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The P2X(3) receptor is an ATP-gated ion channel predominantly expressed in nociceptive neurons from the dorsal root ganglion. P2X(3) receptor channels are highly expressed in sensory neurons and probably contribute to the sensation of pain. Kinetics of P2X(3) currents are characterized by rapid desensitization (20 s). Thus, any mechanism modulating rate of desensitization and/or recovery may have profound effect on susceptibility of nociceptive neurons expressing P2X(3) to ATP. Here we show that currents mediated by P2X(3) receptor channels and the heteromeric channel P2X(2/3) composed of P2X(2) and P2X(3) subunits are potentiated by the neuropeptides substance P and bradykinin, which are known to modulate pain perception. The effect is mediated by the respective neuropeptide receptors, can be mimicked by phorbol ester and blocked by inhibitors of protein kinases. Together with data from site-directed mutagenesis our results suggest that inflammatory mediators sensitize nociceptors through phosphorylation of P2X(3) and P2X(2/3) ion channels or associated proteins.

Reverse transcription-polymerase chain reaction (RT-PCR) was used to characterize the expression of P2X receptor subunits (P2X1-P2X7) in different inner ear tissues. The present study revealed the presence of P2X2, P2X3, P2X4 and P2X7-mRNA in rat organ of Corti, vestibular organ and spiral ganglion at different postnatal developmental stages (PD1-PD16), with slight differences in the onset of expression. Expression of P2X1, P2X5 and P2X6-mRNA was not detectable in the inner ear tissues. In addition, single cell RT-PCR experiments with outer hair cells (OHC) revealed the expression of either the P2X2 or the P2X2-2 splice variant or coexpression of both isoforms in individual cells. Our data suggest that extracellular adenosine-5'-triphosphate (ATP) may play an important role in signal transduction in the inner ear.

Despite the considerable evidence of signaling by extracellular nucleotides in other sensory systems, few studies have been undertaken in the eye. Molecular and immunohistochemical methods were used to demonstrate the expression and cellular localization of the P2X7 receptor subunit in the retina and choroid. RT-PCR was used for the detection of P2X7 subunit mRNA in the rat of different postnatal developmental stages (P23-P210) and revealed the presence of P2X7-mRNA in the retina, but not in the choroid. In the adult rat retina, immunolabelling for P2X7 was detected in a number of cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), suggesting different types of amacrine cells and ganglion cells. These results demonstrate for the first time the expression of the P2X7 receptor in the mammalian retina and furthermore in distinct neuronal cell populations. Our data suggest that extracellular ATP may provide both neuromodulatory and trophic influences on visual processing.

The authors describe a study in progress to identify Turkish families with hereditary hearing loss and isolate possible responsible disease genes. Due to extreme genetic heterogeneity and limited audiological differentiation of hereditary hearing loss, it is necessary to identify large or small families from genetic isolates to locate loci responsible for hearing loss on a chromosome. To accomplish this goal, the medical records of 3800 children were examined at the ENT Clinic of Ege University between 1975 and 1994. All were suspected of having various hearing impairments. Additionally, students from two schools for the hearing impaired in Izmir and Eskisehir, Turkey were examined. To date, 16 families with syndromal deafness and 55 families with non-syndromal hereditary hearing loss involving two or more affected individuals have been identified and categorized according to the mode of inheritance. The majority (66%) of the non-syndromal families showed an autosomal recessive pattern, 29% an autosomal dominant inheritance and 5% an X-linked mode of inheritance. In the study presented there has been a predominance of affected males versus females and the consanguinity rate was 22%.

Molecular–biological methods were used to demonstrate the expression of six P2X receptor subunits (P2X 1 –P2X 6 ) in retina and choroid. Despite the considerable evidence for signalling by extracellular nucleotides in other sensory systems, few studies have been undertaken in the eye. RT-PCR for the detection of P2X subunit mRNA in the rat of different postnatal developmental stages (P23–P210) revealed the presence of P2X 2 and P2X 4 mRNA in the retina and choroid; P2X 3 , and P2X 5 were detected only in the retina. There was no evidence for P2X 1 and P2X 6 mRNA in the ocular tissue under investigation. Our data suggest that extracellular ATP may have influences on visual processing.

