Calcium Channels: Critical Targets of Toxicants and Diseases.
The structure and regulation of voltage-dependent calcium channels and their role in inherited and toxicant-induced neurologic disorders was the topic of a conference sponsored and hosted by the National Institute of Environmental Health Sciences The National Institute of Environmental Health Sciences (NIEHS) is one of 27 Institutes and Centers of the National Institutes of Health (NIH),which is a component of the Department of Health and Human Services (DHHS). The Director of the NIEHS is Dr. David A. Schwartz. , the U.S. Environmental Protection Agency Environmental Protection Agency (EPA), independent agency of the U.S. government, with headquarters in Washington, D.C. It was established in 1970 to reduce and control air and water pollution, noise pollution, and radiation and to ensure the safe handling and , Sibia Neurosciences, and the Muscular Dystrophy Association The Muscular Dystrophy Association (MDA) is an organization founded in 1950 which combats muscular dystrophy and diseases of the nervous system and muscular system in general by funding research, providing medical and community services, and educating health professionals on 6-8 December 1999. The conference, "Calcium Channels: Critical Targets of Toxicants and Diseases," brought together experts in a variety of disciplines to discuss our current knowledge of calcium channel structure and function, channel regulation by intracellular signaling molecules, and the effects of mutations, aging, and toxicants on channel functions.
Calcium channels play important and diverse roles in neurologic function. It has been known for many years that calcium channels are likely targets of a variety of organic and inorganic toxicants. More recently, calcium channel mutations have been implicated in several inherited neurologic disorders, including migraine headaches, various movement disorders, and some types of malignant hyperthermia. The last decade has seen great strides in determining the subunit structure of calcium channels, how the genetic diversity of subunits generates a variety of functionally distinct channels, and how intracellular signaling pathways regulate channel function. The meeting was convened to guide future research by applying this knowledge of calcium channel biology to further our understanding of how toxicants and mutations alter channel functions.
The presentations discussed four major aspects of calcium channels that are relevant for understanding their roles in genetic and environmental neurologic disorders: the genetics and molecular biology of channel structure; interactions between calcium channels and intracellular signaling pathways; the effects of toxicants on calcium channel function, both through direct interactions with channel subunits and by altering intracellular signaling; and the genetics, molecular biology, and physiology of inherited disorders caused by mutations in the genes encoding calcium channel subunits or from autoimmune disorders caused by the generation of antibodies against subunits of those proteins.
Structure and Function of Calcium Channels
Voltage-dependent calcium channels are composed of four principal subunits: the transmembrane transmembrane /trans·mem·brane/ (trans-mem´bran) extending across a membrane, usually referring to a protein subunit that is exposed on both sides of a cell membrane.
adj. , pore-forming [Alpha]1 subunits and three accessory subunits that modulate channel function--the glycosylated [Alpha]2[Delta] subunits, the integral membrane subunits, and the cytoplasmic cytoplasmic
pertaining to or included in cytoplasm.
include secretory inclusions (enzymes, acids, proteins, mucosubstances), nutritive inclusions (glycogen, lipids), pigment granules (melanin, lipofuscin, [Beta] subunits. There are several isoforms of each of these channel subunits, and the composition of the channel complex determines its expression level, localization Customizing software and documentation for a particular country. It includes the translation of menus and messages into the native spoken language as well as changes in the user interface to accommodate different alphabets and culture. See internationalization and l10n. , kinetics, and pharmacology.
Ten genes on nine different chromosomes encode the l subunits. Diversity of [Alpha]1 subunits gives rise to the familiar classification of calcium channels (i.e., L, N, T, P/Q, R) on the basis of channel kinetics, cation cation (kăt'ī`ən), atom or group of atoms carrying a positive charge. The charge results because there are more protons than electrons in the cation. permeability, and pharmacology. Several human disorders have been traced to mutations in the l subunit genes, including familial hemiplegic migraine familial hemiplegic migraine
A rare inherited migraine disorder characterized by a temporary paralysis of one side of the body followed by severely throbbing headaches and nausea; it occasionally can cause coma. , several types of ataxia ataxia (ətăk`sēə), lack of coordination of the voluntary muscles resulting in irregular movements of the body. Ataxia can be brought on by an injury, infection, or degenerative disease of the central nervous system, e.g. , hypokalemic periodic paralysis hy·po·ka·le·mic periodic paralysis
An inherited form of periodic paralysis characterized by attacks in which the serum potassium level is low and respiratory paralysis may occur. Onset usually occurs between the ages of 7 and 21 years. , and malignant hyperthermia. In most cases, it remains to be established how the mutations produce pathology.
