The G. L. Brown Prize Lecture. Hypoxic regulation of ion channel function and expression

Acute hypoxia regulates the activity of specific ion channels in a rapid and reversible manner. Such effects underlie appropriate cellular responses to hypoxia which are designed to initiate cardiorespiratory reflexes and contribute importantly to other tissue responses, all of which are designed to improve tissue O2 supply. These responses include excitation of chemoreceptors as well as pulmonary vasoconstriction and systemic vasodilatation. However, such responses may also contribute to the adverse responses to hypoxia, such as excitotoxicity in the central nervous system. Whilst numerous ion channel types are known to be modulated by acute hypoxia, the nature of the O2 sensor in most tissues remains to be identified. Prolonged (chronic) hypoxia regulates functional expression of ion channels, and so remodels excitability of various cell types. Whilst this may contribute to adaptive responses such as high-altitude acclimatization, such altered channel expression may also contribute to the onset of pathological disorders, including Alzheimer's disease. Indeed, evidence is emerging that production of pathological peptides associated with Alzheimer's disease is increased during prolonged hypoxia. Such effects may account for the known increased incidence of this disease in patients who have previously endured hypoxic episodes, such as congestive heart failure and stroke. Identification of the mechanisms coupling hypoxia to the increased production of these peptides is likely to be of therapeutic benefit.     

Ferric ion, ferrocyanide, and inorganic phosphate as cytochemical reactants at peripheral nodes of Ranvier.

Ferric ion (Fe3+) and ferrocyanide (Fe(CN)64-) were used under a variety of conditions to stain nodes of Ranvier in mammalian peripheral nerves. Principal findings are: 1. Ferric ion will bind to the extracellular nodal gap substance if nerves are pretreated with a phosphate buffer; or, it will bind to the cytoplasmic surface of the nodal axolemma if pretreatment is with cacodylate or veronal--acetate buffer. 2. Ferrocyanide will bind to the inner surface of the nodal axolemma, where it may react with ferric ion to form a blue stain, or with an osmium compound to form a black stain. 3. Ferric ion and ferrocyanide are bound to nodes as colloidal precipitates, and may migrate away from their sites of formation. 4. Not all nodes in a single piece of tissue, or in a single fibre have identical staining properties. It is concluded that ferric ion, ferrocyanide, and inorganic phosphate are valuable as cytochemical reactants for peripheral nodes of Ranvier, but they must be used in carefully controlled experimental situations in order to avoid spurious results.     

Physicochemical properties of a liquid ion exchanger microelectrode and its application to biological fluids.

Double-barreled potassium or chloride ion-selective microelectrodes were constructed using a liquid ion exchanger. Technical details of the fabrication of double-barreled microelectrodes, having a PD sensor as one barrel and an ionic sensor as the other, are described. The sensitivity of K+ or CI- ion-selective microelectrodes exhibited an approximately Nernstian response over a temperature range of 7 degrees-37 degrees C, and the electromotive force (EMF) was stable within +/- mV for a few hours. The rise time was less than 1 sec. The effect of pH on the electrode response was negligible over a physiological range of pH 5.6 to 7.8. The selectivity constants of the K+ microelectrode to other cations were 0.011 for Na+, 0.200 for NH+4, and less than 0.002 and 0.001 for Ca++ and Mg++, respectively, while that of the CI- microelectrode was 0.067 for HCO-3. Glucose or urea has no effect on the EMF. Protein has a significant effect on ion exchanger membrane only when the concentration of the tested ion is low and protein is high. On the basis of this background the determination of K+ and CI- activity was carried out both in vivo and in vitro on several biological samples, such as serum, tissue and cellular fluids, and other protein-containing fluids. The values obtained with the microelectrode were consistent with those obtained with the other conventional methods or with the current theory on electrolyte solutions. These results were taken to assure the practical application of these electrodes to biological studies in many fields.     

Metabolic flux analysis: a powerful tool for monitoring tissue function.

In recent years, metabolic flux analysis has been widely used in bioprocess engineering to monitor cell viability and improve strain activity. Metabolic flux analysis refers to a methodology for investigating cellular metabolism whereby intracellular fluxes are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of reaction networks. By considering the stoichiometry of biochemical reactions, it is possible to estimate the degree of engagement of each pathway participating in overall cellular activity, and hence obtain a comprehensive view of a cell s metabolic state. Given the potential impact of cellular energy metabolism on the function of engineered tissues, such comprehensive analysis of metabolic activity can be an extremely useful tool for tissue engineers. Estimates of intracellular fluxes under various environmental conditions could be used to optimize function in vivo as well as culture conditions in vitro. In this review, we provide a brief theoretical background of metabolic flux analysis and summarize the most widely used experimental approaches to obtain flux data. This review is intended as an overview of the field and as a starting point for tissue engineers wishing to learn about and eventually employ this methodology.     

