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Biomedical subjects

R Penner

Publications and source records attributed to R Penner.

At least 19 recordsLinked to original sources
PubMed2007

The Mg2+ and Mg(2+)-nucleotide-regulated channel-kinase TRPM7.

TRPM7 is a member of the melastatin-related subfamily of TRP channels and represents a protein that contains both an ion channel and a kinase domain. The protein is ubiquitously expressed and represents the only ion channel known that is essential for cellular viability. TRPM7 is a divalent cation-selective ion channel that is permeable to Ca2+ and Mg2+, but also conducts essential metals such as Zn2+, Mn2+, and Co2+, as well as nonphysiologic or toxic metals such as Ni2+, Cd2+, Ba2+, and Sr2+. The channel is constitutively open but strongly downregulated by intracellular levels of Mg2+ and MgATP and other Mg-nucleotides. Reducing the cellular levels of these regulators leads to activation of TRPM7-mediated currents that exhibit a characteristic nonlinear current-voltage relationship with pronounced outward rectification due to divalent influx at physiologically negative voltages and monovalent outward fluxes at positive voltages. TRPM7 channel activity is also actively regulated following receptor-mediated changes in cyclic AMP (cAMP) and protein kinase A activity. This regulation as well as that by Mg-nucleotides requires a functional endogenous kinase domain. The function of the kinase domain is not completely understood, but may involve autophosphorylation of TRPM7 as well as phosphorylation of other target proteins such as annexin and myosin IIA heavy chain. Based on these properties, TRPM7 is currently believed to represent a ubiquitous homeostatic mechanism that regulates Ca2+ and Mg2+ fluxes based on the metabolic state of the cell. Physiologically, the channel may serve as a regulated transport mechanism for these ions that could affect cell adhesion, cell growth and proliferation, and even cell death under pathological stress such as anoxia.

Adenosine Triphosphate↗
PubMed2006

CRACM1 is a plasma membrane protein essential for store-operated Ca2+ entry.

Store-operated Ca2+ entry is mediated by Ca2+ release-activated Ca2+ (CRAC) channels following Ca2+ release from intracellular stores. We performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that inhibit store-operated Ca2+ influx. A secondary patch-clamp screen identified CRACM1 and CRACM2 (CRAC modulators 1 and 2) as modulators of Drosophila CRAC currents. We characterized the human ortholog of CRACM1, a plasma membrane-resident protein encoded by gene FLJ14466. Although overexpression of CRACM1 did not affect CRAC currents, RNAi-mediated knockdown disrupted its activation. CRACM1 could be the CRAC channel itself, a subunit of it, or a component of the CRAC signaling machinery.

Animals↗
PubMed2001

CaT1 and the calcium release-activated calcium channel manifest distinct pore properties.

The calcium release-activated calcium channel (CRAC) is a highly Ca(2+)-selective ion channel that is activated on depletion of inositol triphosphate (IP(3))-sensitive intracellular Ca(2+) stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na(+) and Cs(+); effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg(2+) inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg(2+)-gated channel not directly related to CRAC.

Calcium Channels↗
PubMed2001

LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability.

The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg.ATP and Mg.GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg.ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.

Adenosine Triphosphate↗
PubMed2001

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

Adenosine Diphosphate Ribose↗
PubMed2000

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase.

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.

Animals↗
PubMed1999

Differential modulation of voltage-dependent Ca2+ currents by EGTA and BAPTA in bovine adrenal chromaffin cells.