The accessory subunits ([Alpha]2[Delta], [Beta], and [Gamma]) regulate the expression, localization, kinetics, and modulation of calcium channels, but relatively little is known of the molecular mechanisms involved. In mice, mutations in the [Beta]4 (lethargic) and [Gamma]2 (stargazer stargazer, common name for any of several species of marine fishes of the family Uranoscopidae, found in southern waters, and having the mouth, nostrils, and eyes set high in the head. Stargazers lie buried in the sand, waiting for their prey of small crustaceans. ) subunits, prominently expressed in cerebellar cerebellar /cer·e·bel·lar/ (ser?e-bel´ar) pertaining to the cerebellum.
Involving the part of the brain (cerebellum), which controls walking, balance, and coordination. Purkinje neurons, produce ataxia. Recently, Escayg et al. (1) reported that mutations in the [Beta]4 subunit are associated with some forms of epilepsy and episodic ataxia. It thus appears likely that a variety of human neurologic disorders may be attributable to mutations in the accessory subunits.
Studies of mice with mutant channel subunits are providing clues both to normal channel assembly and to the effects of altered channel function. Of particular importance for the theme of this meeting is that alterations in the expression of one channel subunit are often partially compensated for by changes in expression of other subunits. For example, the absence of the [Beta]4 subunit in the lethargic mutant mouse causes increased expression of the [Beta]1B subunit despite a decrease in the total pool of [Beta] subunit protein. The absence of [Alpha]1A subunits results in decreased expression of [Beta]4 subunits and increases in [Beta]1B subunits. Nevertheless, animals with either mutation show clear pathologic phenotypes, suggesting that compensatory changes in expression of calcium channel subunits are limited in their ability to restore normal neuronal function. Further, at least some of these compensatory changes may themselves cause cellular dysfunction. This principle, that a primary defect in calcium channel function, either genetic, autoimmune, or toxicant-induced, may produce both "primary" symptoms and a constellation of "secondary" symptoms caused by other, possibly compensatory, changes in neuronal physiology, was repeated several times in various contexts during the presentations.
Calcium channel expression also changes with normal aging. For example, in hippocampal neurons, the expression of L-type channels increases with age. This increase in L-type channels is correlated with increased failure of excitatory postsynaptic potentials as well as increased susceptibility to cell death. At the whole-animal level, these changes are correlated with decreased performance on spatial learning tasks. Up-regulation of the expression of the [Alpha]1D subunit appears to underlie the increased expression of L-type channels in hippocampal neurons with age; much smaller increases in expression of [Alpha]1C, and no change in the expression of [Beta]1B subunits are observed. This age-related change in channel expression may make the nervous system more susceptible to both pathologic and toxicologic insult.
Interactions between Calcium Channels and Intracellular Signaling
The activity of calcium channels is regulated by a wide variety of intracellular signaling pathways. Probably the three most well understood are binding and activation of calmodulin calmodulin /cal·mod·u·lin/ (kal-mod´u-lin) a calcium-binding protein present in all nucleated cells; it mediates a variety of cellular reponses to calcium.
n. , phosphorylation phosphorylation, chemical process in which a phosphate group is added to an organic molecule. In living cells phosphorylation is associated with respiration, which takes place in the cell's mitochondria, and photosynthesis, which takes place in the chloroplasts. by several protein kinases, and binding of G protein [Beta][Gamma] subunits. These pathways not only modulate calcium channel activity, all are themselves modulated in various ways by calcium influx through calcium channels, and many interact with one another. Thus, complex feedback mechanisms among calcium channels and intracellular signaling exist and are likely targets for both environmental and genetic disorders.