Mechanisms of ion transport across the choroid plexus

1. Mechanisms of ion transport across the choroidal epithelium were investigated using an in vitro preparation of the frog choroid plexus.2. Sodium was actively transported across the plexus from the vascular to the ventricular surface by an ouabain sensitive electrically silent pump. As in other epithelial membranes the rate of sodium transport was stimulated by the presence of bicarbonate ions in the Ringer solutions. Chloride and bicarbonate ions accompany the net flux of sodium across this tissue.3. Some experiments suggest that potassium is actively transported from the ventricular to the serosal surface, and that the rate of transport is a function of the extracellular potassium concentration.4. No evidence was obtained to suggest that calcium is actively transported across this tissue in either direction.5. Diamox, ethoxyzolamide, pitocin, pitressin, hydrocortisone, amiloride, spironolactone and anoxia all failed to influence sodium transport.6. The sequence of passive ion permeation across the plexus was P(Rb) approximately P(K) > P(Cs) approximately P(Na) approximately P(Cl) approximately P(HCO3) > P(Li) as deduced from diffusion potential measurements. At least for Na, K and Cl there was a good correlation between the permeability coefficients derived from unidirectional flux measurements and from electrical parameters. This indicates that exchange diffusion is unimportant as a mechanism for passive ion transport.7. The instantaneous current-voltage curves were linear in both symmetrical and asymmetrical salt solutions and the choroid plexus conductance was found to be directly proportional to the external salt concentration. These and other lines of evidence suggest that the major route of passive ion permeation across this epithelium is via the tight junction route and not through the cell interior.8. These results are discussed in relation to the in vivo studies of c.s.f. secretion and the mechanisms of active and passive ion transport across other epithelial membranes such as the gall-bladder, intestine and renal proximal tubule.     

Effects of ion channel blockade on the distribution of Na, K, Ca and other elements in oxygen-glucose deprived CA1 hippocampal neurons

The pathophysiology of brain ischemia and reperfusion injury involves perturbation of intraneuronal ion homeostasis. To identify relevant routes of ion flux, rat hippocampal slices were perfused with selective voltage- or ligand-gated ion channel blockers during experimental oxygen-glucose deprivation and subsequent reperfusion. Electron probe X-ray microanalysis was used to quantitate water content and concentrations of Na, K, Ca and other elements in morphological compartments (cytoplasm, mitochondria and nuclei) of individual CA1 pyramidal cell bodies. Blockade of voltage-gated channel-mediated Na+ entry with tetrodotoxin (1 microM) or lidocaine (200 microM) significantly reduced excess intraneuronal Na and Ca accumulation in all compartments and decreased respective K loss. Voltage-gated Ca2+ channel blockade with the L-type antagonist nitrendipine (10 microM) decreased Ca entry and modestly preserved CA1 cell elemental composition and water content. However, a lower concentration of nitrendipine (1 microM) and the N-, P-subtype Ca2+ channel blocker omega-conotoxin MVIIC (3 microM) were ineffective. Glutamate receptor blockade with the N-methyl-D-aspartate (NMDA) receptor-subtype antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP; 100 microM) or the alpha-amino-3-hydroxy-5-methyl-4-isoazole propionic acid (AMPA) receptor subtype blocker 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM/100 microM glycine) completely prevented Na and Ca accumulation and partially preserved intraneuronal K concentrations. Finally, the increase in neuronal water content normally associated with oxygen-glucose deprivation/reperfusion was prevented by Na+ channel or glutamate receptor blockade.Results of the present study demonstrate that antagonism of either postsynaptic NMDA or AMPA glutaminergic receptor subtypes provided nearly complete protection against ion and water deregulation in nerve cells subjected to experimental ischemia followed by reperfusion. This suggests activation of ionophoric glutaminergic receptors is involved in loss of neuronal osmoregulation and ion homeostasis. Na+ channel blockade also effectively diminished neuronal ion and water derangement during oxygen-glucose deprivation and reperfusion. Prevention of elevated Nai+ levels is likely to provide neuroprotection by decreasing presynaptic glutamate release and by improving cellular osmoregulation, adenosine triphosphate utilization and Ca2+ clearance. Thus, we suggest that voltage-gated tetrodotoxin-sensitive Na+ channels and glutamate-gated ionotropic NMDA or AMPA receptors are important routes of ion flux during nerve cell injury induced by oxygen-glucose deprivation/reperfusion.     

