TRP channels in disease.

The transient receptor potential (TRP) channels are a large family of proteins with six main subfamilies termed the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) groups. The sheer number of different TRPs with distinct functions supports the statement that these channels are involved in a wide range of processes ranging from sensing of thermal and chemical signals to reloading intracellular stores after responding to an extracellular stimulus. Mutations in TRPs are linked to pathophysiology and specific diseases. An understanding of the role of TRPs in normal physiology is just beginning; the progression from mutations in TRPs to pathophysiology and disease will follow. In this review, we focus on two distinct aspects of TRP channel physiology, the role of TRP channels in intracellular Ca2+ homeostasis, and their role in the transduction of painful stimuli in sensory neurons.

Nuclear and cytosolic calcium are regulated independently.

Nuclear calcium (Ca(2+)) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca(2+) signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca(2+) release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP(3)R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca(2+) signals induced by low InsP(3) concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca(2+) stores for InsP(3). Nuclear InsP(3)R channels were active at lower InsP(3) concentrations than InsP(3)R from cytosol. Enriched expression of type II InsP(3)R in the nucleus results in greater sensitivity of the nucleus to InsP(3), thus providing a mechanism for independent regulation of Ca(2+)-dependent processes in this cellular compartment.

Characterization of the calcium-release channel/ryanodine receptor from zebrafish skeletal muscle.

Calcium (Ca2+)-mediated signaling is fueled by two sources for Ca2+: Ca2+ can enter through Ca2+ channels located in the plasma membrane and can also be released from intracellular stores. In the present study the intracellular Ca2+ release channel/ryanodine receptor (RyR) from zebrafish skeletal muscle was characterized. Two RyR isoforms could be identified using immunoblotting and single-channel recordings. Biophysical properties as well as the regulation by modulators of RyR, ryanodine, ruthenium red and caffeine, were measured. Comparison with other RyRs showed that the zebrafish RyRs have features observed with all RyRs described to date and thus, can serve as a model system in future genetic and physiological studies. However, some differences in the biophysical properties were observed. The slope conductance for both isoforms was higher than that of the mammalian RyR type 1 (RyR1) measured with divalent ions. Also, inhibition by millimolar Ca2+ concentrations of the RyR isoform that is inhibited by high Ca2+ concentrations (teleost alpha RyR isoform) was attenuated when compared to mammalian RyRs. Due to the widespread expression of RyR these findings have important implications for the interpretation of the role of the RyR in Ca2+ signaling when comparing zebrafish with mammalian physiology, especially when analyzing mutations underlying physiological changes in zebrafish.

Regulation of Ins(1,4,5)P3 receptor isoforms by endogenous modulators.

Three isoforms of the inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor have been identified. Each receptor isoform has been functionally characterized using many different techniques. Although these receptor isoforms possess high homology, interesting differences in their Ca2+ dependence, Ins(1,4,5)P3 sensitivity and subcellular distribution exist, implying distinct cellular roles. Indeed, interplay among the isoforms might be necessary for a cell to control spatial and temporal aspects of cytosolic Ca2+ signals, which are important for many cellular processes. In this review isoform-specific functions, primarily at the single-channel level, will be highlighted and these properties will be correlated with Ca2+ signals in intact cells.

Conservation of localization patterns of IP(3) receptor type 1 in cerebellar Purkinje cells across vertebrate species.

The distribution of inositol 1,4,5-trisphosphate (IP(3)) receptor type 1 (IP(3)R1) protein was studied in the adult cerebella of six different vertebrate species, zebrafish, skate, claw frog, rat, hamster, and mouse. The receptor was found at high expression levels in Purkinje cells in all species examined using a subtype-specific polyclonal antiserum against IP(3)R1 and fluorescence immunocytochemistry. The immunoreactivity for IP(3)R1 was found intracellularly at high concentrations in dendrites and somata and at lower levels in axons of these cells. Despite the morphological and functional differences of the cerebella the staining patterns of IP(3)R1 labeling in Purkinje cells was preserved. This is notable because the cerebella were taken from organisms representing a large segment of vertebrate phylogenetic development. The high expression levels of IP(3)R1 in Purkinje cells were found independent of the degree of the formation of fissures and folia and of the degree of branching of Purkinje cell dendrites. The conservation of cerebellar structures not only at the cellular level but more importantly at the molecular level suggests that identical intracellular calcium signaling mechanisms are used in a number of species that represent different areas of phylogenetic development and specialization.

