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James W Putney

Publications and source records attributed to James W Putney.

32 records · Page 2Linked to original sources
PubMed2004

Obligatory role of Src kinase in the signaling mechanism for TRPC3 cation channels.

Members of the canonical transient receptor potential (TRPC) subfamily of cation channels are candidates for capacitative and non-capacitative Ca2+ entry channels. When ectopically expressed in cell lines, TRPC3 can be activated by phospholipase C-mediated generation of diacylglycerol or by addition of synthetic diacylglycerols, independently of Ca2+ store depletion. Apart from this mode of regulation, little is known about other receptor-dependent signaling events that modulate TRPC3 activity. In the present study the role of tyrosine kinases in receptor- and diacylglycerol-dependent activation of TRPC3 was investigated. In HEK293 cells stably expressing TRPC3, pharmacological inhibition of tyrosine kinases, and specifically of Src kinases, abolished activation of TRPC3 by muscarinic receptor stimulation and by diacylglycerol. Channel regulation was lost following expression of a dominant-negative mutant of Src, or when TRPC3 was expressed in an Src-deficient cell line. In both instances, wild-type Src restored TRPC3 regulation. We conclude that Src plays an obligatory role in the mechanism for receptor and diacylglycerol activation of TRPC3.

Blotting, Western↗
PubMed2004

Store-operated calcium channels: how do we measure them, and why do we care?

A major mechanism whereby calcium entry into cells is regulated is the store-operated or capacitative calcium entry pathway. In this article, two basic issues are discussed: (i) the methods investigators use to measure store-operated entry, and (ii) the role played by the store-operated pathway in responses to hormones and neurotransmitters under physiological conditions. The two topics are considered together because they are closely interrelated; as we begin to ask questions about calcium movements at low concentrations of agonists, the technology to measure these movements becomes increasing challenging.

Calcium Channels↗
PubMed2004

Mechanisms of phospholipase C-regulated calcium entry.

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.

Calcium↗
PubMed2004

Signalling mechanisms for TRPC3 channels.

The putative ion channel subunits TRPC3, TRPC6 and TRPC7 comprise a structurally related subgroup of the family of mammalian TRPC channels. As is the case for the founding member of the TRPC family, Drosophila TRP, the ion channels formed by these proteins appear to be activated in some manner downstream of phospholipase C (PLC). Earlier studies indicating that TRPC3 could be activated by depletion of intracellular stores (i.e. that it is a store-operated channel, SOC) were subsequently shown to be attributable to constitutive activity of the channels. Studies on the mechanism of activation of TRPC6 and TRPC7 indicated that PLC-dependent activation involved diacylglycerol and was independent of G proteins or inositol 1,4,5-trisphosphate (IP3). Although TRPC3 can also be activated by diacylglycerols, there is evidence suggesting that these channels can be activated by IP3 and the IP3 receptor through a conformational coupling mechanism. We have re-examined the activation mechanism for TRPC3 in mammalian cells by using HEK293 cell lines stably expressing human TRPC3. Our data indicate that, like TRPC6 and TRPC7, TRPC3 is activated by PLC-generated diacylglycerol and is independent of G proteins or IP3. However, in an avian pre-B cell line, TRPC3 can function either as a diacylglycerol-activated channel, or as a SOC. The mechanism of regulation of TRPC3 in this cell line appears to be related to the level of expression of the protein.

Animals↗
PubMed2003

A calmodulin/inositol 1,4,5-trisphosphate (IP3) receptor-binding region targets TRPC3 to the plasma membrane in a calmodulin/IP3 receptor-independent process.

Conformational coupling with the inositol 1,4,5-trisphosphate (IP3) receptor has been suggested as a possible mechanism of activation of TRPC3 channels and a region in the C terminus of TRPC3 has been shown to interact with the IP3 receptor as well as calmodulin (calmodulin/IP3 receptor-binding (CIRB) region). Here we show that internal deletion of 20 amino acids corresponding to the highly conserved CIRB region results in the loss of diacylglycerol and agonist-mediated channel activation in HEK293 cells. By using confocal microscopy to examine the cellular localization of Topaz fluorescent protein fusion constructs, we demonstrate that this loss in activity is caused by faulty targeting of CIRB-deleted mutants to intracellular compartments. Wild type TRPC3 and mutants lacking a C-terminal predicted coiled coil region downstream of CIRB were targeted to the plasma membrane correctly in HEK293 cells and exhibited TRPC3-mediated calcium entry in response to agonist activation. Mutation of conserved YQ and MKR motifs to alanine within the CIRB region in TRPC3-Topaz, which would be expected to interfere with IP3 receptor and/or calmodulin binding, had no effect on channel function or targeting. Additionally, TRPC3 targets to the plasma membrane of DT40 cells lacking all three IP3 receptors and forms functional ion channels. These findings indicate that the previously identified CIRB region of TRPC3 is involved in its targeting to the plasma membrane by a mechanism that does not involve interaction with IP3 receptors.