Combination of ion beam stabilisation, plasma etching and plasma deposition for the development of tissue engineering micropatterned supports

The performance of biomedical assays at both molecular and cellular level depends greatly on the ability to design new polymer surfaces. Patterns can be created by using materials with contrasted surface properties. In this work we describe in detail the preparation of micropatterned surfaces to be used as tissue engineering supports. Poly(ethylene glycol) (PEG) was used as the 'anti-fouling' polymer in opposition to functional regions covered by acrylic acid (AAc). Since spin-casted PEG films are unstable, ion beam stabilization (IBS) treatment was applied in order to render it insoluble. On the other hand, AAc films were deposited by low-power plasma chemical vapour deposition. Chemical properties of both polymers were monitored by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy while topographic features were followed by atomic force microscopy. Finally, a micropattern was produced by using a mask, which isolated the IBS-PEG from the AAc-deposited regions. Endothelial cells cultured on the surface were observed to follow the micropatterns. In fact, for a certain surface density it was observed that the cells present tensile or compressive stresses when forced to remain in the anti-fouling or the functionalised regions, respectively.     

Combination of ion beam stabilisation,plasma etching and plasma deposition for the development of tissue engineering micropatterned supports

The performance of biomedical assays at both molecular and cellular level depends greatly on the ability to design new polymer surfaces. Patterns can be created by using materials with contrasted surface properties. In this work we describe in detail the preparation of micropatterned surfaces to be used as tissue engineering supports. Poly(ethylene glycol) (PEG) was used as the 'anti-fouling' polymer in opposition to functional regions covered by acrylic acid (AAc). Since spin-casted PEG films are unstable, ion beam stabilization (IBS) treatment was applied in order to render it insoluble. On the other hand, AAc films were deposited by low-power plasma chemical vapour deposition. Chemical properties of both polymers were monitored by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy while topographic features were followed by atomic force microscopy. Finally, a micropattern was produced by using a mask, which isolated the IBS-PEG from the AAc-deposited regions. Endothelial cells cultured on the surface were observed to follow the micropatterns. In fact, for a certain surface density it was observed that the cells present tensile or compressive stresses when forced to remain in the anti-fouling or the functionalised regions, respectively.     

Kinetic analysis of bacterial clearance in mice using the ESTRIPc and KINET microcomputer programs

Two BASIC microcomputer programs, ESTRIP^c and KINET, were used to analyze the kinetics of bacterial clearance from the blood and mesenteric lymph nodes of mice. Because of the similarities between the clearance of bacteria and the clearance of drugs from tissue, blood pharmacokinetic techniques were applied to the analysis of bacterial clearance data. The ESTRIP^c program, developed for pharmacokinetic analysis and modified for the study of bacterial clearance, was employed to fit the experimental data of bacterial survival versus time to a polyexponential equation with 1, 2, or 3 terms. The KINET program, written specifically for kinetic analysis of bacteria clearance, uses the biexponential equation constants derived with ESTRIP^c to calculate half-life values, rate constants, and other useful kinetic parameters. The combined use of these programs permits precise comparisons of the clearance rates of different bacterial species from the blood or tissues of experimental animals.     

Effect of silver, copper, mercury, and nickel ions on cellular proliferation during extended, low-dose exposures

Previous studies have demonstrated and quantified the cytotoxicity of metal ions in vitro, but the data from these reports have been limited to short-term exposures of metal ions to cells (24-72 h). Yet, the longer-term, low-dose effects of metal ions are most relevant to the clinical use of dental and other biomedical alloys. Thus, the purpose of the current study was to assess longer-term effects of ions of silver, copper, mercury, and nickel - four metal ions known to be released from dental alloys - on monocytes. THP-1 human monocytes were exposed to the metal ions for up to 4 weeks. Concentrations of the metal ions were 1-10% of those known to cause cytotoxicity with 24-h exposures. Cellular proliferation and cellular viability were measured weekly. Ag(1+) and Hg(2+) did not alter the percentage of nonviable cells, but Cu(2+) and Ni(2+) increased the nonviable component as a function of metal concentration. These effects were cumulative over the 4 weeks only for Ni(2+). All metal ions caused a significant reduction in cellular proliferation, but the pattern of the effect was unique to each metal ion, and the effects were often not evident until 3 or 4 weeks of exposure. The results of the current study indicate that metal ions released from metallic biomaterials may have adverse biological effects at concentrations lower than previously reported.     

