Adhesion forces measured at the level of a terminal plate of the fly's seta.

The attachment pads of fly legs are covered with setae, each ending in small terminal plates coated with secretory fluid. A cluster of these terminal plates contacting a substrate surface generates strong attractive forces that hold the insect on smooth surfaces. Previous research assumed that cohesive forces and molecular adhesion were involved in the fly attachment mechanism.

Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imaging.

Resonance energy transfer (RET) has been extensively used to estimate the distance between two different fluorophores. This study demonstrates how protein-protein interactions can be visualized and quantified in living cells by time-correlated single-photon counting (TCSPC) imaging techniques that exploit the RET between appropriate fluorescent labels. We used this method to investigate the association of the potassium inward rectifier channel Kir2.

[Atomic force microscope (AFM). A nanomanipulator for biophysical studies of stereocilia of the cochlear hair cells].

Objectives: Studies of the mechanoelectrical sensor system of the hair cell bundle in the cochlea require a manipulation device that enables controlled force application and movement of individual stereocilia in the nanometer range.

Methods: In our atomic force microscope (AFM) setup, the scan is directly controlled in an upright differential interference contrast (DIC) infrared video microscope with a water immersion objective and in the measured AFM image. Here we present studies on hair cells of the mammalian cochlea.

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).

Molecular and functional characterization of acid-sensing ion channel (ASIC) 1b.

Acid-sensing ion channels (ASICs) are activated by extracellular protons and are involved in neurotransmission in the central nervous system, in pain perception, as well as in mechanotransduction. Six different ASIC subunits have been cloned to date, which are encoded by four genes (ASIC1-ASIC4). Proton-gated currents have been described in isolated neurons from sensory ganglia as well as from central nervous system.

K(+)-dependent gating of K(ir)1.1 channels is linked to pH gating through a conformational change in the pore.

1. We have used giant patch-clamp recording to investigate the interaction between pH gating and K(+)-dependent gating in rat K(ir)1.1 (ROMK) channels heterologously expressed in Xenopus oocytes.

Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein.

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate.

Memantine inhibits efferent cholinergic transmission in the cochlea by blocking nicotinic acetylcholine receptors of outer hair cells.

Memantine is a blocker of Ca(2+)-permeable glutamate and nicotinic acetylcholine receptors (nAChR). We investigated the action of memantine on cholinergic synaptic transmission at cochlear outer hair cells (OHCs). At this inhibitory synapse, hyperpolarization of the postsynaptic cell results from opening of SK-type Ca(2+)-activated K(+) channels via a highly Ca(2+)-permeable nAChR containing the alpha 9 subunit.

Lateral mechanical coupling of stereocilia in cochlear hair bundles.

For understanding the gating process of transduction channels in the inner ear it is essential to characterize and examine the functional properties of the ultrastructure of stereociliary bundles. There is strong evidence that transduction channels in hair cells are gated by directly pulling at the so-called tip links. In addition to these tip links a second class of filamentous structures was identified in the scanning and transmission electron microscope: the side-to-side links.

Phospholipids as modulators of K(ATP) channels: distinct mechanisms for control of sensitivity to sulphonylureas, K(+) channel openers, and ATP.

Recent work has established membrane phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP(2)) as potent regulators of K(ATP) channels controlling open probability and ATP sensitivity. We here investigated the effects of phospholipids on the pharmacological properties of cardiac type K(ATP) (Kir6.2/SUR2A) channels.

A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K(+) channels.

Integral membrane proteins are sorted via the secretory pathway. It was proposed that this pathway is non-selective provided that the cargo protein is properly assembled and lacks an endoplasmic reticulum (ER) retention signal. However, recent experimental evidence suggests that efficient export of proteins from the ER to the Golgi complex is not simply a default pathway.

Developmental expression of the small-conductance Ca(2+)-activated potassium channel SK2 in the rat retina.