Amino Acid Sequence↗
PubMed2003

Expression level of the canonical transient receptor potential 3 (TRPC3) channel determines its mechanism of activation.

Studies on the mechanism of activation of canonical transient receptor potential (TRPC) channels have often yielded conflicting results. In the current study, we have investigated the influence of expression level on the mode of regulation of TRPC3 channels. At relatively low levels of expression in DT40 chicken B-lymphocytes, TRPC3 was activated by the depletion of Ca2+ stores. Expression was increased by either transfecting with a 10-fold greater concentration of plasmid or transfecting with TRPC3 under control of a more efficient avian beta-actin promoter. At higher levels of expression, TRPC3 was no longer store-operated but could be activated through receptor-coupled phospholipase C. Under these expression conditions, TRPC3 was efficiently activated in DT40 cells lacking inositol 1,4,5-trisphosphate receptors. The Ca2+ store-operated channels formed upon expression of TRPC3 at limited levels were blocked by gadolinium; the receptor-activated channels formed upon expression of higher levels of TRPC3 were insensitive to gadolinium. These findings indicate that a single ion channel protein can form or contribute to the formation of channels regulated in two very distinct ways, i.e. either by phospholipase C-derived messengers or Ca2+ store-depletion. The mechanism of regulation of the channels depends on their level of expression.

Animals↗
PubMed2003

Signaling mechanism for receptor-activated canonical transient receptor potential 3 (TRPC3) channels.

Canonical transient receptor potential 3 (TRPC3) is a receptor-activated, calcium permeant, non-selective cation channel. TRPC3 has been shown to interact physically with the N-terminal domain of the inositol 1,4,5-trisphosphate receptor, consistent with a "conformational coupling" mechanism for its activation. Here we show that low concentrations of agonists that fail to produce levels of inositol 1,4,5-trisphosphate sufficient to induce Ca(2+) release from intracellular stores substantially activate TRPC3. By several experimental approaches, we demonstrate that neither inositol 1,4,5-trisphosphate nor G proteins are required for TRPC3 activation. However, diacylglycerols were sufficient to activate TRPC3 in a protein kinase C-independent manner. Surface receptor agonists and exogenously applied diacylglycerols were not additive in activating TRPC3. In addition, inhibition of metabolism of diacylglycerol slowed the reversal of receptor-dependent TRPC3 activation. We conclude that receptor-mediated activation of phospholipase C in intact cells activates TRPC3 via diacylglycerol production, independently of G proteins, protein kinase C, or inositol 1,4,5-trisphosphate.

Calcium↗
PubMed2003

2-aminoethoxydiphenyl borane activates a novel calcium-permeable cation channel.

The membrane-permeable, noncompetitive inositol 1,4,5-trisphosphate (IP3)-receptor inhibitor 2-aminoethoxydiphenyl borane (2-APB) has been widely used to probe for IP3-receptor involvement in calcium signaling pathways. However, a number of recent studies in different cell types revealed other sites of action of 2-APB. In this study, we examined the influence of 2-APB on capacitative calcium entry and intracellular Ca2+ concentrations in rat basophilic leukemia (RBL-2H3 m1) cells. 2-APB was found to inhibit capacitative calcium entry, but at concentrations greater than 50 microM, a new effect of 2-APB was observed. When capacitative calcium entry was blocked with Gd3+, 2-APB caused an increase in cytoplasmic Ca2+. This increase in intracellular Ca2+ was not caused by altered buffering of cytoplasmic Ca2+ and was not caused by or in any way affected by the depletion of intracellular Ca2+ stores. Associated with the increase in intracellular Ca2+, in the presence of 2 mM Ca2+, 2-APB activated single channels in the plasma membrane with a conductance of approximately 50 pS. These channels seem to be nonselective cation channels; monovalent cations are the major carriers of current, but finite permeability to Ca2+ leads to a significant intracellular Ca2+ signal. Experiments with excised patches indicate that 2-APB activates these channels from the outer aspect of the cell membrane. This effect of 2-APB further illustrates the complex actions of this compound and reveals the presence in RBL-2H3 m1 cells of a novel, ligand-gated calcium-permeable channel.