Whole-cell patch-clamp recordings were made to examine the effects of the Ca2+ chelators EGTA and BAPTA on the biophysical properties of voltage-operated Ca2+ currents in bovine adrenal chromaffin cells. Ca2+ currents in the presence of either EGTA or BAPTA over a concentration range of 0.1-60 mM were recorded under otherwise identical conditions. Analysis of current-voltage relationships yielded unexpected differences in several important parameters such as the voltage dependence of activation, kinetics, slope, and reversal potential, which seemed to be unrelated to the Ca(2+)-binding properties of these chelators. Increasing concentrations of BAPTA augmented the peak Ca2+ current amplitude while current amplitudes in the presence of EGTA remained constant over the entire concentration range tested. Increasing concentrations of BAPTA shifted the voltage sensitivity of Ca2+ currents by about 15 mV towards positive voltages. EGTA, over the same concentration range, did not affect the voltage sensitivity. The shift in voltage sensitivity observed with BAPTA was unrelated to its faster Ca92+)-binding kinetics, as it was also observed when substituting Ca2+ with Ba2+ as the charge carrier. The mechanism by which BAPTA affects Ca2+ channel voltage dependence also seems unrelated to kinase-mediated modulation of Ca2+ channels, since the protein-kinase-C- (PKC-) specific drugs bisindolylmaleimide and phorbol ester (PMA) neither mimicked nor prevented the action of BAPTA. The less specific kinase inhibitor staurosporine, however, augmented Ca2+ currents similarly to BAPTA, but without affecting the voltage sensitivity. The BAPTA-mediated shift in voltage sensitivity was partially suppressed by non-hydrolysable analogs of GTP (GDP[beta-S] and GTP[gamma-S]). Lowering [Mg2+]i mimicked the BAPTA-induced shift in voltage sensitivity and prevented further shifts in voltage sensitivity by BAPTA. The results demonstrate that BAPTA and EGTA, despite their similarities in terms of Ca2+ buffering, have disparate effects on the voltage dependence of Ca2+ channels and careful selection of the chelator is required to quantitatively assess Ca2+ currents.

Algorithms↗
PubMed1998

Calcium release-activated calcium current (ICRAC) is a direct target for sphingosine.

Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current (ICRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits ICRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, including N,N-dimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphingosine (C2-ceramide) also block ICRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block ICRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit ICRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits ICRAC, and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that ICRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks ICRAC, suggesting that the sphingomyelin pathway is involved in ICRAC regulation.

Animals↗
PubMed1998

Near-visible ultraviolet light induces a novel ubiquitous calcium-permeable cation current in mammalian cell lines.

1. We studied the immediate and short-term effects of UV light in the near-visible range at the cellular and membrane level using the whole-cell patch-clamp technique in combination with digital fluorescence imaging. 2. Illumination with monochromatic UVA light (340-380 nm) induced a sustained non-saturable increase in membrane conductance dependent on wavelength and light intensity in several different mammalian cell types including RBL, mast, HEK, PC12 and 3T3 cells. 3. The current was non-selective for cations and permeable to Ca2+, but was inhibited by trivalent cations and was not due to the activation of an endogenous ion channel. We termed this novel current ILiNC for light-induced non-selective cation current. 4. A similar current was evoked by chemical peroxidants such as hydrogen peroxide and tertbutylhydroperoxide, but not by cytosolic oxidized glutathione. 5. The free-radical scavengers tocopherol (vitamin E) and ascorbic acid (vitamin C) significantly reduced the UV light effect. 6. The generation of the current was membrane delimited since it could be induced by the same UVA treatment in cell-free membrane patches showing a similar wavelength dependence. 7. These results suggest that ILiNC is activated by UVA light-induced generation of free radicals acting through lipid or protein peroxidation, and may represent a ubiquitous mechanism by which Na+ and Ca2+ can enter cells after phototoxic or free radical-induced membrane damage.

Animals↗
PubMed1997

The store-operated calcium current I(CRAC): nonlinear activation by InsP3 and dissociation from calcium release.

Patch-clamp experiments aimed at determining the relationship between intracellular Ca2+ release and activation of store-operated calcium current I(CRAC) reveal that both agonist and InsP3-mediated activation of I(CRAC) are highly nonlinear, occurring over a narrow concentration range. Ca2+ release and Ca2+ influx can be dissociated, as they possess differential sensitivities to InsP3: low concentrations induce substantial Ca2+ release without any activation of I(CRAC), whereas micromolar concentrations of InsP3 are required to activate Ca2+ influx. This suggests functionally distinct stores controlling Ca2+ release and influx and enables cells to switch between sources of Ca2+ to fit best their current needs.

Animals↗
PubMed1997

Ca2+-induced Ca2+ release in Chinese hamster ovary (CHO) cells co-expressing dihydropyridine and ryanodine receptors.