Reversible block of the calcium release channel/ryanodine receptor by protamine, a heparin antidote.

Channel activity of the calcium release channel from skeletal muscle, ryanodine receptor type 1, was measured in the presence and absence of protamine sulfate on the cytoplasmic side of the channel. Single-channel activity was measured after incorporating channels into planar lipid bilayers. Optimally and suboptimally calcium-activated calcium release channels were inactivated by the application of protamine to the cytoplasmic side of the channel. Recovery of channel activity was not observed while protamine was present. The addition of protamine bound to agarose beads did not change channel activity, implying that the mechanism of action involves an interaction with the ryanodine receptor rather than changes in the bulk calcium concentration of the medium. The block of channel activity by protamine could be reversed either by removal by perfusion with buffer or by the addition of heparin to the cytoplasmic side of the channel. Microinjection of protamine into differentiated C(2)C(12) mouse muscle cells prevented caffeine-induced intracellular calcium release. The results suggest that protamine acts on the ryanodine receptor in a similar but opposite manner from heparin and that protamine can be used as a potent, reversible inhibitor of ryanodine receptor activity.

Regulation of the type III InsP(3) receptor by InsP(3) and ATP.

Many hormones and neurotransmitters raise intracellular calcium (Ca(2+)) by generating InsP(3) and activating the inositol 1,4, 5-trisphosphate receptor (InsP(3)R). Multiple isoforms with distinct InsP(3) binding properties () have been identified (). The type III InsP(3)R lacks Ca(2+)-dependent inhibition, a property that makes it ideal for signal initiation (). Regulation of the type III InsP(3)R by InsP(3) and ATP was explored in detail using planar lipid bilayers. In comparison to the type I InsP(3)R, the type III InsP(3)R required a higher concentration of InsP(3) to reach maximal channel activity (EC(50) of 3.2 microM versus 0.5 microM for the types III and I InsP(3)R, respectively). However, the type III InsP(3)R did reach a 2.5-fold higher level of activity. Although activation by InsP(3) was isoform-specific, regulation by ATP was similar for both isoforms. In the presence of 2 microM InsP(3), low ATP concentrations (<6 mM) increased the open probability and mean open time. High ATP concentrations (>6 mM) decreased channel activity. These results illustrate the complex nature of type III InsP(3)R regulation. Enhanced channel activity in the presence of high InsP(3) may be important during periods of prolonged stimulation, whereas allosteric modulation by ATP may help to modulate intracellular Ca(2+) signaling.

Regulation of the type III InsP3 receptor and its role in beta cell function.

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.

Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP3 and calcium: Simulation of single channel kinetics based on ligand binding and electrophysiological analysis.

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751-754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443-446; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561-17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding approximately 3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529-538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.

Calcium signaling molecules in human cerebellum at midgestation and in ataxia.

A fundamental question in brain development is how neurons make the precise topographic connections necessary for function. The hypothesis that transient expression of calcium (Ca2+) signaling molecules may have a role in this process was tested by studying human cerebella at midgestation. In addition, four adult brains, two controls and two from patients with ataxia, were studied as well. The temporal and spatial distribution of intracellular Ca2+ channel/receptors, inositol trisphosphate receptor type 1 (IP3R1) and ryanodine receptor (RyR) and three Ca2+ binding proteins were examined with immunocytochemical methods. A positive immune reaction with all markers of Ca2+ signaling was found in the Purkinje cell layer starting from 17 g.w. (gestational weeks), the youngest age studied. The immune reactions were not homogeneous throughout the extent of the Purkinje cell layer, but instead displayed a 'patchy' appearance in all intrauterine stages. In the adult cerebellum the expression of Ca2+ signaling molecules was homogenous. In the two cerebella obtained from patients suffering from ataxia, a several-fold reduction of immunostaining with IP3R1 was found. Our findings suggest that transient and differential mobilization of intracellular Ca2+ in seemingly homogenous neuronal types may play a role in development of highly organized projection maps of the cerebellar cortex. Moreover, lack of IP3R1 in the diseased brains suggests that internal stores of Ca2+ play an important role in normal function of the cerebellum.