Inward-rectifier, G-protein-regulated and ATP-dependent K$^{+}$ channels form a novel gene family of related proteins which share two transmembrane segments as a common structural feature. These K$^{+}$ channels are only distantly related to the voltage-gated Shaker-type K$^{+}$ channels comprising six transmembrane segments. Although the quaternary structure of voltage-gated K$^{+}$ channels has been extensively studied in the past, little is known about subunit assembly of inward-rectifier K$^{+}$ channels. Differential sensitivity of inward-rectifier K$^{+}$ channels to voltage-dependent pore block by spermine was used to analyse subunit assembly. It is shown that inward-rectifier K$^{+}$ channel proteins are composed of four subunits whose assembly obeys the rules of a binomial distribution. \`Strong' and \`mild' inward-rectifier K$^{+}$ channel subunits (BIR10 and ROMK1) which are co-expressed in individual auditory hair cells form hetero-tetramers. Distribution of these hetero-tetramers, however, is not binomial. Hetero- and homo-oligomeric channels form with similar probabilities resulting in independent channel populations with distinct functional properties.

Inward rectifier K+ channels mediate the K+ conductance at resting potential in many types of cell. Since these K+ channels do not pass outward currents (inward rectification) when the cell membrane is depolarized beyond a trigger threshold, they play an important role in controlling excitability. Both a highly voltage-dependent block by intracellular Mg2+ and an endogenous gating process are presently assumed to underly inward rectification. It is shown that strong voltage dependence of rectification found under physiological conditions is predominantly due to the effect of intracellular spermine. Physiological concentrations of free spermine mediate strong rectification of IRK1 inward rectifier K+ channels even in the absence of free Mg2+ and in IRK1 mutant channels that have no endogenous rectification.

The recently manifested important role of the Ca 2+ -activated K + channels, especially of the Slo gene-coded channels, for the cochlea function of the chicken raised the question of homolog expression in mammalian inner ear tissue. Molecular biological methods were used to demonstrate the expression of Ca 2+ -activated K + channel subunits and splice variants of the Slo gene in the rat organ of Corti. RT-PCR experiments for the detection of rat Slo α subunit mRNA revealed the presence of several already known splice variants including variants which appeared to be typical for the organ of Corti (+58 aa) and for the brain (+61 aa). To detect the accessory β subunit we used Southern blot hybridization. Our data support the hypothesis that Ca 2+ -activated K + channel subunits (i.e. Slo variants) are also involved in the hearing of mammals in the organ of Corti.

Publisher Summary This chapter provides an overview of the multiple modes of intracellular regulation of inwardly rectifying potassium (Kir) channels and provides some new insights into mechanisms and subunit specificity of the underlying molecular processes. Some types of intracellular regulation of Kir channels are complex: one example is dependence on adenosine triphosphate (ATP). While ATP dependence of Kir (KATP) channels is blocked by ATP without hydrolyzing it, in Kir2.l ATP hydrolysis reverses the spontaneous rundown. A similar process has also been described for Kir6.2 channels where it is independent of the presence of the regulatory sulfonylurea receptor (SUR) subunit. Chimeric channels constructed from Kir2.1 and Kirl.1 are analyzed in the chapter to define domains involved in this regulation. Even more complex is the regulation by oxidation and the effects of antioxidants that have been studied in Kir2.1 channels.

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X2 receptor splice variant (P2X2-2) carrying a 207 bp deletion in the intracellular C-terminus and the analysis of the corresponding genomic structure of the P2X2 gene. P2X2-2 is as highly expressed as the original P2X2 sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X2 or P2X2-2 receptors showed significant differences in desensitization time constants and steady-state currents in the continuous presence of ATP. These results imply functional differences between cells differentially expressing these P2X2 isoforms. © 1997 Federation of European Biochemical Societies.

The channels that control K+ homeostasis by mediating K+ secretion across the apical membrane of renal tubular cells have recently been cloned and designated ROMK1, -2, and -3. Native apical K+ channels are indirectly regulated by the K+ concentration at the basolateral membrane through a cascade of intracellular second messengers. It is shown here that ROMK1 (Kir1.1) channels are also directly regulated by the extracellular (apical) K+ concentration, and that this K+ regulation is coupled to intracellular pH. The K+ regulation and its coupling to pH were assigned to different structural parts of the channel protein. K+ regulation is determined by the core region, which comprises the two hydrophobic segments M1 and M2 and the P region. Decoupling from pH was achieved by exchanging the N terminus of ROMK1 by that of the pH-insensitive channel IRK1 (Kir2.1). These results suggest an allosteric regulation of ROMK1 channels by extracellular K+ and intracellular pH, which may represent a novel link between K+ homeostasis and pH control.