Calcium channels play key, but far from exclusive, roles in regulating intracellular calcium homeostasis homeostasis
Any self-regulating process by which a biological or mechanical system maintains stability while adjusting to changing conditions. Systems in dynamic equilibrium reach a balance in which internal change continuously compensates for external change in a feedback . Both pathologic conditions (stroke, ischemia, AIDS, etc.) and toxicologic insult (e.g., by methylmercury, lead) produce alterations in calcium homeostasis that can lead to neuronal death. Calcium influx through both voltage- and ligand-gated channels (e.g., the N-methyl-D-aspartate receptor) contributes to alterations in calcium homeostasis, but other intracellular organelles and calcium ATPases also are part of the machinery responsible for regulation of calcium homeostasis. These intracellular calcium stores and ATPases interact with channel-mediated calcium entry to give rise to locally confined calcium increases, or responses such as calcium spikes or waves. The amplitude, time course, and intracellular distribution of calcium signals are important determinants of the cellular response. All aspects of cellular calcium homeostasis are likely targets for diseases and toxicant-induced disorders.
Calmodulin, a ubiquitous calcium-binding protein, has long been known to mediate many of the intracellular effects of calcium influx through calcium channels, especially L-type channels. For example, calcium entry via the L-type channel appears to be critical for activation of a number of different transcriptional pathways, including those mediated by CREB CREB CAMP Response Element Binding Protein
CREB Calgary Real Estate Board
CREB Clean Renewable Energy Bond
CREB Certified Real Estate Broker [cyclic AMP response element binding (protein)], NF-AT (nuclear factor of activated T-cell), and p38 MAP (mitogen-activated protein) kinase. More recently, calmodulin has also been found to act in the feedback regulation of calcium entry through L-type channels. Calmodulin binds to the intracellular carboxy terminal of the [Alpha]1C subunit. Calcium entering through the channel binds to this calmodulin and reduces further calcium influx. Thus calmodulin can act as a key molecule both in stimulation of gene transcription and other cellular events and in feedback inhibition of calcium channels.
Calcium channels may be phosphorylated by many protein kinases, including calcium/calmodulin-dependent protein kinases and protein kinases A and C. The effects of such phosphorylation depend on the channel subtype under study, the protein kinase, and other signaling pathways that may be active. In recent years considerable progress has been made in understanding the molecular mechanisms of G protein modulation of calcium channels (particularly the N-type, or [Alpha]1B, channels). The intracellular amino terminal of the 1B subunit has been shown to be essential for G protein effects, but [Beta] channel subunits are also required for regulation by G protein [Beta][Gamma] subunits. Interactions among all of these intracellular pathways also abound. For example, G protein binding to calcium channels may be modulated by channel phosphorylation.
The Effects of Toxicants on Channel Function
There are two fundamentally different mechanisms whereby neurotoxicants might alter calcium channel function: by direct interaction with channel subunits (e.g., occluding the pore) or by interaction with intracellular signaling pathways that modulate channel activity. Interactions with the channel subunits themselves are well known for example, heavy metals blocking the pore. Although there is a substantial literature describing effects of neurotoxicants on intracellular signaling, relatively little is known about how such effects might modulate calcium channel activity. The time appears to be ripe for a significant research effort to study the interactions among toxicants, intracellular signaling pathways, and calcium channel activity.
Several papers presented at the meeting discussed the effects of specific toxicants [methylmercury, inorganic lead, polychlorinated biphenyls (PCBs), and ethanol] on calcium channels. Interestingly, all of these toxicants, with the possible exception of PCBs, appear to have effects both directly on channels and indirectly via intracellular signaling. Unfortunately, studies of toxicant toxicant /tox·i·cant/ (tok´si-kant)
1. A poison or poisonous agent.
2. An intoxicant.
adj. effects on calcium channels have used a variety of cell types and experimental approaches, so that unambiguous comparisons among toxicants are often difficult or impossible. Nevertheless, several generalizations can be drawn from the presentations at the meeting.