A new scenario for the evolutionary origin of hair, feather, and avian scales

In zoology it is well known that birds are characterized by the presence of feathers, and mammals by hairs. Another common point of view is that avian scales are directly related to reptilian scales. As a skin embryologist, I have been fascinated by the problem of regionalization of skin appendages in amniotes throughout my scientific life. Here I have collected the arguments that result from classical experimental embryology, from the modern molecular biology era, and from the recent discovery of new fossils. These arguments shape my view that avian ectoderm is primarily programmed toward forming feathers, and mammalian ectoderm toward forming hairs. The other ectoderm derivatives – scales in birds, glands in mammals, or cornea in both classes – can become feathers or hairs through metaplastic process, and appear to have a negative regulatory mechanism over this basic program. How this program is altered remains, in most part, to be determined. However, it is clear that the regulation of the Wnt/beta‐catenin pathway is a critical hub. The level of beta‐catenin is crucial for feather and hair formation, as its down‐regulation appears to be linked with the formation of avian scales in chick, and cutaneous glands in mice. Furthermore, its inhibition leads to the formation of nude skin and is required for that of corneal epithelium. Here I propose a new theory, to be further considered and tested when we have new information from genomic studies. With this theory, I suggest that the alpha‐keratinized hairs from living synapsids may have evolved from the hypothetical glandular integument of the first amniotes, which may have presented similarities with common day terrestrial amphibians. Concerning feathers, they may have evolved independently of squamate scales, each originating from the hypothetical roughened beta‐keratinized integument of the first sauropsids. The avian overlapping scales, which cover the feet in some bird species, may have developed later in evolution, being secondarily derived from feathers.     

Frozen sections of cellular lymphoid proliferations provide adequate DNA for routine gene rearrangement analysis

Optimal use of frozen tissue procured as part of a thorough diagnostic workup of suspected lymphoma is important, and conservation of similar samples is a prerequisite for maintaining a large and varied frozen archive repository. The authors have evaluated a simple tissue-conserving method for the preparation of cellular lymphoid specimens for immunoglobulin and T-cell receptor gene rearrangement analysis. Initially, 16-microns-thick frozen tonsil sections were examined to determine adequacy for DNA extraction. Specimens containing three, six, and nine sections each were evaluated separately. DNA quantitation disclosed yields ranging from 84 to 204 micrograms (mean, 156 micrograms). The authors have used this technique on 24 cellular lymphoid proliferations from their frozen archives. Six to ten 16-microns sections were used, depending on tissue size. DNA quantitation ranged from 0 to 520 micrograms (mean, 135 micrograms). Twenty-one of 24 cases yielded adequate DNA for analysis; each showed appropriate germline or rear-ranged bands with respect to the particular morphologic diagnosis. Attempts to obtain adequate DNA with the use of this technique on skin biopsy specimens with lymphoid infiltrates resulted in overall poor yields; this may be because of dermal collagen or small sample size. This method of sample preparation provides adequate DNA for routine Southern blot hybridization analysis of cellular lymphoid tissues and offers the additional advantage of allowing preservation of frozen tissue for future study.     

Quality management of medical physics issues at the German heavy ion therapy project

For the commissioning and operation of the German Heavy Ion Therapy Project a quality assurance program was developed and successfully applied. The complete radiotherapy process using heavy ions was carefully analyzed and divided into three areas related to beam delivery and control, safety-interlock system and medical physics issues. In this paper, the medical physics issues are addressed. Since the irradiation with heavy ions is a nonstandard modality, new concepts and ion specific tests were developed. As far as possible, national and international standard specifications for radiotherapy were adopted. For each aspect, a performance characteristic and a corresponding acceptance test were introduced. In addition, test characteristics for the constancy tests were established. For all tests, intervention thresholds and test frequencies were specified. Using the described protocol of acceptance tests, the commissioning was passed successfully. The heavy ion irradiation facility was approved by the governmental authorities on the basis of these test results. During clinical operation, constancy tests are performed at the beginning of each treatment period, in order to maintain the quality found during the acceptance tests. Up to now, 48 patients have been treated within 6 treatment periods of 4 weeks each. The concepts used and the tests developed for the quality assurance program may serve as an example of how to introduce systematically a quality assurance program for a new treatment modality.     

