Expression of Ca(2+)-activated K(+) channel subunits and splice variants in the rat cochlea.

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 alpha 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 beta 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.

Inflammatory mediators potentiate ATP-gated channels through the P2X(3) subunit.

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 (<100 ms) and slow recovery (>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.

Gene expression of P2X-receptors in the developing inner ear of the rat.

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.

[Hereditary deafness in Turkey. Initial results]. / Hereditäre Schwerhörigkeit in der Türkei. Erste Ergebnisse.

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%.

Expression of the P2X7-receptor subunit in neurons of the rat retina.

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.

Gene expression of the P2X receptors in the rat retina.

Molecular-biological methods were used to demonstrate the expression of six P2X receptor subunits (P2X1-P2X6) 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 P2X2 and P2X4 mRNA in the retina and choroid; P2X3, and P2X5 were detected only in the retina. There was no evidence for P2X1 and P2X6 mRNA in the ocular tissue under investigation. Our data suggest that extracellular ATP may have influences on visual processing.

Desensitization of the P2X(2) receptor controlled by alternative splicing.

P2X receptors are ion channels gated by extracellular ATP. We report here cloning of a P2X(2) receptor splice variant (P2X(2-2)) carrying a 207 bp deletion in the intracellular C-terminus and the analysis of the corresponding genomic structure of the P2X(2) gene. P2X(2-2) is as highly expressed as the original P2X(2) sequence in various tissues. ATP-activated currents mediated by heterologous expressed P2X(2) or P2X(2-2) 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 P2X(2) isoforms.

Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH.

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.

Extracellular K+ and intracellular pH allosterically regulate renal Kir1.1 channels.

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.

Evolution of the creative kinases. The chicken acidic type mitochondrial creatine kinase gene as the first nonmammalian gene.

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.

Cell-specific expression of the alpha 9 n-ACh receptor subunit in auditory hair cells revealed by single-cell RT-PCR.

Single-cell reverse transcription polymerase chain reaction was carried out in three different cell types from the organ of Corti of the four-day old rat. For this purpose, pieces of the organ of Corti were mounted under a differential-interference contrast video microscope. Two different mounting configurations were used to allow imaging of cells from two almost orthogonal angles. This method afforded unequivocal recognition of various cell types in the vital tissue, and extraction of nucleus and cytoplasm of specified individual cells with a patch pipette. Messenger RNA encoding the alpha 9 acetylcholine (ACh) receptor subunit was detected and sequenced from individual outer hair cells and inner hair cells, but was not found in Deiters' cells. The identical Deiters' cells were positive for a P2x receptor subunit. This indicates cell-specific expression of the alpha 9 subunit in inner hair cells and outer hair cells and supports the hypothesis that this subunit contributes to calcium (Ca2+) permeable ionotropic ACh receptors (ACh-R). ACh-dependent Ca2+ concentration increase has been observed in both outer hair cells and inner hair cells.

Subunit-specific inhibition of inward-rectifier K+ channels by quinidine.

Distinct inward-rectifier K+ channel subunits were expressed in Xenopus oocytes and tested for their sensitivity to the channel blocker quinidine. The 'strong' inward-rectifier K+ channel IRK1 was inhibited by quinidine with an EC50 of 0.7 mM, while the 'weak' rectifier channel ROMK1 was only moderately inhibited. ROMK1(N171D)-IRK1C-term chimeric channels, which carry both sites for strong rectification of IRK1 channels (the negatively charged D171 in the second transmembrane domain and the IRK1-C-terminus including E224), displayed strong rectification like IRK1, but showed weak sensitivity to quinidine-like ROMK1, suggesting independence of quinidine binding and rectification mechanisms. Moreover, BIR10 and BIR11, two strong rectifier subunits originally cloned from rat brain, exerted subunit-specific sensitivity to quinidine, being much higher for BIR11. Quinidine blockade of IRK1 was not voltage-dependent, but strongly dependent on the pH in the superfusate. These results strongly suggest a subunit-specific interaction of inward-rectifier K+ channels with neutral quinidine within membrane lipid bilayers.

Subunit-dependent assembly of inward-rectifier K+ channels.

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.

Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine.

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.

A structural determinant of differential sensitivity of cloned inward rectifier K+ channels to intracellular spermine.

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.

Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis.

