Overexpression of a Shaker-type potassium channel in mammalian central nervous system dysregulates native potassium channel gene expression

The nervous system maintains a delicate balance between excitation and inhibition, partly through the complex interplay between voltage-gated sodium and potassium ion channels. Because K+ channel blockade or gene deletion causes hyperexcitability, it is generally assumed that increases in K+ channel gene expression should reduce neuronal network excitability. We have tested this hypothesis by creating a transgenic mouse that expresses a Shaker-type K+ channel gene. Paradoxically, we find that addition of the extra K+ channel gene results in a hyperexcitable rather than a hypoexcitable phenotype. The presence of the transgene leads to a complex deregulation of endogenous Shaker genes in the adult central nervous system as well as an increase in network excitability that includes spontaneous cortical spike and wave discharges and a lower threshold for epileptiform bursting in isolated hippocampal slices. These data suggest that an increase in K+ channel gene dosage leads to dysregulation of normal K+ channel gene expression, and it may underlie a mechanism contributing to the pathogenesis of human aneuploidies such as Down syndrome.

Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin

A finely tuned Ca2+ signaling system is essential for cells to transduce extracellular stimuli, to regulate growth, and to differentiate. We have recently cloned CaT-like (CaT-L), a highly selective Ca2+ channel closely related to the epithelial calcium channels (ECaC) and the calcium transport protein CaT1. CaT-L is expressed in selected exocrine tissues, and its expression also strikingly correlates with the malignancy of prostate cancer. The expression pattern and selective Ca2+ permeation properties suggest an important function in Ca2+ uptake and a role in tumor progression, but not much is known about the regulation of this subfamily of ion channels. We now demonstrate a biochemical and functional mechanism by which cells can control CaT-L activity. CaT-L is regulated by means of a unique calmodulin binding site, which, at the same time, is a target for protein kinase C-dependent phosphorylation. We show that Ca2+-dependent calmodulin binding to CaT-L, which facilitates channel inactivation, can be counteracted by protein kinase C-mediated phosphorylation of the calmodulin binding site.

Tetrodotoxin-resistant Na+ currents and inflammatory hyperalgesia

Several mechanisms have been identified that may underlie inflammation-induced sensitization of high-threshold primary afferent neurons, including the modulation of voltage- and Ca2+-dependent ion channels and ion channels responsible for the production of generator potentials. One such mechanism that has recently received a lot of attention is the modulation of a tetrodotoxin (TTX)-resistant voltage-gated Na+ current. Evidence supporting a role for TTX-resistant Na+ currents in the sensitization of primary afferent neurons and inflammatory hyperalgesia is reviewed. Such evidence is derived from studies on the distribution of TTX-resistant Na+ currents among primary afferent neurons and other tissues of the body that suggest that these currents are expressed only in a subpopulation of primary afferent neurons that are likely to be involved in nociception. Data from studies on the biophysical properties of these currents suggest that they are ideally suited to mediate the repetitive discharge associated with prolonged membrane depolarizations. Data from studies on the effects of inflammatory mediators and antinociceptive agents on TTX-resistant Na+ currents suggest that modulation of these currents is an underlying mechanism of primary afferent neuron sensitization. In addition, the second-messenger pathways underlying inflammatory mediator-induced modulation of these currents appear to underlie inflammatory mediator-induced hyperalgesia. Finally, recent antisense studies have also yielded data supporting a role for TTX-resistant Na+ currents in inflammatory hyperalgesia. Although data from these studies are compelling, data presented at the Neurobiology of Pain colloquium raised a number of interesting questions regarding the role of TTX-resistant Na+ currents in inflammatory hyperalgesia; implications of three of these questions are discussed.

Signaling in plants

Higher plants are sessile organisms that perceive environmental cues such as light and chemical signals and respond by changing their morphologies. Signaling pathways utilize a complex network of interactions to orchestrate biochemical and physiological responses such as flowering, fruit ripening, germination, photosynthetic regulation, and shoot or root development. In this session, the mechanisms of signaling systems that trigger plant responses to light and to the gaseous hormone, ethylene, were discussed. These signals are first sensed by a receptor and transmitted to the nucleus by a complex network. A signal may be transmitted to the nucleus by any of several systems including GTP binding proteins (G proteins), which change activity upon GTP binding; protein kinase cascades, which sequentially phosphorylate and activate a series of proteins; and membrane ion channels, which change ionic characteristics of the cells. The signal is manifested in the nucleus as a change in the activity of DNA-binding proteins, which are transcription factors that specifically interact and modulate the regulatory regions of genes. Thus, detection of an environmental signal is transmitted through a transduction pathway, and changes in transcription factor activity may coordinate changes in the expression of a portfolio of genes to direct new developmental programs.