In both mammals and birds, the creatine kinase (CK) family consists of four types of genes: cytosolic brain type (B-CK); cytosolic muscle type (M-CK); mitochondrial ubiquitous, acidic type (Mia-CK); and mitochondrial sarcomeric, basic type (Mib-CK). We report here the cloning of the chicken Mia-CK cDNA and its gene. Amino acid sequences of the mature chicken Mi-CK proteins show about 90% identity to the homologous mammalian isoforms. The leader peptides, however, which are isoenzyme-specifically conserved among the mammalian Mi-CKs, are quite different in the chicken with amino acid identity values compared with the mammalian leader peptides of 38.5-51.3%. The chicken Mia-CK gene spans about 7.6 kilobases and contains 9 exons. The region around exon 1 shows a peculiar base composition, with more than 80% GC, and has the characteristics of a CpG island. The upstream sequences lack TATA or CCAAT boxes and display further properties of housekeeping genes. Several transcription factor binding sites known from mammalian Mi-CK genes are absent from the chicken gene. Although the promoter structure suggests a ubiquitous range of expression, analysis of Mia-CK transcripts in chicken tissues shows a restricted pattern and therefore does not fulfill all criteria of a housekeeping enzyme.

Large subtype-specific differences in the sensitivity of cloned inward-rectifier K+ channels of the IRK1, BIR10 and ROMK1 subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIR10 channels compared to ROMK1 channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIR10, is homologous to the residue in IRK1 and ROMK1 which has previously been shown to change gating properties and Mg2+ sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K+ channels.

Summary Second messenger regulation of IRK1 (K ir 2.1) inward rectifier K + channels was investigated in giant inside-out patches from Xenopus oocytes. K ir 2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with 2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATPS, this suggests an ATPase-like mechanism. In addition to ATP, the catalytic subunit of cAMP-dependent protein kinase (PKA) induced an increase in current amplitude, which could, however, only be observed if channels were previously or subsequently stimulated by Mg-ATP and free Mg 2+ . This indicates that functional activity of K ir 2.1 channels requires both phosphorylation by PKA and ATP hydrolysis. Moreover, currents could be down-regulated by N-heptyl-5-chloro-1-naphthalenesulfonamide, a specific stimulator of protein kinase C (PKC), suggesting that PKA and PKC mediate inverse effects on K ir 2.1 channels. Regulation of K ir 2.1 channels described here may be an important mechanism for regulation of excitability.

Gamma-Aminobuttersaure, kurz GABA genannt, stellt den wichtigsten inhibitorischen Transmitter des Vetrebratengehirns dar. Dieser hemmende Effekt wird durch zwei Klassen von Rezeptoren vermittelt: Eine Gruppe ist mit einem G-Protein-vermittelten Steuerungsprozes verbunden. Fur die cochleare Neurotransmission gilt GABA, neben L-Glutamat und Azetylcholin, als wichtige Effektorsubstanz, wie vielfaltige biochemische und elektrophysiologische Untersuchungen zeigten.

Der sog. Kainatrezeptorkomplex bildet eine Unterform der Glutamatrezeptorfamilie (Tabelle 1, Seeburg et al. 1990), d.h. er ist Glutamat-sensibel, und Kainat (Ka), eine exzitatorische Aminsosaure (EAA), bindet spezifisch. Kainat gilt als neurotoxisch, und es wird vermutet, das diese Toxizitat durch einen ionotropen Rezeptortyp vermittelt wird. Kainat wird mit hoher Affinitat im nanomolaren Bereich am Rezeptor spezifisch gebunden (Werner et al. 1991). Fehlsteuerungen im System der EAA, zu denen neben Kainat auch AMPA und NMDA gerechnet werden, konnen als pathophysiologische Grundlage fur verschiedene neurologische Krankheiten postuliert werden. Dazu zahlen z.B. die Chorea Huntington, M. Alzheimer, Formen der Epilepsie, M. Parkinson, die amyotrophe Lateralsklerose und der Komplex der Demenz (Gasic u. Hollmann 1992). Fur den Menschen ist ein Kainatrezeptorprotein des Gehirns charakterisiert worden, sog. humEAA-1, welches in hoher Homologie zum Kainatrezeptor der Ratte steht (Ka-1, Werner et al. 1991) und eine Kainatbindungscharakteristik zeigt, die zur Annahme berechtigt, das es sich um eine Untereinheit des Kainatrezeptorkomplexes handelt (Kamboj et al. 1994). Homologien in der Aminosauresequenz zu anderen Untereinheiten der Glutamatrezeptorfamilie bestatigen diese Vermutung.