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca2+ release channel (in the membrane of internal Ca2+ stores) and the dihydropyridine receptor (DHPR)-Ca2+ channel (in the plasma membrane). To ensure expression of functional L-type Ca+ channels, we expressed alpha2, beta, and gamma DHPR subunits and a chimeric DHPR alpha(i) subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca2+ transient (CaT) in the absence of extracellular Ca2+ ([Ca2+]o). However, in the presence of [Ca2+]o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca2+ concentration ([Ca2+]i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca2+]i which lasted for 5-10 s (the second component). Our results suggest that the first component primarily reflected Ca2+ influx through Ca2+ channels, whereas the second component resulted from Ca2+ release through the RyR expressed in the membrane of internal Ca2+ stores. However, the onset and the rate of Ca2+ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca2+ current. These results suggest that the skeletal muscle RyR isoform supports Ca2+-induced Ca2+ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional "close coupling" like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.

Animals↗
PubMed1997

Store depletion and calcium influx.

Calcium influx in nonexcitable cells regulates such diverse processes as exocytosis, contraction, enzyme control, gene regulation, cell proliferation, and apoptosis. The dominant Ca2+ entry pathway in these cells is the store-operated one, in which Ca2+ entry is governed by the Ca2+ content of the agonist-sensitive intracellular Ca2+ stores. Only recently has a Ca2+ current been described that is activated by store depletion. The properties of this new current, called Ca2+ release-activated Ca2+ current (ICRAC), have been investigated in detail using the patch-clamp technique. Despite intense research, the nature of the signal that couples Ca2+ store content to the Ca2+ channels in the plasma membrane has remained elusive. Although ICRAC appears to be the most effective and widespread influx pathway, other store-operated currents have also been observed. Although the Ca2+ release-activated Ca2+ channel has not yet been cloned, evidence continues to accumulate that the Drosophila trp gene might encode a store-operated Ca2+ channel. In this review, we describe the historical development of the field of Ca2+ signaling and the discovery of store-operated Ca2+ currents. We focus on the electrophysiological properties of the prototype store-operated current ICRAC, discuss the regulatory mechanisms that control it, and finally consider recent advances toward the identification of molecular mechanisms involved in this ubiquitous and important Ca2+ entry pathway.

Animals↗
PubMed1996

Absence of Ca2+ current facilitation in skeletal muscle of transgenic mice lacking the type 1 ryanodine receptor.

1. Whole-cell patch-clamp recordings were used to study voltage-dependent facilitation of Ca2+ currents and excessive Ca2+ tail current in skeletal myoballs cultured from wild-type and transgenic mice expressing a null mutation of the ryanodine receptor (RyR) type 1 (dyspedic myoballs). 2. Ca2+ current density in dyspedic myoballs was reduced by about 60% compared with wild-type cells, with dihydropyridine-binding capacity largely retained. 3. Strong and long-lasting depolarizations (+80 mV and 600 ms), which normally produce excessive tail currents upon repolarization in control cells, failed to do so in dyspedic myoballs. 4. Dyspedic myoballs also failed to produce both Ca2+ current facilitation and the left shift of the current-voltage (I-V) curve induced by paired-pulse stimulation. 5. We propose that excessive tail currents and facilitation arise from silent Ca2+ channels acting as the voltage sensors in excitation-contraction coupling.

Animals↗
PubMed1996

Silent calcium channels generate excessive tail currents and facilitation of calcium currents in rat skeletal myoballs.