Second messenger regulation of IRK1 (Kir2.1) inward rectifier K+ channels was investigated in giant inside-out patches from Xenopus oocytes. Kir2.1-mediated currents that run down completely within minutes upon excision of the patches could be partly restored by application of Mg-ATP together with > 10 microM free Mg2+ to the cytoplasmic side of the patch. As restoration could not be induced by the ATP analogs AMP-PNP or ATP gamma S, 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 Mg2+. This indicates that functional activity of Kir2.1 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 Kir2.1 channels. Regulation of Kir2.1 channels described here may be an important mechanism for regulation of excitability.

Conservative evolution of the Mbc-DP region in anthropoid primates.

To determine the organization of the DP region in the Mbc of anthropoid primates, we constructed contig maps from cosmid clones of the chimpanzee and orangutan, representatives of the infraorder Catarrhini, as well as of the cotton-top tamarin, a representative of the infraorder Platyrrhini. We found the maps to be remarkably similar to each other and to the previously published map of the human DP region. In each of the four species, the DP region consists of four loci arranged in the same order (DPB2 . . . DPA2 . . . DPB1 . . . DPA1) and in the same transcriptional orientation (tail-to-tail). The regions in the four species are of approximately the same length and many of the restriction sites are shared between species. The inserts of most Alu elements, of a ribosomal protein pseudogene, and of an IgC epsilon-like pseudogene are found in corresponding positions in all four species. The data indicate that the human-type organization of the DP region was established before the divergence of the Catarrhini and Platyrrhini lines more than 37 million years ago and that it has remained principally intact since that time. This conservation of the DP region is in striking contrast to the evolutionary instability of certain other Mbc regions, in particular those occupied by the DRB or C4 and CYP21 loci. We interpret the stability of the DP region as an indication that the region is being phased out functionally.

Differentiation of isolates of Discula umbrinella (Teleomorph: Apiognomonia errabunda) from beech, chestnut, and oak using randomly amplified polymorphic DNA markers.

Genetic variation in 30 isolates of Discula umbrinella derived from beech, chestnut, and oak was assessed using randomly amplified polymorphic DNA (RAPD) and restriction fragment length polymorphic markers. Polymerase chain reaction amplifications with 17 primers produced 134 different DNA fragments. Three RAPD fragments were subsequently used for Southern hybridization. By these techniques up to four different individuals could be detected in the same leaf. The presence of several individuals within a single leaf indicates a finely tuned balance between the endophyte and its host. Cluster analysis of all arbitrary primed amplified DNA fragments showed that the isolates could be placed into four groups corresponding to their host origin. The high percentage of private RAPD variants within groups is consistent with low gene flow.

Trans-species evolution of Mhc-DRB haplotype polymorphism in primates: organization of DRB genes in the chimpanzee.

The DRB region of the human major histocompatibility complex displays length polymorphism: Five major haplotypes differing in the number and type of genes they contain have been identified, each at appreciable frequency. In an attempt to determine whether this haplotype polymorphism, like the allelic polymorphism, predates the divergence of humans from great apes, we have worked out the organization of the DRB region of the chimpanzee Hugo using a combination of chromosome walking, pulsed-field gel electrophoresis, and sequencing. Hugo is a DRB homozygote whose single DRB haplotype is some 440 kilobases (kb) long and contains five genes. At least one and possibly two of these are pseudogenes, while three are presumably active genes. The genes are designated DRB*A0201, DRB2*0101, DRB3*0201, DRB6*0105, and DRB5*0301, and are arranged in this order on the chromosome. The DRB2 and DRB3 genes are separated by approximately 250 kb of sequence that does not seem to contain any additional DRB genes. The DRB*A0201 gene is related to the DRB1 gene of the human DR2 haplotype; the DRB2*0101 and DRB3*0201 genes are related to the DRB2 and DRB3 genes of the human DR3 haplotype, respectively; the DRB6*0105 and DRB5*0301 genes are related to the DRBVI and DRB5 genes of the human DR2 haplotype, respectively. Thus the Hugo haplotype appears to correspond to the entire human DR2 haplotype, into which a region representing a portion of the human DR3 haplotype has been inserted. Since other chimpanzees have their DRB regions organized in different ways, we conclude that, first, the chimpanzee DRB region, like the human DRB region, displays length polymorphism; second, some chimpanzee DRB haplotypes are longer than the longest known human DRB haplotypes; third, in some chimpanzee haplotypes at least, the DRB genes occur in combinations different from those of the human haplotypes; fourth, and most importantly, certain DRB gene combinations have been conserved in the evolution of chimpanzees and humans from their common ancestors. These data thus provide evidence that not only allelic but also haplotype polymorphism can be passed on from one species to another in a given evolutionary lineage.