Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia.

Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system.

Cell cycle-related changes in the conducting properties of r-eag K+ channels.

Release from arrest in G2 phase of the cell cycle causes profound changes in rat ether-à-go-go (r-eag) K+ channels heterologously expressed in Xenopus oocytes. The most evident consequence of the onset of maturation is the appearance of rectification in the r-eag current. The trigger for these changes is located downstream of the activation of mitosis-promoting factor (MPF). We demonstrate here that the rectification is due to a voltage-dependent block by intracellular Na+ ions. Manipulation of the intracellular Na+ concentration indicates that the site of Na+ block is located approximately 45% into the electrical distance of the pore and is only present in oocytes undergoing maturation. Since the currents through excised patches from immature oocytes exhibited a fast rundown, we studied CHO-K1 cells permanently transfected with r-eag. These cells displayed currents with a variable degree of block by Na+ and variable permeability to Cs+. Partial synchronization of the cultures in G0/G1 or M phases of the cell cycle greatly reduced the variability. The combined data obtained from mammalian cells and oocytes strongly suggest that the permeability properties of r-eag K+ channels are modulated during cell cycle-related processes.

Voltage-dependent sodium channels in an invertebrate striated muscle.

Striated skeletal muscles from the planktonic arrowworm Sagitta elegans (phylum Chaetognatha) were voltage-clamped. The muscles displayed classical voltage-dependent sodium channels that (i) showed peak transient currents when the membrane was depolarized 90 millivolts from rest, (ii) opened rapidly with peak currents flowing within 0.4 milliseconds at 4 degrees C, (iii) showed voltage-dependent inactivation with 50 percent inactivation at +25 millivolts from rest, and (iv) were blocked by 500 nanomolar tetrodotoxin.

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain.

Eag1 (K(V)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K(+) channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed.

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.

Effects of hydrostatic pressure on membrane processes. Sodium channels, calcium channels, and exocytosis.

A patch-clamp study under high hydrostatic pressure was performed by transferring cells or membrane patches into a pressure vessel (Heinemann, S. H., W. Stühmer, and F. Conti, 1987, Proceedings of the National Academy of Sciences, 84:3229-3233). Whole-cell Na currents as well as Ca currents were measured at pressures up to 40 MPa (approximately 400 atm; 1 MPa = 9.87 atm) in bovine adrenal chromaffin cells. Ca currents were found to be independent of pressure within experimental resolution. The mean amplitude and the gating kinetics of Na currents were affected by less than 20% at 10 MPa. This lack of a pronounced effect is surprising since the high-pressure nervous syndrome (HPNS), a disorder at high pressures known to result from impaired nervous transmission, manifests itself at pressures as low as 5 MPa. The results show that ion channels involved in transmission cannot be implicated in HPNS. However, when exocytosis was studied at high pressure by monitoring the cell capacitance (Neher, E., and A. Marty, 1982, Proceedings of the National Academy of Sciences, 79:6712-6716), more drastic effects were seen. The degranulation evoked by dialyzing the cell with 1 microM free Ca2+ could be slowed by a factor of 2 by application of 10 MPa. The same effect was observed for the degranulation of rat peritoneal mast cells stimulated with 40 microM of the GTP analogue GTP-gamma-S. According to these results, the process of exocytosis is the most likely site at which hydrostatic pressure can act to produce nervous disorders. Furthermore, we demonstrate that pressure can be a useful tool in the investigation of other cellular responses, since we were able to separate different steps occurring during exocytosis owing to their different activation volumes.

Mobility of voltage-dependent ion channels and lectin receptors in the sarcolemma of frog skeletal muscle.

The mobility of lectin receptors and of two types of ion channels was studied in skeletal muscles of the frog Rana temporaria. Lectin receptors were labeled with fluorescent derivatives of succinyl-concanavalin A (Con A) or wheat germ agglutinin (WGA), and their mobility was measured by fluorescence recovery after photobleaching. Of the receptors for WGA, approximately 53% were free to diffuse in the plane of the membrane, with an average diffusion coefficient as found in other preparations (D = 6.4 X 10(-11) cm2/s). Con A receptors were not measurably mobile. The mobility of voltage-dependent Na and K (delayed rectifier) channels was investigated with the loose-patch clamp method, coupled with through-the-pipette photodestruction of channels by ultraviolet (UV) light. Na channels were not measurably mobile (D less than or equal to 10(-12) cm2/s). With K channels, photodestruction was followed by a small but consistent recovery of K current, which suggested that some K channels diffused in the plane of the membrane. Our results with K currents are best fit if 25% of the K channels diffuse with D = 5 X 10(-11) cm2/s, with the remainder being immobile. For both Na and K channels, photodestruction by UV was most effective at a wavelength of approximately 289 nm. At this wavelength, the energy density required for an e-fold reduction in the number of functional channels was 0.40 J/cm2 for Na channels and 0.94 J/cm2 for K channels. Irradiation at this wavelength and dose did not measurably diminish the mobility of WGA receptors; hence, the immobility of Na and most K channels is not due to UV irradiation. It is concluded that mobile and immobile membrane proteins coexist in the sarcolemma of frog skeletal muscle, and that voltage-dependent Na and K channels are singled out for immobilization.

