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. The main elements that contribute to the overall attachment force, however, remained unknown. Multiple local force-volume measurements were performed on individual terminal plates by using atomic force microscopy. It was shown that the geometry of a single terminal plate had a higher border and considerably lower centre. Local adhesion was approximately twice as strong in the centre of the plate as on its border. Adhesion of fly footprints on a glass surface, recorded within 20 min after preparation, was similar to adhesion in the centre of a single attachment pad. Adhesion strongly decreased with decreasing volume of footprint fluid, indicating that the layer of pad secretion covering the terminal plates is crucial for the generation of a strong attractive force. Our data provide the first direct evidence that, in addition to Van der Waals and Coulomb forces, attractive capillary forces, mediated by pad secretion, are a critical factor in the fly's 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 Kir2.1 and the neuronal PDZ protein PSD-95, which has been implicated in subcellular targeting and clustering of ion channels. Our data show that the two proteins not only colocalize within clusters but also interact with each other. Moreover, the data allow a spatially resolved quantification of this protein-protein interaction with respect to the relative number and the proximity between interacting molecules. Depending on the subcellular localization, a fraction of 20 to 60% of PSD-95 molecules interacted with Kir2.1 channels, approximating their fluorescent labels by less than 5 nm.

[Atomic force microscope (AFM). A nanomanipulator for biophysical studies of stereocilia of the cochlear hair cells]. / Das Rasterkraftmikroskop (AFM). Ein Nanomanipulator für biophysikalische Untersuchungen an Stereozilien der Sinneszellen der Kochlea.

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. RESULTS AND CONCLUSIONS: The resulting images revealed the tips of individual stereocilia of living sensory cells of the organ of Corti and the typical shape of the ciliary bundle. Scanning electron-microscopic (SEM) images of the identical hair bundles obtained after AFM investigation demonstrated that up to four AFM manipulations on the same cell did not cause obvious damage to the surface morphology of the stereocilia.

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.

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. 2. Gating by intracellular protons (pH gating) and extracellular K(+) ions (K(+)-dependent gating) is a hallmark of K(ir)1.1 channels that mediate K(+) secretion and control NaCl reabsorption in the kidney. pH gating is driven by protonation of an intracellular lysine residue (K80 in K(ir)1.1). K(+)-dependent gating occurs upon withdrawal of K(+) ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterically. 3. K(+)-dependent gating was shown to be strictly coupled to pH gating; it only occurred when channels were in the pH-inactivated closed state, but not in the open state. Moreover, K(+)-dependent gating was absent in the non-pH-gated mutant K(ir)1.1(K80 M). 4. Channels inactivated by K(+)-dependent gating were reactivated upon addition of permeant ions to the extracellular side of the membrane, while impermeant ions failed to induce channel reactivation. Moreover, mutagenesis identified two residues in the P-helix (L136 and V140 in K(ir)1.1) that are crucial for K(+)-dependent gating. Replacement of these residues with the ones present in the non-K(+)-gated K(ir)2.1 abolished K(+)-dependent gating of K(ir)1.1 channels without affecting pH gating. 5. The results indicate that pH gating and K(+)-dependent gating are coupled to each other via structural rearrangements in the inner pore involving the P-helix.

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. However, it is largely unclear which of the cloned ASIC subunits underlie these native proton-gated currents. Recently, a splice variant, ASIC-beta, has been described for ASIC1a. In this variant about one-third of the protein is exchanged at the N terminus. Here we show that ASIC-beta has a longer N terminus than previously reported, extending the sequence divergence between ASIC1a and this new variant (ASIC1b). We investigated in detail kinetic and selectivity properties of ASIC1b in comparison to ASIC1a. Kinetics is similar for ASIC1b and ASIC1a. Ca(2+) permeability of ASIC1a is low, whereas ASIC1b is impermeable to Ca(2+). Currents through ASIC1a resemble currents, which have been described in sensory and central neurons, whereas the significance of ASIC1b remains to be established. Moreover, we show that a pre-transmembrane 1 domain controls the permeability to divalent cations in ASIC1, contributing to our understanding of the pore structure of these channels.