Type III InsP3 receptor channel stays open in the presence of increased calcium.

The inositol 1,4,5-trisphosphate receptor (InsP3R) is the main calcium(Ca2+) release channel in most tissues. Three isoforms have been identified, but only types I and II InsP3R have been characterized. Here we examine the functional properties of the type III InsP3R because this receptor is restricted to the trigger zone from which Ca2+ waves originate and it has distinctive InsP3-binding properties. We find that type III InsP3R forms Ca2+ channels with single-channel currents that are similar to those of type I InsP3R; however, the open probability of type III InsP3R isoform increases monotonically with increased cytoplasmic Ca2+ concentration, whereas the type I isoform has a bell-shaped dependence on cytoplasmic Ca2+. The properties of type III InsP3R provide positive feedback as Ca2+ is released; the lack of negative feedback allows complete Ca2+ release from intracellular stores. Thus, activation of type III InsP3R in cells that express only this isoform results in a single transient, but global, increase in the concentration of cytosolic Ca2+. The bell-shaped Ca2+-dependence curve of type I InsP3R is ideal for supporting Ca2+ oscillations, whereas the properties of type III InsP3R are better suited to signal initiation.

Characterization of the ryanodine receptor/channel of invertebrate muscle.

Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161-173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM-10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner, J. Biol. Chem. 267: 15893-15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.

Inositol 1,4,5-trisphosphate (InsP3) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling.

The inositol 1,4,5-trisphosphate (InsP3)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754; Finch, E.A., T. J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443-446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642-29649; Iino, M. 1990. J. Gen. Physiol. 95:1103-1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591-598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 microM InsP3, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751-754). We report here that, when we used lower InsP3 concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP3 concentrations, channel activity persisted at Ca concentrations as high as 30 microM. To explore this unexpected response of the channel, we measured InsP3 binding over a broad range of InsP3 concentrations. We found the well-characterized high affinity InsP3 binding sites (with Kd < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61-67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192-195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468-470) and a low affinity InsP3 binding site (Kd = 10 microM). Using these InsP3 binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP3 levels and the maintained channel activity at high Ca and InsP3 levels. The observed Ca dependence of the InsP3-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP3, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.

Regulation of intracellular calcium release channel function by arachidonic acid and leukotriene B4.

Arachidonic acid has been shown to affect the intracellular calcium concentration in many cell types (1-5), but the target of this regulation was unclear. Here we show that two types of intracellular calcium release channel, the inositol 1,4,5-trisphosphate-gated channel (IP3R) and the ryanodine receptor (RyR) are modulated in an opposing manner by arachidonic acid and its product leukotriene B4 (LTB4). The IP3R was inhibited by arachidonic acid (Ki = 27 nM), whereas the RyR was unaffected by this compound. In contrast, 100 nM LTB4 fully activated the RyR but did not influence the IP3R. The concerted action of arachidonic acid and LTB4 could provide specific mobilization of stored calcium by terminating IP3-induced release and activating the RyR/calcium release channel by its newly identified agonist.

Methanethiosulfonate derivatives inhibit current through the ryanodine receptor/channel.

To identify regions of the ryanodine receptor (RyR) important for ion conduction we modified the channel with sulfhydryl-reacting compounds. After addition of methanethiosulfonate (MTS) compounds channel conductance was decreased while other channel properties, including channel regulation by ATP, caffeine, or Ca, were unaffected. The site of action was accessible to the MTS compounds from the cytoplasmic, but not the luminal, side of the channel. In addition, the hydrophilic MTS compounds were only effective when the channel was open, suggesting that the compounds covalently modify the channel from within the water-filled ion conducting pathway. The decrease in channel current amplitude occurred in a step-wise fashion and was irreversible and cumulative over time, eventually leading to the complete block of channel current. However, the time required for each consecutive modification during continuous exposure to the MTS compounds increased, suggesting that successive modification by the MTS compounds is not independent. These results are consistent with the hypothesis that the channel forms a wide vestibule on the cytoplasmic side and contains a much smaller opening on the luminal side. Furthermore, our results indicate that the MTS compounds can serve as functional markers for specific residues of the RyR to be identified in molecular studies.