1. Whole-cell patch-clamp recording were employed to study facilitation of Ca2+ currents and excessive Ca2+ tail currents evoked by strong and long-lasting conditioning depolarizations in skeletal myoballs cultured from newborn rats. 2. Paired-pulse facilitation and excessive tail currents showed the same voltage dependence, becoming prominent at conditioning potentials above +30 mV. 3. Recruitment of excessive tail currents and facilitation occurred with the same time dependence (time constant (tau), approximately 200 ms to approximately 1 s), accelerating with the depolarization strength of conditioning pulses. 4. Reversal of Ca2+ current facilitation during the repolarization period between conditioning and test pulses was time- and voltage dependent. The time window of recruitment of facilitated Ca2+ currents narrowed considerably at more negative repolarization potentials (tau: approximately 10 ms at -100 mV, but approximately 1.5 at 0 mV). 5. Neither omission of internal ATP nor perfusion of the cells with the peptide inhibitor of protein kinase A (PKI) had significant effects on Ca2+ current facilitation, although internal perfusion with ATP gamma S slowly suppressed the facilitation currents by about 30%. External application of either ryanodine or caffeine under control conditions selectively and significantly suppressed the facilitated Ca2+ currents by about 30-40%. 6. We propose that facilitation of Ca2+ currents and excessive tail currents are consequences of a common mechanism linked to ryanodine receptors.

Animals↗
PubMed1996

Acceleration of membrane recycling by axotomy of cultured aplysia neurons.

The rapid transition of a stationary axon into a motile growth cone requires the recruitment of membrane and its strategic insertion into the neurolemma. The source of membrane to support the initial rapid growth postaxotomy is not known. Using membrane capacitance measurements, we examined quantitative aspects of membrane dynamics following axotomy of cultured Aplysia neurons. Axotomy activates two processes in parallel: membrane retrieval and exocytosis. Unexpectedly, membrane retrieval is the dominant process in the majority of the experiments. Thus, while a growth cone is vigorously extending, the total neuronal surface area decreases. We suggest that the initial rapid extension phase of the newly formed growth cone postaxotomy is supported by a pool of intracellular membrane that is rapidly retrieved from the neurolemma.

Animals↗
PubMed1996

Characterization of the Ca2+ current in isolated terminals of crustacean peptidergic neurons.

Ca2+ currents (ICa) were recorded from the neurosecretory terminals of the crab X-organ-sinus gland under voltage-clamp conditions. ICa was detectable at command potentials above -40mV, with maximum currents at approximately +20mV. No differences were observed between current-voltage (I/V) relationships from holding potentials of -50 or -90mV, indicating that there were no low-voltage-activated Ca2+ channels present in the terminals. The decay of ICa was best fitted with a single exponential, the extent of inactivation over 50 ms averaging 53%. The rate of decay of ICa was reduced by the substitution of Ca2+ with Sr2+ in the external solution and was eliminated by substitution with Ba2+. The effect of varying prepulse potential on the amplitude of ICa at +20mV was tested. ICa declined with increasing prepulse depolarization up to +20mV and then showed partial recovery at more depolarized prepulse potentials. Inactivation curves in solutions containing Sr2+ and Ba2+ showed much less current-dependent inactivation. Removing Ca2+ chelators from the internal solution significantly increased ICa decay. ICa was insensitive to nifedipine at a concentration of 1 mumol l-1. Pretreatment of the isolated sinus gland containing the intact terminals with a combination of omega-conotoxin (omega-Ctx) GVIA, omega-Ctx MVIIC and omega-agatoxin IVA had no effect on the levels of K+-induced peptide release.

Animals↗
PubMed1995

Activation of store-operated calcium influx at resting InsP3 levels by sensitization of the InsP3 receptor in rat basophilic leukaemia cells.

1. Patch clamp and fura-2 AM measurements were performed to study the effects of sensitizing the inositol 1,4,5-trisphosphate (InsP3) receptor to InsP3 on the activation of Ca2+ release-activated Ca2+ current (ICRAC) in rat basophilic leukaemia (RBL) cells. 2. The sensitizing agent thimerosal (1 microM) triggered Ca2+ release, and this was followed by Ca2+ influx. With no added InsP3 in the patch pipette, thimerosal activated ICRAC; this was prevented by heparin. ICRAC activated by thimerosal was very similar to that evoked by InsP3 or ionomycin. 3. Dialysing cells either for short (30 s) or long (600 s) periods of time prior to application of thimerosal did not affect the subsequent activation of ICRAC, even though no InsP3 was included in the patch pipette. 4. These results suggest that sensitizing the InsP3 receptor can result in large Ca2+ influx in the presence of resting InsP3, and that stores closer to the membrane may contribute more to activation of ICRAC than stores further away.

Animals↗