Animals↗
PubMed2002

An inositol 1,4,5-trisphosphate receptor-dependent cation entry pathway in DT40 B lymphocytes.

We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.

Amino Acid Substitution↗
PubMed2002

Comparison of human TRPC3 channels in receptor-activated and store-operated modes. Differential sensitivity to channel blockers suggests fundamental differences in channel composition.

Capacitative calcium entry or store-operated calcium entry in nonexcitable cells is a process whereby the activation of calcium influx across the plasma membrane is signaled by depletion of intracellular calcium stores. Transient receptor potential (TRP) proteins have been proposed as candidates for store-operated calcium channels. Human TRPC3 (hTRPC3), an extensively studied member of the TRP family, is activated through a phospholipase C-dependent mechanism, not by store depletion, when expressed in HEK293 cells. However, store depletion by thapsigargin is sufficient to activate hTRPC3 channels when expressed in DT40 avian B-lymphocytes. To gain further insights into the differences between hTRPC3 channels generated in these two expression systems and further understand the role of hTRPC3 in capacitative calcium entry, we examined the effect of two well characterized inhibitors of capacitative calcium entry, Gd3+ and 2-aminoethoxydiphenyl borane (2APB). We confirmed that in both DT40 cells and HEK293 cells, 1 microm Gd3+ or 30 microm 2APB completely blocked calcium entry due to receptor activation or store depletion. In HEK293 cells, 1 microm Gd3+ did not block receptor-activated hTRPC3-mediated cation entry, whereas 2APB had a partial (approximately 60%) inhibitory effect. Interestingly, store-operated hTRPC3-mediated cation entry in DT40 cells was also partially inhibited by 2APB, whereas 1 microm Gd3+ completely blocked store-operated hTRPC3 activity in these cells. Furthermore, the sensitivity of store-operated hTRPC3 channels to Gd3+ in DT40 cells was similar to the endogenous store-operated channels, with essentially 100% block of activity at concentrations as low as 0.1 microm. Finally, Gd3+ has a rapid inhibitory effect when added to fully developed hTRPC3-mediated calcium entry, suggesting a direct action of Gd3+ on hTRPC3 channels. The distinct action of these inhibitors on hTRPC3-mediated cation entry in these two cell types may result from their different modes of activation and may also reflect differences in basic channel structure.

Boron Compounds↗
PubMedDate not supplied

The TRPC3/6/7 subfamily of cation channels.

The mammalian transient receptor potential (TRP) proteins consist of a superfamily of Ca2+-permeant non-selective cation channels with structural similarities to Drosophila TRP. The TRP superfamily can be divided into three major families, among them the "canonical TRP" family (TRPC). The seven protein products of the mammalian TRPC family of genes (designated TRPC1-7) share in common the activation through PLC-coupled receptors and have been proposed to encode components of native store-operated channels in different cell types. In addition, the three members of the TRPC3/6/7 subfamily of TRPC channels can be activated by diacylglycerol analogs, providing a possible mechanism of activation of these channels by PLC-coupled receptors. This review summarizes the current knowledge about the mechanism of activation of the TRPC3/6/7 subfamily, as well as the potential role of these proteins as components of native Ca2+-permeant channels.

Animals↗
PubMedDate not supplied

Capacitative calcium entry in the nervous system.

Capacitative calcium entry is a process whereby the depletion of Ca(2+) from intracellular stores (likely endoplasmic or sarcoplasmic reticulum) activates plasma membrane Ca(2+) channels. Current research has focused on identification of capacitative calcium entry channels and the mechanism by which Ca(2+) store depletion activates the channels. Leading candidates for the channels are members of the transient receptor potential (TRP) superfamily, although no single gene or gene product has been definitively proven to mediate capacitative calcium entry. The mechanism for activation of the channels is not known; proposals fall into two general categories, either a diffusible signal released from the Ca(2+) stores when their Ca(2+) levels become depleted, or a more direct protein-protein interaction between constituents of the endoplasmic reticulum and the plasma membrane channels. Capacitative calcium entry is a major mechanism for regulated Ca(2+) influx in non-excitable cells, but recent research has indicated that this pathway plays an important role in the function of neuronal cells, and may be important in a number of neuropathological conditions. This review will summarize some of these more recent findings regarding the role of capacitative calcium entry in normal and pathological processes in the nervous system.

Animals↗