Regional variability, processing methods, and biophysical properties of human fascia lata extracellular matrix

This study aims to assess the regional variability, processing methods, mechanical, biochemical, and cellular properties of human fascia lata as a scaffold for soft tissue repair and tissue engineering applications. Ten pairs of fascia lata (donor age 18-55) were used. One fascia patch from each pair was used to assess the geometric and biomechanical variability of fresh fascia. The other from each pair was subjected to 1 of 2 allograft processing methods: antibiotic soak alone or acellularization plus antibiotic soak. Stiffness, modulus, hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan (CSDS GAG), and DNA content were quantified in fascia from fresh and treated groups. The effect of location was not significant for thickness or stiffness within a 6 x 12 cm2 region of the iliotibial tract of fresh human fascia lata. Processing did not significantly change the stiffness, modulus, or CSDS GAG content of fascia ECM. However, hydroxyproline (collagen) content is significantly reduced in acellularized fascia, probably reflecting a removal of soluble collagen during the treatment (p < 0.02). Processing reduced the DNA content of fresh fascia approximately 10-fold (p < 0.001). The mechanical, chemical and ultrastructural similarities between fascia lata and tendon may make fresh or processed fascia an attractive ECM scaffold for soft tissue, particularly tendon, repair.     

The quality of maximum likelihood estimates of ion channel rate constants

Properties of maximum likelihood estimators of rate constants for channel mechanisms are investigated, to see what can and cannot be inferred from experimental results. The implementation of the HJCFIT method is described; it maximises the likelihood of an entire sequence of apparent open and shut times, with the rate constants in a specified reaction mechanism as free parameters. The exact method for missed brief events is used. Several methods for testing the quality of the fit are described. The distributions of rate constants, and correlations between them, are investigated by doing sets of 1000 fits to simulated experiments. In a standard nicotinic receptor mechanism, all nine free rate constants can be estimated even from one single channel recording, as long as the two binding sites are independent, even when the number of channels in the patch is not known. The estimates of rate constants that apply to diliganded channels are robust; good estimates can be obtained even with erroneous assumptions (e.g. about the value of a fixed rate constant or the independence of sites). Rate constants that require distinction between the two sites are less robust, and require that an EC₅₀ be specified, or that records at two concentrations be fitted simultaneously. Despite the complexity of the problem, it appears that there exist two solutions with very similar likelihoods, as in the simplest case. The hazards that result from this, and from the strong positive correlation between estimates of opening and shutting rates, are discussed.     

Increased cell proliferation and R.Msp1 fragmentation induced by 5-aza-2'-deoxycytidine in rat testes related to the gene imprinting mechanism.

DNA methylation is one of the crucial mechanisms for cellular and tissue differentiation during developmental stages in mammals. 5-aza-2'-deoxycytidine, a specific cytosine DNA Methyltransferase inhibitor, is known to cause DNA hypomethylation in CpG, CpNpG and CCGG sequences. Therefore the present study was designed to determine the effects of 5-aza-2'-deoxycytidine on the germinal cells of the adult rat testicular tissue. Rat testicular tissues from the 5-aza-2'-deoxycytidine treated experimental and non-treated control groups were processed for light microscopy and also for genomic DNA isolation assays. The isolated genomic DNAs were digested with R.Msp1 in order to determine the methyl pattern differences in the enzyme cognate CCGG sequence. Testicular tissues from treated rats showed increased cell proliferation when investigated at the light microscopical level. On the other hand, genomic DNA of these proliferative tissue showed high fragmentation sizes of R.Msp1 digestion when compared to controls. While the R.Msp1 digested control group DNA fragmentation condensed at approximately 4700-5100 bps size, the experimental group DNA fragmentation was condensed at 700-900 bps size. In addition, 5-aza-2'-deoxycytidine has effects on increased cell proliferation via the loss of somatic de novo gene imprinting. These results imply that abnormally imprinted normal somatic cells in mammals are susceptible to epigenetic modification. These results also suggest that the genomic DNA of testicular tissues from control rats is resistant to R.Msp1 while DNA from the experimental group testicular cells demonstrating high proliferation rate could not resist to R.Msp1 digestion due to DNA hypomethylation in CCGG sequence. In conclusion, it could be suggested that the reversal of gene imprinting in germinal cells may cause an increased cellular proliferation and R.Msp1 fragmentation when induced by 5-aza-2'-deoxycytidine.     

