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).

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).

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.

[Hereditary deafness in Turkey. Initial results].

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

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.

A unique chicken B-creatine kinase gene gives rise to two B-creatine kinase isoproteins with distinct N termini by alternative splicing.

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.

Renal papillary morphology in adults.

Renal papillary morphology and distribution have been analysed endoscopically in 50 adult autopsy kidneys. Concave papillae of the refluxing type were found with greater frequency in the adult kidneys than had been reported by others in children. A further type of calyx lacking a true papilla, not previously described in children, was occasionally observed.