In the last few years research efforts have succeeded in detectury the genetic background of several hereditary hearing disorders by molecular biological methods. The genetic code was been decoded for Neurofibromatosis types 1 and 2, as well as for such X-linked diseases as Alport's syndrome or Norrie's disease. Besides the classic genetic tools as chromosomal analysis, molecular biological techniques and methods have become important clinically for the ENT-specialist. In the present review we show the principles and applications of DNA-and RNA-analysis with hybridization techniques in Southern- and Northern-blot techniques, as well as in-situ hybridization and polymerase chain reaction (PCR). These molecular biological techniques will help improve the detection and analysis of hereditary inner ear disorders, but also be able to study in greater detail tumor carcinogenesis and mutagenesis. The various techniques are explained and the applications are demonstrated.

Isolates of the barley mildew pathogen from the air spora over a large part of Europe and from fields of variety mixtures, were tested for virulence against 12 host resistance alleles. Subsamples were tested for their response to triadimenol fungicide and analyzed for 10 DNA loci using RAPD markers and PCR. There was a large range of haplotypes spread over Europe; irregularity in the distribution was probably due mainly to non-uniform use of the corresponding host resistances and fungicides. A large range of variation was also detectable within individual fields. Positive gametic disequilibria distorted the distribution of virulence alleles among haplotypes and reduced the number of haplotypes detectable in the sample. Analysis of the spread of the newly selected Val3 allele into different European sub-populations indicated that gene flow throughout the population may be rapid for alleles that have a selective advantage.

Isolates of Erysiphe graminis f. sp. hordei derived from airborne conidia and progeny of specific crosses were screened for random amplified polymorphic DNA (RAPD) variation detected with 10-base primers. Using two DNA samples, 58 of 76 primers yielded good amplification products. Twenty-seven primers were screened further on a test set of 16 E. g. hordei isolates collected from throughout Europe. Approximately 119 resolvable bands were reproducibly amplified, 56 bands were variable, and each primer yielded at least one polymorphism. A subset of 10 of these primers detected 30 polymorphisms in the European test set but only 19 variable bands in 48 isolates collected with a stationary spore trap

11B NMR chemical shifts and linewidths have been measured in very dilute aqueous solutions of boric acid and borates. The results can be explained bv taking pH dependent weighted averages over the species B(OH)3 and B(OH)4 −. The11B−10B primary isotope effect on the magnetic shielding is smaller then 3 · 10-8. The H2O - D2O solvent isotope effect on T1 has been established for 11B and 10B in the species mentioned, and from the ratios of T1 the quadrupolar origin of the relaxation mechanism has been inferred.

11B NMR studies have been performed in dilute solutions of boric acid resp. borate and some typical hydroxy-carboxylic acids as a function of pH. Typical chemical shifts have been found for the esters occurring at the different ranges of pH. The origin of the two 11B NMR signals observable in wine could be determined.

In the chicken, a high degree of heterogeneity at the protein level has been reported for the creatine kinase-type B (B-CK). Here we show that the two B-CK isoproteins, Ba- and Bb-CK, are encoded by two mRNAs, which are derived from a single copy gene by a stochastic alternative splicing mechanism. The transcription of the single hnRNA is directed by a complex promoter region containing a stretch of sequences which is highly conserved among all the B-CK genes known to date. This stretch encompasses a putative binding site for the TA-rich DNA-binding protein (Hobson, G. M., Mitchell, M. T., Molloy, G. R., and Pearson, M. L. (1988) Nucleic Acids Res. 16, 8925-8944) which is located in the distal part of the promoter region, while the proximal portion containing the TATA-box used in vivo is not conserved between chicken and mammals. The two isoproteins arising from this gene contain distinct N-terminal portions. According to comparative analysis, Bb-CK is the form which is homologous to the mammalian B-CKs, whereas Ba-CK shows some sequence features unique among all other vertebrate cytosolic creatine kinases characterized so far.

Potassium (K+) homeostasis is controlled by the secretion of K+ ions across the apical membrane of renal collecting duct cells through a low-conductance inwardly rectifying K+ channel. The sensitivity of this channel to intracellular pH is particularly high and assumed to play a key role in K+ homeostasis. Recently, the apical K+ channel has been cloned (ROMK1,2,3 = Kir1.1a, Kir1.1b and Kir1.1c) and the pH dependence of ROMK1 was shown to resemble closely that of the native apical K+ channel. It is reported here that the steep pH dependence of ROMK channels is determined by a single amino acid residue located in the N-terminus close to the first hydrophobic segment M1. Changing lysine (K) at position 80 to methionine (M) removed the sensitivity of ROMK1 channels to intracellular pH. In pH-insensitive IRK1 channels, the reverse mutation (M84K) introduced dependence on intracellular pH similar to that of ROMK1 wild-type. A detailed mutation analysis suggests that a shift in the apparent pKalpha of K80 underlies the pH regulation of ROMK1 channels in the physiological pH range.