A molecular determinant for submillisecond desensitization in glutamate receptors.

The decay of excitatory postsynaptic currents in central neurons mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is likely to be shaped either by receptor desensitization or by offset after removal of glutamate from the synaptic cleft. Native AMPA receptors show desensitization time constants of 1 to about 10 milliseconds, but the underlying molecular determinants of these large differences are unknown. Cloned AMPA receptors carrying the "flop" splice variants of glutamate receptor subtype C (GluR-C) and GluR-D are shown to have desensitization time constants of around 1 millisecond, whereas those with the "flip" variants are about four times slower. Cerebellar granule cells switch their expression of GluR-D splice variants from mostly flip forms in early stages to predominantly flop forms in the adult rat brain. These findings suggest that rapid desensitization of AMPA receptors can be regulated by the expression and alternative splicing of GluR-D gene transcripts.

Control by asparagine residues of calcium permeability and magnesium blockade in the NMDA receptor.

The N-methyl-D-aspartate (NMDA) receptor forms a cation-selective channel with a high calcium permeability and sensitivity to channel block by extracellular magnesium. These properties, which are believed to be important for the induction of long-term changes in synaptic strength, are imparted by asparagine residues in a putative channel-forming segment of the protein, transmembrane 2 (TM2). In the NR1 subunit, replacement of this asparagine by a glutamine residue decreases calcium permeability of the channel and slightly reduces magnesium block. The same substitution in NR2 subunits strongly reduces magnesium block and increases the magnesium permeability but barely affects calcium permeability. These asparagines are in a position homologous to the site in the TM2 region (Q/R site) of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that is occupied by either glutamine (Q) or arginine (R) and that controls divalent cation permeability of the AMPA receptor channel. Hence AMPA and NMDA receptor channels contain common structural motifs in their TM2 segments that are responsible for some of their ion selectivity and conductance properties.

PIP2 and PIP as determinants for ATP inhibition of KATP channels.

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of KATP channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4, 5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned KATP channels (Kir6.2 and SUR1). These phospholipids acted on the Kir6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of KATP-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

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.

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.

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.

Argiotoxin detects molecular differences in AMPA receptor channels.

Argiotoxin, a component of the spider venom from Argiope lobata, blocks AMPA receptor channels expressed in homomeric and heteromeric configuration in Xenopus oocytes. Argiotoxin acts as an open channel blocker in a voltage-dependent manner and discriminates between the functionally diverse AMPA receptors. Importantly, a transmembrane region 2 determinant for divalent cation permeability also determines argiotoxin sensitivity. Subunit-specific differences in the time courses of block and recovery demonstrate that heteromeric AMPA receptors can assemble in variable ratios. Thus, argiotoxin can be used as a tool in analyzing the subunit composition of AMPA receptors in native membranes.

Genetic analysis of vertebrate sensory hair cell mechanosensation: the zebrafish circler mutants.

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

Imaging of cell membraneous and cytoskeletal structures with a scanning tunneling microscope.

The first observation of unstained cell membraneous structures by a scanning tunneling microscope is reported. An adhesive preparation method was used for imaging human medulloblastoma cells from the cell line TE 671 and oocytes from the clawed toad Xenopus laevis. The images show filaments, stacks of molecules and hilly structures. The possible identify of the filamentous structures is discussed, although the observed structures cannot yet be fully characterized. The work suggests possible future experiments on various biological structures in their natural environment.

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.

Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain.

A rat brain cDNA (Raw3) related to the Drosophila Shaw K+ channel family has been characterized. Raw3 cRNA leads to the formation of TEA-insensitive, fast inactivating (A-type) K+ channels when injected into Xenopus laevis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A-type K+ channel RCK4, Raw3 channels operate in the positive voltage range.

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.

Subunit-specific block of cloned NMDA receptors by argiotoxin636.

Cloned NMDA receptor channels of the NR1-NR2A, NR1-NR2B and NR1-NR2C type show differences in argiotoxin636 block. Mutations of an asparagine residue located at a homologous position in the TM2 region of all NMDA receptor subunits, which corresponds to the Q/R site of the AMPA receptors, alters the argiotoxin636-induced block. The results suggest that the toxin interacts at this amino acid position with the putative pore forming TM2 region of the NMDA receptor subunits. Sequence differences in the TM2 segment of NR2A and NR2C subunits are not responsible for the subtype-specific sensitivity to argiotoxin636 as revealed by site-directed mutagenesis.

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.

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.

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.

Complexity of the regulation of Kir2.1 K+ channels.

Regulation of native inward-rectifier K+ channels by second-messenger systems is important for the control of membrane potential and excitability in various types of cell. Regulation of cloned IRK1 (Kir2.1) channels was investigated in inside-out patches from Xenopus oocytes. Fast run down of Kir2.1 channels was induced by application of GTP-gamma S to the cytoplasmic side of the patches and reversed by application of PKA plus Mg-ATP. This effect of GTP-gamma S was inhibited by the phosphatase inhibitor microcystin. These results suggest regulation of Kir2.1 channels by a G-protein-controlled phosphatase. A similar effect was observed upon application of G-protein beta gamma subunits. This effect, however, was most likely mediated by contamination of this preparation by the antioxidant DTT, since DTT by itself was also able to induce a fast run-down of Kir2.1 channels. On the other hand, DTT was found to reverse the run-down induced by GTP-gamma S, similar to PKA. This results indicate that DTT mediates two opposite effects on Kir2.1 channels. It is concluded that regulation of Kir2.1 channels is complex and most likely involves other proteins endogenously expressed in Xenopus oocytes, such as ABC-proteins, G-proteins and phosphatases, which are sensitive to oxidation.

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.

Mechanically and ATP-induced currents of mouse outer hair cells are independent and differentially blocked by d-tubocurarine.

Mechano-electrical transducer channels (MET) and ATP-gated ion channels (P2X receptors) of hair cells have several properties in common: they share the same location at the apex of the cell, both channels are non-selective for cations and blocked by aminoglycosides and pyrazinecarboxamides (amiloride-related compounds). In this study, we test the relationship and possible identity of these two channel types. Using whole-cell patch-clamp recordings of outer hair cells (OHCs) of the cultured neonatal mouse cochlea and a fluid jet to stimulate their hair bundles mechanically, we show that d-tubocurarine, a blocker of P2X2 receptors, blocks MET channels with a half-blocking concentration of 2.3 microM. In contrast, the KD for the P2X2 receptors was 90 microM and 84 microM measured in hair cells and Xenopus oocytes, respectively. When hair bundles of OHCs were simultaneously stimulated with saturating mechanical stimuli and superfused by 100-300 microM ATP, transducer currents and ATP-activated currents were elicited simultaneously. Their amplitudes were additive, however. We conclude that MET- and ATP-activated currents are mediated by two distinct channel populations in hair cells.

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.