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. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

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. We show that inhibitory postsynaptic currents recorded from OHCs were reversibly blocked by memantine with an IC(50) value of 16 microM. RT-PCR revealed that a newly cloned nAChR subunit, alpha 10, is expressed in OHCs. In contrast to homomeric expression, coexpression of alpha 9 and alpha 10 subunits in Xenopus laevis oocytes resulted in robust acetylcholine-induced currents, indicating that the OHC nAChR may be an alpha 9/alpha 10 heteromer. Accordingly, nAChR currents evoked by application of the ligand to OHCs and currents through alpha 9/alpha 10 were blocked by memantine with a similar IC(50) value of about 1 microM. Memantine block of alpha 9/alpha 10 was moderately voltage dependent. The lower efficacy of memantine for inhibition of inhibitory postsynaptic currents (IPSCs) most probably results from a blocking rate that is slow with respect to the short open time of the receptor channels during an IPSC. Thus, synaptic transmission in OHCs is inhibited by memantine block of Ca(2+) influx through nAChRs. Importantly, prolonged receptor activation and consequently massive Ca(2+) influx, as might occur under pathological conditions, is blocked at low micromolar concentrations, whereas the fast IPSCs initiated by short receptor activation are only blocked at concentrations above 10 microM.

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. These links laterally connect stereocilia of the same row of a hair bundle. This study concentrates on mechanical coupling of stereocilia of the tallest row connected by side-to-side links. Atomic Force microscopy (AFM) was used to investigate hair bundles of outer hair cells (OHCs) from postnatal rats (day 4). Although hair bundles of postnatal rats are still immature at day 4 and interconnecting cross-links do not show preferential direction yet, hair bundles of investigated OHCs already showed the characteristic V-shape of mature hair cells. In a first experiment, the stiffness of stereocilia was investigated scanning individual stereocilia with an AFM tip. The spring constant for the excitatory direction was 2.5 +/- 0.6 x 10(-3) N/m whereas a higher spring constant (3.1 +/- 1.5 x 10(-3) N/m) was observed in the inhibitory direction. In a second set of experiments, the force transmission between stereocilia of the tallest row was measured using AFM in combination with a thin glass fiber. This fiber locally displaced a stereocilium while the force laterally transmitted to the neighboring untouched taller stereocilia was measured by AFM. The results show a weak force interaction between tallest stereocilia of postnatal rats. The force exerted to an individual stereocilium declines to 36% at the nearest adjacent stereocilium of the same row not touched with the fiber. It is suggested that the amount of force transmitted from a taller stereocilium to an adjacent one of the same row depends on the orientation of links. Maximum force transmission is expected to appear along the axis of interconnecting side links. In our studies it is suggested that transmitted forces are small because connecting side links are oriented very close to an angle of 90 degrees with respect of the scan direction (excitatory-inhibitory direction).

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. In excised membrane patches K(ATP) channels showed considerable variability in sensitivity to glibenclamide and ATP. Application of the phosphatidylinositol phosphates (PIPs) phosphatidylinositiol-4-phosphate, PIP(2), and phosphatidylinositol-3,4,5-trisphosphate reduced sensitivity to ATP and glibenclamide closely resembling the native variability. Insertion of the patch back into the oocyte (patch-cramming) restored high ATP and glibenclamide sensitivity, indicating reversible modulation of K(ATP) channels via endogenous PIPs-degrading enzymes. Thus, the observed variability seemed to result from differences in the membrane phospholipid content. PIP(2) also diminished activation of K(ATP) channels by the K(+) channel openers (KCOs) cromakalim and P1075. The properties mediated by the sulphonylurea receptor (sensitivity to sulfonylureas and KCOs) seemed to be modulated by PIPs via a different mechanism than ATP inhibition mediated by the Kir6.2 subunits. First, polycations abolished the effect of PIP(2) on ATP inhibition consistent with an electrostatic mechanism but only weakly affected glibenclamide inhibition and activation by KCOs. Second, PIP(2) had clearly distinct effects on the concentration-response curves for ATP and glibenclamide. However, PIPs seemed to mediate the different effects via the Kir6.2 subunits because a mutation in Kir6.2 (R176A) attenuated simultaneously the effects of PIP(2) on ATP and glibenclamide inhibition. Finally, experiments with various lipids revealed structural features necessary to modulate K(ATP) channel properties and an artificial lipid (dioleoylglycerol-succinyl-nitriloacetic acid) that mimicked the effects of PIPs on K(ATP) 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. Here we demonstrate a novel sequence motif (FxYENEV) in the cytoplasmic C-terminus of mammalian inward rectifier potassium (Kir) channels which determines ER export. This motif is found to be both necessary and sufficient for efficient export from the ER that eventually leads to efficient surface expression of Kir2.1 channels.

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.