Aluminum,calcium ion and radiofrequency synergism in acceleration of lymphomagenesis

This study that was done on lymphomagene-bearing mice indicates a synergism aluminum-radiofrequency which induces an early increase in mortality that is in concomitance with lymphoid elements proliferation and infiltration of spleen and liver. These two last phenomena were assesed by determination of the hypertrophic index (Growth Index) which is the organ weight to to the body weight ratio, as well as by the histopathological examination of the organ tissue. The importance of this synergism appears to be determined by the ionization at the physiological pH of the used aluminum complexes: much higher with lactate complex than with the citrate one. On the other hand, this dissociation appears to induce a remarkable acceleration of the mortality and the lymphoid elements-related hypertrophy of the spleen and liver at early age. Aluminum complexes are known as modifiers of the intracellular calcium homeostasis, and to verify if such process could be implicated in this synergism, the effects of calcium chloride were assayed, in this case the calcium-overload had no effects in the presence of a workable cellular control of intracellular calcium homeostasis. This finding support the hypothesis that ionized aluminum provided by lactate may be implicated in the inhibition of the buffering and extruding extracellular calcium system.     

Purinergic signalling in epithelial ion transport: regulation of secretion and absorption

Intracellular ATP, the energy source for many reactions, is crucial for the activity of plasma membrane pumps and, thus, for the maintenance of transmembrane ion gradients. Nevertheless, ATP and other nucleotides/nucleosides are also extracellular molecules that regulate diverse cellular functions, including ion transport. In this review, I will first introduce the main components of the extracellular ATP signalling, which have become known as the purinergic signalling system. With more than 50 components or processes, just at cell membranes, it ranks as one of the most versatile signalling systems. This multitude of system components may enable differentiated regulation of diverse epithelial functions. As epithelia probably face the widest variety of potential ATP-releasing stimuli, a special attention will be given to stimuli and mechanisms of ATP release with a focus on exocytosis. Subsequently, I will consider membrane transport of major ions (Cl(-) , HCO(3)(-) , K(+) and Na(+) ) and integrate possible regulatory functions of P2Y2, P2Y4, P2Y6, P2Y11, P2X4, P2X7 and adenosine receptors in some selected epithelia at the cellular level. Some purinergic receptors have noteworthy roles. For example, many studies to date indicate that the P2Y2 receptor is one common denominator in regulating ion channels on both the luminal and basolateral membranes of both secretory and absorptive epithelia. In exocrine glands though, P2X4 and P2X7 receptors act as cation channels and, possibly, as co-regulators of secretion. On an organ level, both receptor types can exert physiological functions and together with other partners in the purinergic signalling, integrated models for epithelial secretion and absorption are emerging.     

Acid-sensing ion channel (ASIC) 4 gene: physical mapping, genomic organisation, and evaluation as a candidate for paroxysmal dystonia

Acid-sensing ion channels (ASICs) are protongated Na(+) channels. They have been implicated with synaptic transmission, pain perception as well as mechanoperception. ASIC4 is the most recent member of this gene family. It shows expression throughout the central nervous system with strongest expression in pituitary gland. ASIC4 is inactive by itself and its function is unknown. Mutations in ion channel subunits, which are homologues of ASICs lead to neurodegeneration in Caenorhabditis elegans. It has, therefore, been speculated that similar mutations in ASICs may be responsible for neurodegeneration in humans. Here, we show that ASIC4 maps to the long arm of chromosome 2 in close proximity to the locus for paroxysmal dystonic choreoathetosis (PDC), a movement disorder with unknown cause. Ion channel genes have been shown to cause several other paroxysmal neurologic disorders and are important candidate genes for PDC. We established the genomic organisation of the ASIC4 gene and screened a PDC pedigree for mutations in the coding region. Although we identified three polymorphisms in the Cterminal part of the ASIC4 protein, these were not present in each affected subject in the PDC kindred we analysed. Therefore, although the ASIC4 gene is physically mapped to the PDC locus, our data indicates that ASIC4 gene mutation is not the cause of PDC. It remains to be established if mutations in ASIC4 or other ASIC subunits may cause neurological disorders.