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.

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.

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.