First, it has long been known that many heavy metals impair current flow through calcium channels, mostly by blocking the pore. However, many metals, in both inorganic and perhaps especially in organic form, have other effects, presumably pre·sum·a·ble
That can be presumed or taken for granted; reasonable as a supposition: presumable causes of the disaster. often mediated through intracellular signaling. Lead is perhaps the best studied of all toxicants with actions on calcium channels. Inhibition of channel current has been demonstrated for acute exposure to low micromolar concentrations of lead in virtually every preparation in which it has been examined, presumably due to blocking the pore. However, lead may also enter the cell through some types of calcium channels (particularly the L-type channel). Intracellular lead may promote channel activity by attenuating the normal channel inactivation inactivation /in·ac·ti·va·tion/ (in-ak?ti-va´shun) the destruction of biological activity, as of a virus, by the action of heat or other agent. by intracellular calcium. This effect occurs at much lower lead concentrations (picomolar to nanomolar) than are required for blocking the pore. Intracellular lead may also activate protein kinase C Protein kinase C ('PKC', EC 220.127.116.11) is a family of protein kinases consisting of ~10 isozymes. They are divided into three subfamilies: conventional (or classical), novel, and atypical based on their second messenger requirements. and various calmodulin-dependent enzymes, such as calmodulin-dependent protein kinase, phosphodiesterase phosphodiesterase /phos·pho·di·es·ter·ase/ (-di-es´ter-as) any of a group of enzymes that catalyze the hydrolytic cleavage of an ester linkage in a phosphoric acid compound containing two such ester linkages. , and calcineurin, at picomolar concentrations. The impacts of such actions on calcium channel activity, or on the pathways that are normally activated by calcium influx through calcium channels, remain largely unknown.
Methylmercury provides another example of complex interactions of toxicants with calcium channels. Unlike most inorganic heavy metals, methylmercury is lipophilic lipophilic,
adj/n the ability to dissolve or attach to lipids.
adj 1. showing a marked attraction to, or solubility in, lipids.
2. and may permeate cells directly through the phospholipid phospholipid (fŏs'fōlĭp`ĭd), lipid that in its simplest form is composed of glycerol bonded to two fatty acids and a phosphate group. bilayer bilayer /bi·lay·er/ (bi´la-er) a membrane consisting of two molecular layers.
A structure, such as a film or membrane, consisting of two molecular layers. or as a cysteine cysteine (sĭs`tēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of mammalian protein. complex and exert intracellular effects in addition to blocking the channel pore. For example, although acute, externally applied methylmercury blocks calcium channels, the block is slowly and incompletely reversible. The lack of reversibility may be the result of prolonged residence in the phospholipid bilayer or the cytoplasm cytoplasm: see protoplasm.
Portion of a eukaryotic cell outside the nucleus. The cytoplasm contains all the organelles (see eukaryote). due to the lipophilicity of this organometal, or due to the relatively greater reactivity of methylmercury with sulfhydryl groups on calcium channels. Prolonged exposure (6 days) of PC12 cells to nanomolar concentrations of methylmercury reduces whole cell calcium currents; it is not known whether this decrease in current is caused by direct effects of methylmercury on calcium channels, or to indirect effects, such as altering channel density.
Voltage-gated calcium channels are also prominent targets of ethanol. Acute exposure to ethanol inhibits the function of both Land N-type calcium channels in several neuronal systems. The mechanisms associated with impaired function of L-type channels involve decreased open channel probability and enhanced inactivation of the channel. With chronic exposure to ethanol, the density of L- and N-type calcium channels increases in PC12 cells and in rodent brain. Up-regulation of L-type channels is thought to contribute to the enhanced neuronal excitability excitability
readiness to respond to a stimulus; irritability. seen during ethanol withdrawal; up-regulation of N-type channels would be expected to enhance transmitter release. Both effects could contribute to the behavioral hyperexcitability seen during ethanol withdrawal. The up-regulation of L- and N-type calcium channels in PC12 cells by ethanol is due in part to effects on protein kinase C (PKC PKC Protein Kinase C (biochemistry)
PKC Public Key Cryptography
PKC Public Key Certificate
PKC PaKua Chang (Chinese martial art)
PKC Paroxysmal Kinesigenic Choreoathetosis ); chronic exposure to ethanol increases PKC-mediated phosphorylation and the abundance of PKC [Delta] and isozymes. Interestingly, inhibiting PKC [Delta] prevents upregulation of L-type channels by ethanol, while inhibiting PKC [Epsilon] prevents up-regulation of N-type channels. Thus acute exposure to ethanol inhibits the function of two classes of voltage-gated calcium channels, whereas chronic exposure induces up-regulation of these channels by PKC-dependent mechanisms that involve different PKC isozymes.