Small-conductance Ca(2+)-activated potassium (SK) channels are present in most central neurons, where they mediate the afterhyperpolarizations (AHPs) following action potentials. SK channels integrate changes in intracellular Ca(2+) concentration with membrane potential and thus play an important role in controlling firing pattern and excitability. Here, we characterize the expression pattern of the apamin-sensitive SK subunits, SK2 and SK3, in the developing and adult rat retina using in situ hybridization and immunohistochemistry.

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).

Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat.

Endolymphatic ion composition in the adult inner ear is characterized by high K(+) and low Na(+) concentration. This unique ion composition is essential for proper functioning of sensory processing. Although a lot has been learned in recent years about molecules involved in K(+) transport in inner ear, the molecules involved in Na(+) transport are only beginning to emerge.

Intracellular regulation of inward rectifier K+ channels.

Inward rectifier potassium (Kir) channels comprise a relatively young gene family of ion channels whose first member was isolated in 1993. A common property its members share is a strong dependence on intracellular regulators such as polyamines, nucleotides, phospholipids, kinases, pH and guanosine-triphosphate-binding proteins (G-proteins). The physiological role of Kir channels is to modulate the excitability and secretion of potassium (K+) to maintain K+ homeostasis, under the control of various intracellular second messengers.

Expression of AMPA receptor subunit flip/flop splice variants in the rat auditory brainstem and inferior colliculus.

The expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit mRNAs and their flip/flop splice variants was evaluated in the rat auditory brainstem and inferior colliculus employing in situ hybridization with radiolabeled oligonucleotide probes. A differential expression of AMPA receptor subunits in auditory nuclei was observed. In general, neurons in all nuclei of the auditory brainstem express high levels of GluR-C flop and GluR-D flop mRNA, but low to very low levels of GluR-A and GluR-B mRNA.

Regulation of GIRK channel deactivation by Galpha(q) and Galpha(i/o) pathways.

G protein regulated inward rectifying potassium channels (GIRKs) are activated by G protein coupled receptors (GPCRs) via the G protein betagamma subunits. However, little is known about the effects of different GPCRs on the deactivation kinetics of transmitter-mediated GIRK currents. In the present study we investigated the influence of different GPCRs in the presence and absence of RGS proteins on the deactivation kinetics of GIRK channels by coexpressing the recombinant protein subunits in Xenopus oocytes.

Gating of Ca2+-activated K+ channels controls fast inhibitory synaptic transmission at auditory outer hair cells.

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively.

A new member of acid-sensing ion channels from pituitary gland.

Acid-sensing ion channels (ASICs) constitute a branch of the super-gene family of amiloride-sensitive sodium channels. So far five different ASICs have been cloned from mammalian tissues. They are activated by a drop of extracellular pH but differ with respect to effective agonist concentration, desensitization and mRNA expression pattern.

Mechanical stimulation of individual stereocilia of living cochlear hair cells by atomic force microscopy.

This paper describes the investigation of elastical properties and imaging of living cochlear hair bundles of inner (IHC) and outer hair cells (OHC) on the level of individual stereocilia. A custom-made AFM-setup was used, allowing to scan the mechano-sensitive structures of the inner ear under direct control of an upright differential interference contrast (DIC) microscope with a water-immersion objective. Scanning electron microscopy (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that forces up to 1.

pH gating of ROMK (K(ir)1.1) channels: control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome.

Inward-rectifier K(+) channels of the ROMK (K(ir)1.1) subtype are responsible for K(+) secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range.

NMR structure and functional characteristics of the hydrophilic N terminus of the potassium channel beta-subunit Kvbeta1.1.

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming alpha-subunit or certain auxiliary beta-subunits. Upon coexpression, Kvbeta1.

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.

Expression pattern of aquaporin water channels in the inner ear of the rat. The molecular basis for a water regulation system in the endolymphatic sac.

Mammalian aquaporins constitute a family of so far 10 related water channel proteins which mediate osmotically driven water fluxes across the plasma membrane. Because regulation of the ionic composition and osmolality of inner ear fluids is of great functional significance, we investigated the expression patterns of aquaporins in five defined areas of the rat inner ear by RT-PCR. The tissues used were stria vascularis, endolymphatic sac, Reissner's membrane, vestibulum and organ of Corti.

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.