-347 1. To elucidate the possible involvement of a G protein in ATP-evoked Ca(2+)-permeable channel activity, membrane currents of rat peritoneal macrophages were recorded using inside-out and cell-attached configurations of the patch clamp technique. 2. In inside-out experiments with a pipette solution containing 105 mM Ba2+, application of 100 microM GTP or GTP gamma S to the internal surface of the membrane elicited a rise in channel activity. This effect was observed in 49% of the patches investigated (n = 69). The mean value of NPo (N, number of open channels; Po, channel open probability) was equal to 0.49 +/- 0.27 (mean +/- S.E.M.; n = 16). The delay in the activity development was 21 +/- 8 s (n = 18) with 200 microM ATP added to the pipette solution and about 4 min (n = 5) without agonist in the pipette. Similar results were obtained with 10 mM Ca2+ as the only permeant cation. 3. Properties of GTP gamma S-evoked channels were identical to those of channels activated by extracellular application of ATP. The channels exhibited at least four conductance sublevels, the 4th one being the least frequent. With 105 mM Ba2+ as a permeant cation, sublevel conductances were 3.5, 7, 10 and 15 pS. Corresponding values for 10 mM Ca2+ were about 4, 9, 13 and 17 pS. Extrapolated reversal potential (Er) values were about +40 and +25 mV for Ba2+ and Ca2+, respectively. 4. The activity of channels with similar characteristics could be induced by the extracellular application of fluoride in cell-attached experiments without any agonist in the pipette solution.(ABSTRACT TRUNCATED AT 250 WORDS)
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