The effects of PCBs on the ryanodine receptor provide an illustration of complex effects of toxicants on calcium channels. Ryanodine receptors mediate calcium-induced calcium release Calcium-induced calcium release (CICR) is a mechanism of calcium release from muscle sarcoplasmic reticulum that was proposed in the 1970s. Originally proposed for skeletal muscle, subsequent research has revealed that it is actually the predominant mechanism in from the endoplasmic endoplasmic
pertaining to or arising from endoplasm.
small, cytoplasmic granules consisting of approximately 60% RNA and 40% protein. reticulum reticulum /re·tic·u·lum/ (re-tik´u-lum) pl. retic´ula [L.]
1. a small network, especially a protoplasmic network in cells.
2. reticular tissue. . Calcium influx through plasma membrane calcium channels is an important trigger for calcium-induced calcium release; the calcium released from intracellular stores may then cause calcium-induced inactivation of the channels (perhaps via calmodulin; see above). Some non-coplanar PCBs impair the function of the ryanodine receptor and alter intracellular calcium regulation. This would be expected to alter calcium channel function as well, but such effects, if they occur, remain unknown.
Neurologic Disorders Mediated by Calcium Channels
A diverse set of clinical syndromes is associated with mutations in the genes encoding the various calcium channel subunits. Some of the disorders affect skeletal muscle or the neuromuscular junction, such as hypokalemic periodic paralysis (HypoKPP) and LambertEaton myasthenic syndrome (LEMS LEMS Lambert-Eaton Myasthenic Syndrome
LEMS Laboratory for Engineering Man/Machine Systems
LEMS Low Energy Magnetic Spectrometer
LEMS Link Elimination Via Matching Scheme
LEMS Low Energy Molecular Scattering
LEMS Linear Econometric Modeling System ). Other diseases affect the central nervous system, such as familial hemiplegic migraine (FHM FHM For Him Magazine
FHM Fachhochschule München (Munich University of Applied Sciences, Germany)
FHM Forest Health Monitoring
FHM Familial Hemiplegic Migraine
FHM Funeral Home Marker (genealogy) ), episodic ataxia type 2 (EA-2), and spinocerebellar ataxia type 6 (SCA (Single Connector Attachment) An 80-pin plug and socket used to connect peripherals. With a SCSI drive, it rolls three cables (power, data channel and ID configuration) into one connector for fast installation and removal. 6). Interestingly, some features seem to be shared by most of the clinical syndromes`
* Most syndromes have episodic features: muscle weakness, ataxia, or migraine attacks
* All syndromes have some features that are chronic and/or progressive, such as muscle damage or cerebellar atrophy
* There seems to be a gradual superimposition In graphics, superimposition is the placement of an image or video on top of an already-existing image or video, usually to add to the overall image effect, but also sometimes to conceal something (such as when a different face is superimposed over the original face in a of permanent deficits on initially episodic deficits. One exception seems to be SCA6, which lacks episodic features
* All genetic syndromes show reduced penetrance penetrance /pen·e·trance/ (pen´i-trins) the frequency with which a heritable trait is manifested by individuals carrying the principal gene or genes conditioning it.
n. , in that not all carriers of a given mutation are affected clinically, and there is considerable variation in the symptoms. Without doubt, other genetic and/or environmental factors are involved in all of the channelopathies
* Several syndromes are responsive to azetozolamide. Somehow, changing the pH in the extracellular environment and/or blocking the channel pores by the binding of protons seems to affect these channelopathies
* Some general triggers such as stress, fatigue, or certain foods seem to precipitate attacks in different syndromes.