A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II.

A single point mutation of the rat sodium channel II reduces its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude. The mutation replaces glutamic acid 387 with a glutamine and has only slight effects on the macroscopic current properties, as measured under voltage-clamp in Xenopus oocytes injected with the corresponding cDNA-derived mRNA.

Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product.

Mammalian voltage-activated Shaker K+ channels associate with at least three cytoplasmic proteins: Kv beta 1, Kv beta 2 and Kv beta 3. These beta subunits contain variable N-termini, which can modulate the inactivation of Shaker alpha subunits, but are homologous throughout an aldo-keto reductase core. Human and ferret beta 3 proteins are identical with rat beta 1 throughout the core while beta 2 proteins are not; beta 2 also contains a shorter N-terminus and has no reported physiological role. We report that human beta 1 and beta 3 are derived from the same gene and that beta 2 modulates the inactivation properties of Kv1.4 alpha subunits.

ATP binding site of P2X channel proteins: structural similarities with class II aminoacyl-tRNA synthetases.

The extracellular loop of P2X channel proteins contains a sequence stretch (positions 170-330) that exhibits similarities with the catalytic domains of class II aminoacyl-tRNA synthetases as shown by secondary structure predictions and sequence alignments. The arrangement of several conserved cysteines (positions 110-170) shows similarities with metal binding regions of metallothioneins and zinc finger motifs. Thus, for the extracellular part of P2X channel proteins a metal binding domain and an antiparallel six-stranded beta-pleated sheet containing the ATP binding site are very probable. The putative channel forming H5 part (positions 320-340) shows similarities with the enzyme motif 1 responsible for aggregation of subunits to the holoenzyme.

Molecular cloning and functional expression of a novel rat heart P2X purinoceptor.

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full-length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40-47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK-293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP-activated current reveals little agonist desensitization, is not activated by alpha,beta-meATP and is completely blocked by suramin and PPADS.

Potassium channels expressed from rat brain cDNA have delayed rectifier properties.

Injection into Xenopus oocytes of RNA synthesized in vitro using the rat brain cDNA RCK1 as a template or nuclear injection of the cDNA results in the expression of functional potassium channels. These channels exhibit properties similar to those of the non-inactivating delayed rectifier channel found in mammalian neurons and other excitable cells.

Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II.

The SS2 and adjacent regions of the 4 internal repeats of sodium channel II were subjected to single mutations involving, mainly, charged amino acid residues. These sodium channel mutants, expressed in Xenopus oocytes by microinjection of cDNA-derived mRNAs, were tested for sensitivity to tetrodotoxin and saxitoxin and for single-channel conductance. The results obtained show that mutations involving 2 clusters of predominantly negatively charged residues, located at equivalent positions in the SS2 segment of the 4 repeats, strongly reduce toxin sensitivity, whereas mutations of adjacent residues exert much smaller or no effects. This suggests that the 2 clusters of residues, probably forming ring structures, take part in the extracellular mouth and/or the pore wall of the sodium channel. This view is further supported by our finding that all mutations reducing net negative charge in these amino acid clusters cause a marked decrease in single-channel conductance.

Primary structure and functional expression of a high voltage activated calcium channel from rabbit lung.

The complete amino acid sequence of the receptor for organic calcium channel blockers (CaCB) from rabbit lung has been deduced by cloning and sequence analysis of the cDNA. Synthetic RNA derived from this cDNA induces the formation of a functional CaCB-sensitive high voltage activated calcium channel in Xenopus oocytes.

Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors.

The suramin analogue 8,8'-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023) antagonizes in a competitive fashion P2X receptor-mediated responses in certain vascular and visceral smooth muscles. In the present study, the effect of NF023 on voltage-clamped Xenopus oocytes heterologously expressing homomultimeric P2X1-P2X4 as well as heteromultimeric P2X2/P2X3 receptors has been characterized. P2X1 receptors were most sensitive to inhibition by NF023 with IC50 values of 0.24 and 0.21 microM for the rat and human homologue, respectively. P2X3 receptors have an intermediate sensitivity with IC50 values of 8.5 and 28.9 microM for rat and human subtypes, respectively and P2X2 was the least sensitive subtype (IC50 > 50 microM). P2X4 receptors were insensitive to NF023 at concentrations up to 100 microM. Coexpression of rat P2X3 with rat P2X2 resulted in receptors whose sensitivity to NF023 was identical to that obtained for homomultimeric rat P2X3 receptors (alphabeta meATP as agonist; IC50 = 1.4 and 1.6 microM, respectively). NF023 inhibited P2X1 receptors in a voltage-insensitive manner. In addition, NF023 (5 and 30 microM) caused a shift of the concentration-response curve to the right without affecting the maximal response to ATP (K(B) = 1.1 +/- 0.2 microM). Our results indicate that NF023 is a subtype-selective and surmountable antagonist at P2X1 receptors heterologously expressed in Xenopus oocytes.