Ligand-gated calcium channels inside and out.

Calcium release from intracellular stores occurs through two types of channels associated with intracellular membranes, namely, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. Recently, it has been shown that these channels are regulated by allosteric mechanisms and associated proteins. Release of intracellular calcium induces the opening of calcium-permeable channels on the plasma membrane. Current work has focused on the molecular and functional characterization of these channels which have been identified as store-operated channels or calcium release activated channels.

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+.

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.

Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels from cardiac muscle.

Ryanodine receptors (RyRs) are intracellular channels that regulate the release of Ca2+ from the endoplasmic reticulum of many cell types. The RyRs are physically associated with FK506-binding proteins (FKBPs); immunophilins, with cis-trans peptidyl-prolyl isomerase activity. FKBP12 copurifies with RyR1 (skeletal isoform) and modulates its gating. A different form of FKBP with a slightly higher molecular weight copurifies with RyR2 (cardiac isoform). Previous studies have demonstrated that FKBP stablizes gating of the skeletal Ca(2+)-release channel. In the present study, we measured the activity of cardiac RyRs incorporated into planar lipid bilayers to show that rapamycin, a drug that inhibits the prolyl isomerase activity of FKBP and dissociates FKBP from the RyR, increases the open probability and reduces the current amplitude of cardiac muscle Ca(2+)-release channels. These experiments show for the first time that submicromolar concentrations of rapamycin can alter channel function. Our results provide support for the hypotheses that FKBP functionally associates with the RyR and that the immunosuppressant drug, rapamycin, alters the function of both cardiac and skeletal muscle isoforms of the Ca(2+)-release channel. Our findings suggest that FKBP-dependent modulation of channel function may be generally applicable to all members of the intracellular Ca(2+)-release channel family and that FKBPs may play important regulatory roles in many cell processes, ranging from long-term depression in neurons to contractility in cardiomyocytes.

Single channel properties and calcium conductance of the cloned expressed ryanodine receptor/calcium-release channel.

The calcium-release channel/ryanodine receptor of the sarcoplasmic reticulum is a 2.3 million-D structure required for intracellular calcium release during excitation-contraction coupling in skeletal muscle. This structure is the largest ion channel characterized to date and is composed of four 565,000-D ryanodine receptors plus four molecules of FKBP12. In the present study we describe the single channel properties of the cloned expressed ryanodine receptor, with and without FKBP12, reconstituted into planar lipid bilayers with Ca as the charge carrier. The conductance for Ca (luminal, 53 mM/cytoplasmic, 10 microM) was 103 pS for the cloned expressed RyR and for the native channel from rabbit skeletal muscle. Conductance through the channel was Ca dependent: A decrease in the Ca gradient to luminal 10.6/cytoplasmic 10 microM reduced conductance to 68 pS for both the cloned and native RyR. The recombinant ryanodine receptor consistently behaved like the native skeletal muscle channel in terms of activation by caffeine, calcium, and ATP; inhibition by ruthenium red; and modulation by ryanodine. In the absence of FKBP12, the cloned expressed RyR exhibited multiple subconductance states and addition of FKBP12 reduced the frequency of subconductance states. These results show that with Ca as the charge carrier, the single channel properties of the cloned expressed RyR plus FKBP12 are essentially the same as those of the native channel.

Functional properties of intracellular calcium-release channels.

Two major classes of intracellular calcium-release channels have been identified, the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor. These channels are the largest ion channels identified to date. Recent studies have established that approximately 90% of each of these proteins protrudes into the cytoplasm, presumably exposing many regulatory sites on the channel and allowing functional interactions with other cytoplasmic proteins. Current work is aimed at understanding the molecular mechanisms and cellular roles of these regulatory processes.