Sometimes the same syndrome is caused by mutations in different genes [e.g., mutations in the ryanodine receptor (RYR RyR Ryanodine Receptor
RYR Robert Yates Racing (NASCAR)
RYR Red Yeast Rice
RYR Ryanair Ireland (ICAO code) 1) or the [Alpha] 1S calcium channel cause malignant hyperthermia], and sometimes mutations in the same gene are associated with different clinical syndromes (FHM and EA-2 are both caused by mutations in the [Alpha] 1A calcium channel subunit). This leads to the tantalizing tan·ta·lize
tr.v. tan·ta·lized, tan·ta·liz·ing, tan·ta·liz·es
To excite (another) by exposing something desirable while keeping it out of reach. suggestion that trigger or threshold factors might be shared by all or most channelopathies. It will be a major challenge to reveal those common factors. From various presentations during the meeting, it appears that steroid hormones, alcohol, and heavy metals may be candidates worth pursuing.
Several speakers highlighted the difficulties of determining physiologic mechanisms for calcium channelopathies. For example, some types of FHM appear to be caused by mutations in the gene encoding the [Alpha] 1A (P/Q) subunit. Recent electrophysiologic experiments in embryonic kidney cells transfected with FHM genes show that the mutated genes alter channel kinetics. However, some of the mutations decreased channel density and conductance, leading to reduced calcium influx, whereas other mutations caused an increased channel density and increased open probability, leading to increased calcium influx.
A second example of the difficulty of merging clinical and molecular/physiologic studies is illustrated by EA-2. Until recently, EA-2 was thought to be associated with truncating mutations in the [Alpha] 1A gene. However, missense mutations have now been found as well. Further, the severity of the clinical phenotype can dramatically differ between various mutations. For instance, the Gly293Arg mutation is associated with a severe progressive ataxia, whereas other mutations associated with EA-2 are less progressive. Finally, two apparently different syndromes may be caused by the same mutation (i.e., the same mutation of [Alpha] 1A may cause EA-2 and SCA6).
HypoKPP provided a third example of the promise and difficulties of genetic and physiologic analysis of channelopathies. HypoKPP is caused by three different mutations in the calcium channel subunit [Alpha] 1S (skeletal muscle L-type channel). All three known mutations cause substitutions for positively charged arginine arginine (är`jənĭn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of proteins. residues in the S4 transmembrane domains, which are regions implicated in voltage sensing. These mutations appear to cause reduced current density. It is not understood how reduced calcium currents are related to some of the symptoms of HypoKPP, such as muscle depolarization depolarization /de·po·lar·iza·tion/ (de-po?lahr-i-za´shun)
1. the process or act of neutralizing polarity.
2. in electrophysiology, reversal of the resting potential in excitable cell membranes when stimulated. and reduced serum potassium.
LEMS combines features of both toxicant-induced and inherited channelopathies. It is believed that LEMS begins with an autoimmune response, often evoked by small cell lung cancer Lung Cancer, Small Cell Definition
Small cell lung cancer is a disease in which the cells of the lung tissues grow uncontrollably and form tumors.
Lung cancer is divided into two main types: small cell and non-small cell. . One of the main immunogens is a P/Q-type channel component, probably because P/Q channels are highly expressed on these tumor cells. Circulating autoantibodies recognize P/Q-type channels present at the neuromuscular junction, where they destroy the active zones of nerve terminals, leading to reduced acetylcholine acetylcholine (əsēt'əlkō`lēn), a small organic molecule liberated at nerve endings as a neurotransmitter. It is particularly important in the stimulation of muscle tissue. release. A LEMS-Like disorder can be elicited in mice by repeated administration of serum, plasma, or immunoglobulins from affected patients. In LEMS immunoglobulin-treated mice, there is a reduction in the amplitude of calcium currents and frequency-dependent facilitation. Mostly P/Q and N-type channels normally carry these calcium currents, but in LEMS immunoglobul-intreated mice an additional L-type current has been identified. Thus, there appears to be a down-regulation of normally expressed channels and a compensatory up-regulation of L-type channels. It is not known whether such changes in channel expression occur in human LEMS patients.