Swapping of functional domains in voltage-gated K+ channels.

Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IKA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.

The role of the IsK protein in the specific pharmacological properties of the IKs channel complex.

IKs channels are composed of IsK and KvLQT1 subunits and underly the slowly activating, voltage-dependent IKs conductance in heart. Although it appears clear that the IsK protein affects both the biophysical properties and regulation of IKs channels, its role in channel pharmacology is unclear. In the present study we demonstrate that KvLQT1 homopolymeric K+ channels are inhibited by the IKs blockers 293B, azimilide and 17-beta-oestradiol. However, IKs channels induced by the coexpression of IsK and KvLQT1 subunits have a 6-100 fold higher affinity for these blockers. Moreover, the IKs activators mefenamic acid and DIDS had little effect on KvLQT1 homopolymeric channels, although they dramatically enhanced steady-state currents through heteropolymeric IKs channels by arresting them in an open state. In summary, the IsK protein modulates the effects of both blockers and activators of IKs channels. This finding is important for the action and specificity of these drugs as IsK protein expression in heart and other tissues is regulated during development and by hormones.

Molecular characterization and pharmacological properties of the human P2X3 purinoceptor.

Using PCR and library screening techniques, a cDNA encoding an ATP ligand-gated channel has been isolated from human heart. The full-length cDNA encodes a protein 397 amino acids long which shows a high amino-acid sequence identity with the rat P2X3 purinoceptor (93%). By fluorescence in situ hybridization, the human P2X3 gene has been mapped to region q12 of chromosome 11. Tissue distribution analysis of human P2X3 receptor mRNA shows a restricted expression pattern, i.e. transcripts are limited to the spinal cord and heart. This result contrasts with the distribution of the rat P2X3 receptor which was detected exclusively in sensory neurons of trigeminal, dorsal root and nodose ganglia. Heterologous expression of human P2X3 cRNA in Xenopus oocytes generates a fast desensitizing ATP-activated channel with pharmacological properties resembling the profile of the rat homologue receptor. Thus, the order of agonist potency is 2MeSATP > ATP > alphabeta-meATP > CTP > betagamma-meATP approximately ADP. Moreover, ATP-evoked currents on human P2X3 receptor are efficiently blocked in a reversible manner by the purinoceptor antagonists, suramin and PPADS.

Super-resolution measurements with evanescent-wave fluorescence excitation using variable beam incidence.

The evanescent wave (EW) elicited by total internal reflection of light selectively excites fluorophores in an optical slice above a reflecting dielectric interface. EW excitation eliminates out-of-focus fluorescence present in epiillumination microscopy, and--close to the coverslip--can offer a fivefold enhancement of axial optical sectioning compared to confocal and two-photon microscopy. The decay length of the evanescent field is a function of the refractive indices and light wavelength involved, and is modulated by the beam angle. EW microscopy was used to study the distribution and concentration of fluorophores at or near the interface in the presence of high concentrations in bulk solution. We modified an upright microscope to accommodate the condenser optics needed for EW excitation. Systematic variations of the angle of incidence were attained using an acousto-optical deflector, telecentric optics, and a hemicylindrical prism. The three-dimensional reconstruction of the fluorophore distribution from angle-resolved image stacks results in topographical information with an axial resolution of tens of nanometers. We applied this technique to study the axial position of dye-labeled subcellular storage organelles ('vesicles') of approximately 300 nm diameter in the "footprint" region of living neuroendocrine cells grown on the interface.

Hippocampal ether-à-go-go1 potassium channels blockade: effects in the startle reflex and prepulse inhibition.

Recently, our group described the ether-à-go-go1(Eag1) voltage-gated potassium (K(+)) channel (Kv10.1) expression in the dopaminergic cells indicating that these channels are part of the diversified group of ion channels related to dopaminergic neurons function. The increase of dopamine neurotransmission induces a reduction in the prepulse inhibition (PPI) of the acoustic startle reflex in rodents, which is a reliable index of sensorimotor gating deficits. The PPI response has been reported to be abnormally reduced in schizophrenia patients. The role of Eag1 K(+) channels in the PPI reaction had not been revealed until now, albeit the singular distribution of Eag1 in the dentate gyrus of the hippocampus and the hippocampal regulation of the startle reflex and PPI. The aim of this work was to investigate if Eag1 blockade on hippocampus modifies the PPI-disruptive effects of apomorphine in Wistar rats. Bilateral injection of anti-Eag1 single-chain antibody into the dentate gyrus of hippocampus did not modify apomorphine-disruptive effects in the PPI response. However, Eag1 antibody completely restored the startle amplitude decrease revealed after dentate gyrus surgery. These potentially biological important phenomenon merits further investigation regarding the role of Eag1 K(+) channels, mainly, on startle reflex modulation, since the physiological role of these channels remain obscure.