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. The SK2 subunit showed a distinct and developmentally regulated pattern of expression. It appeared during the first postnatal week and located to retinal ganglion cells and to subpopulations of neurons in the inner nuclear layer. These neurons were identified as horizontal cells and dopaminergic amacrine cells by specific markers. In contrast to SK2, the SK3 subunit was detected neither in the developing nor in the adult retina. These results show cell-specific expression of the SK2 subunit in the retina and suggest that this channel underlies the apamin-sensitive AHP currents described in retinal ganglion cells.

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. The epithelial Na(+) channel (ENaC) is a highly selective Na(+) channel that is expressed in many Na(+)-reabsorbing tissues. The aim of our study was to investigate whether ENaC is expressed in inner ear of rats and could account for Na(+) reabsorption from endolymph. We detected mRNA for the three channel-forming subunits (alpha, beta and gamma ENaC) in cochlea, vestibular system and endolymphatic sac. mRNA abundance increased during the first 12 days of life in cochlea and vestibular system, coinciding with decreasing Na(+) concentration in endolymph. Expression was strongest in epithelial cells lining scala media, most notably Claudius' cells. As these cells are characterized by a very negative resting potential they would be ideally suited for reabsorption of Na(+). mRNA abundance in endolymphatic sac decreased during the first 6 days of life, suggesting that ENaC might be implicated in reabsorption of endolymph in the endolymphatic sac of neonatal animals. Together, our results suggest that the epithelial Na+ channel is a good candidate for a molecule involved in Na(+) homeostasis in inner ear.

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. The strongest GluR-C and -D flop expression is found in the ventral and medial part of the anteroventral cochlear nucleus, the posteroventral cochlear nucleus, and the medial and the lateral superior olive. These nuclei are part of the binaural auditory pathway which is important for sound localization in space. In contrast, neurons in the central nucleus of the inferior colliculus express high levels of GluR-B flip but only low levels of the other AMPA receptor subunits. From our data, we conclude that neurons of nuclei involved in binaural processing exhibit a specific "auditory AMPA receptor" which consists primarily of GluR-C flop and -D flop and often lacks GluR-B subunits; this indicates fast kinetics and high Ca(2+) permeability of AMPA receptor currents. In contrast, neurons in the central nucleus of the inferior colliculus contain large amounts of GluR-B flip subunits resulting in Ca(2+) impermeable AMPA receptors with slow kinetics.

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. The stimulation of both G(i/o)- and G(q)-coupled pathways accelerated GIRK deactivation. GIRK currents deactivated faster upon stimulation of G(i/o)- and G(q)-coupled pathways by P(2)Y(2) receptors (P(2)Y(2)Rs) than upon activation of the G(i/o)-coupled pathway alone via muscarinic acetylcholine receptor M2 (M(2) mAChRs). This acceleration was found to be dependent on phospholipase C (PLC) and protein kinase C (PKC) activities and intracellular calcium. With the assumption that RGS2 has a higher affinity for Galpha(q) than Galpha(i/o), we demonstrated that the deactivation kinetics of GIRK channels can be differentially regulated by the relative amount of RGS proteins. These data indicate that transmitter-mediated deactivation of GIRK currents is modulated by crosstalk between G(i/o)- and G(q)-coupled pathways.

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. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.

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. Here we report cloning of ASIC4, a new protein showing about 45% identity to other ASICs. ASIC4 is 97% identical between rat and human and shows strongest expression in pituitary gland. Moreover, we detected expression throughout the brain, in spinal cord, and inner ear. ASIC4 cannot be activated by a drop of extracellular pH in Xenopus oocytes, suggesting association with other subunits or activation by a ligand different from protons. Our results suggest a role for ASICs also in endocrine glands.

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 1.5 nanonewton (nN) did not cause obvious damage of the surface morphology of the stereocilia. These are the first images of hair bundles of living sensory cells of the organ of Corti by AFM. They display the tips of individual stereocilia and the typical V-shape of ciliary bundles. Since line scans clearly show that slope and force interaction depend on the elastical properties of stereocilia, quantitative stiffness measurements and stimulation of single transduction channels are suggested.

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. Structurally, Kir channels are assembled from four alpha-subunits each carrying the prototypic K+-channel pore region lined by two transmembrane segments with intracellular N- and C-termini. The exact molecular mechanism of Kir channel gating by intracellular second messengers is of considerable biophysical interest. Recent studies have gained significant insight into the molecular mechanism of intracellular regulation by pH. This review illustrates the various modes of regulation of this class of ion channel and the present knowledge of the underlying molecular mechanisms.