Conclusions and Future Directions
Two dominant themes emerged from the meeting. First, we are in an excellent position to apply our knowledge of the molecular biology and physiology of calcium channels to detailed analyses of genetic defects and toxicant effects. Second, the complex interplay among calcium channels, calcium influx, intracellular calcium homeostasis, and intracellular signaling is likely to be important both in clinical syndromes attributable to calcium channel mutations and in the neurologic effects of a variety of toxicants. Although these two themes are not completely independent, it is useful to discuss them separately. Within each theme, implications for future research in understanding clinical syndromes and neurotoxicant effects will be considered.
Molecular biology and physiology of calcium channels. The variety of physiologically defined calcium channels is now becoming understood at the molecular level. Multiple isoforms of each of the subunits have now been identified, cloned, and sequenced (or will be soon). Studies of calcium channel subunit mutations, both in humans and in animal models, will be important in two different ways. First, and most obviously, understanding the electrophysiologic and cellular consequences of subunit mutations provides at least the potential to design novel, rational treatments that may alleviate the symptoms brought on by the inherited channel defects. Such treatments may be either at the level of the channel itself or at "downstream" intracellular effects (e.g., on calcium homeostasis or intracellular signaling pathways). The possible necessity of downstream interventions is highlighted by the fact that similar clinical syndromes may be caused by mutations with different effects on channel physiology, and different syndromes may be elicited by the same mutation. Second, electrophysiologic analyses of cells transfected with known subunit combinations, mutated subunits (either naturally occurring or generated by site-directed mutagenesis), and subunits derived from mutant mouse lines will identify key amino acid residues that regulate voltage dependence, channel kinetics, pharmacologic sensitivity, and modulation by intracellular signaling pathways in normal channels. Such analyses, often initially driven by the need to understand the basis of clinical syndromes, have already provided important information about basic channel physiology and offer great promise for the future.
In neurotoxicologic studies, calcium channel diversity should be viewed as an opportunity, not an obstacle. Studies of channel subunit mutations have clearly shown that different effects, both on the cellular and whole-animal levels, occur when different subunits, or different isoforms of the same subunit (especially the [Alpha] 1 subunits) malfunction. It is quite likely that some toxicants preferentially target certain subunits and/or isoforms. Such toxicant selectivity can now be directly analyzed in cells transfected with cloned subunits, rather than by trying to use pharmacologic tools to tease apart effects in cells expressing many channel types.
Several presenters noted that the progression and severity of clinical syndromes vary considerably even within a population bearing the same mutated subunit gene, suggesting that environmental factors may modulate the threshold at which a mutated subunit produces clinical effects. It would be extremely useful, both for clinicians and for neurotoxicologists, to determine if interactions between toxicants and already-defective channels might hasten the onset or increase the severity of symptoms.
Calcium channel regulation, calcium homeostasis, and intracellular signaling. Several speakers pointed out that calcium channels both regulate and are regulated by intracellular calcium homeostasis and intracellular signaling pathways. Further, channel regulation occurs both acutely (e.g., through changes in channel activity by phosphorylation or G protein binding) and chronically (e.g., up- or down-regulation of channel density). Bidirectional regulation may have profound implications for treating genetic calcium channelopathies. First, in many cases the clinical symptoms are caused, not directly by altered channel function per se, but by altered cellular metabolism caused by channel dysfunction. Therefore, understanding the actual cause of the symptomatology symptomatology /symp·to·ma·tol·o·gy/ (simp?to-mah-tol´ah-je)
1. the branch of medicine dealing with symptoms.
2. the combined symptoms of a disease.
n. and/or designing treatments will require an understanding of the signaling pathways downstream of calcium channel dysfunction. Indeed, it may well be found that some of the symptoms of clinical syndromes not currently thought to be mediated by calcium channels may arise because defective intracellular signaling alters the function of genetically normal channel subunits. Second, cells often up- or down-regulate various channel subunits in response to dysfunction of a given subunit, and the resulting changes in channel types and densities may ameliorate and/or contribute significantly to the clinical symptoms. The mechanisms underlying changes in channel expression, largely unknown at the present time, are likely to be found in the interplay among channel function, intracellular calcium homeostasis, and intracellular signaling pathways.
Neurotoxicants may affect calcium channel function either by directly interacting with the channel subunits or by altering intracellular pathways that regulate channel subunits. A large number of toxicants are known to alter intracellular signaling pathways and/or alter calcium homeostasis, but how these effects impact calcium channel function remains largely unexplored. Such studies are feasible and important. As noted above, in individuals bearing mutated channel subunit genes, toxicants may reduce the threshold at which cellular and organismal dysfunction occurs. These effects may well originate through actions on altered intracellular signaling rather than directly on the channel subunits themselves. It should be noted that aging also changes calcium channel subunit density and susceptibility to insult (e.g., ischemia or excitotoxicity). Although considerable neurotoxicologic research has focused on the developing nervous system, much less attention has been paid to the aging nervous system, which probably also has a unique pattern of susceptibility to toxicants. Interactions between altered intracellular signaling during aging and toxicant exposure, particularly long-term, cumulative exposure, may promote age-related neurologic dysfunction.
REFERENCES AND NOTES
(1.) Escayg A, De Waard M, Lee DD, Bichet D, Wolf P, Mayer T, Johnson J, Baloh R, Sander T, Meisler MH. Coding and noncoding variation of the human calcium-channel [Beta]4-subunit gene CACNB4 in patients with idiopathic generalized epilepsy Idiopathic generalized epilepsy (IGE) is a group of epileptic disorders that are believed to have a strong underlying genetic basis. Patients with an IGE subtype are typically otherwise normal and have no anatomical brain abnormalities. and episodic ataxia. Am J. Hum Genet 06:1531-1539 (2000).
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The School of Medicine has a unique curriculum among American medical schools.
Timothy J. Shafer U.S. Environmental Protection Agency
Janusz Suszkiw University of Cincinnati The University of Cincinnati is a coeducational public research university in Cincinnati, Ohio. Ranked as one of America’s top 25 public research universities and in the top 50 of all American research universities,
Stanley Thayer University of Minnesota Medical School The University of Minnesota Medical School is the medical school of the University of Minnesota. It is a combination of two campuses situated in Minneapolis and Duluth, Minnesota.
Richard W. Tsien Beckman Center
Arn Van den Maagdenberg Leiden University Medical Center
Address correspondence to W. Atchison, Department of Pharmacology, Michigan State University, B331 Life Science Building, East Lansing, MI 48824-1317. Telephone: (517) 353-4947. Fax: (517) 353-8915. E-mail: firstname.lastname@example.org
Received 20 July 2000; accepted 9 August 2000.
Gerald Audesirk,(1) David Armstrong,(2) Arn M.J.M. van den Maagdenberg,(3) William Atchison,(4) Timothy Shafer,s and Colin Fletcher(6)
(1)Biology Department, University of Colorado at Denver
In 1912, the University of Colorado established a downtown Denver campus to meet the needs of the city's rapidly expanding , Denver, Colorado, USA; (2)Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. , North Carolina, USA; (3)Human and Clinical Genetics Department, Leiden University, Leiden, The Netherlands; (4)Michigan State University, Lansing, Michigan, USA; (5)Neurotoxicology Division, Environmental Protection Agency, Research Triangle Park, North Carolina, USA; (6)Genomics Institute of the Novartis Research Foundation The Genomics Institute of the Novartis Research Foundation (GNF) is a research institution specializing in basic biological as well as drug discovery research using high-throughput techniques. , San Diego, California “San Diego” redirects here. For other uses, see San Diego (disambiguation).
San Diego is a coastal Southern California city located in the southwestern corner of the continental United States. As of 2006, the city has a population of 1,256,951. , USA