Two mechanisms for transducer adaptation in vertebrate hair cells

Deflection of the hair bundle atop a sensory hair cell modulates the open probability of mechanosensitive ion channels. In response to sustained deflections, hair cells adapt. Two fundamentally distinct models have been proposed to explain transducer adaptation. Both models support the notion that channel open probability is modulated by calcium that enters via the transduction channels. Both also suggest that the primary effect of adaptation is to shift the deflection-response [I(X)] relationship in the direction of the applied stimulus, thus maintaining hair bundle sensitivity. The models differ in several respects. They operate on different time scales: the faster on the order of a few milliseconds or less and the slower on the order of 10 ms or more. The model proposed to explain fast adaptation suggests that calcium enters and binds at or near the transduction channels to stabilize a closed conformation. The model proposed to explain the slower adaptation suggests that adaptation is mediated by an active, force-generating process that regulates the effective stimulus applied to the transduction channels. Here we discuss the evidence in support of each model and consider the possibility that both may function to varying degrees in hair cells of different species and sensory organs.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

Norepinephrine transporters have channel modes of conduction.

Neurotransmitter transporters couple to existing ion gradients to achieve reuptake of transmitter into presynaptic terminals. For coupled cotransport, substrates and ions cross the membrane in fixed stoichiometry. This is in contrast to ion channels, which carry an arbitrary number of ions depending on the channel open time. Members of the gamma-aminobutyric acid transporter gene family presumably function with fixed stoichiometry in which a set number of ions cotransport with one transmitter molecule. Here we report channel-like events from a presumably fixed stoichiometry [norepinephrine (NE)+, Na+, and Cl-], human NE (hNET) in the gamma-aminobutyric acid transporter gene family. These events are stimulated by NE and by guanethidine, an hNET substrate, and they are blocked by cocaine and the antidepressant desipramine. Voltage-clamp data combined with NE uptake data from these same cells indicate that hNETs have two functional modes of conduction: a classical transporter mode (T-mode) and a novel channel mode (C-mode). Both T-mode and C-mode are gated by the same substrates and antagonized by the same blockers. T-mode is putatively electrogenic because the transmitter and cotransported ions sum to one net charge. However, C-mode carries virtually all of the transmitter-induced current, even though it occurs with low probability. This is because each C-mode opening transports hundreds of charges per event. The existence of a channel mode of conduction in a previously established fixed-stoichiometry transporter suggests the appearance of an aqueous pore through the transporter protein during the transport cycle and may have significance for transporter regulation.

Selection for genes encoding secreted proteins and receptors.

Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

Antifreeze glycoproteins inhibit leakage from liposomes during thermotropic phase transitions.

Antifreeze glycoproteins (AFGPs), found in the blood of polar fish at concentrations as high as 35 g/liter, are known to prevent ice crystal growth and depress the freezing temperature of the blood. Previously, Rubinsky et al. [Rubinsky, B., Mattioli, M., Arav, A., Barboni, B. & Fletcher, G. L. (1992) Am. J. Physiol. 262, R542-R545] provided evidence that AFGPs block ion fluxes across membranes during cooling, an effect that they ascribed to interactions with ion channels. We investigated the effects of AFGPs on the leakage of a trapped marker from liposomes during chilling. As these liposomes are cooled through the transition temperature, they leak approximately 50% of their contents. Addition of less than 1 mg/ml of AFGP prevents up to 100% of this leakage, both during chilling and warming through the phase transition. This is a general effect that we show here applies to liposomes composed of phospholipids with transition temperatures ranging from 12 degrees C to 41 degrees C. Because these results were obtained with liposomes composed of phospholipids alone, we conclude that the stabilizing effects of AFGPs on intact cells during chilling reported by Rubinsky et al. may be due to a nonspecific effect on the lipid components of native membranes. There are other proteins that prevent leakage, but only under specialized conditions. For instance, antifreeze proteins, bovine serum albumin, and ovomucoid all either have no effect or actually induce leakage. Following precipitation with acetone, all three proteins inhibited leakage, although not to the extent seen with AFGPs. Alternatively, there are proteins such as ovotransferrin that have no effect on leakage, either before or after acetone precipitation.

Pentameric subunit stoichiometry of a neuronal glutamate receptor.

Ionotropic glutamate receptors, neurotransmitter-activated ion channels that mediate excitatory synaptic transmission in the central nervous system, are oligomeric membrane proteins of unknown subunit stoichiometry. To determine the subunit stoichiometry we have used a functional assay based on the blockade of two alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunit 1 (GluR1) mutant subunits selectively engineered to exhibit differential sensitivity to the open channel blockers phencyclidine and dizolcipine (MK-801). Coinjection into amphibian oocytes of weakly sensitive with highly sensitive subunit complementary RNAs produces functional heteromeric channels with mixed blocker sensitivities. Increasing the fraction of the highly sensitive subunit augmented the proportion of drug-sensitive receptors. Analysis of the data using a model based on random aggregation of receptor subunits allowed us to determine a pentameric stoichiometry for GluR1. This finding supports the view that a pentameric subunit organization underlies the structure of the neuronal ionotropic glutamate receptor gene family.

Evidence for distinct signaling mechanisms in two mammalian olfactory sense organs.

In mammals, olfactory stimuli are detected by sensory neurons at two distinct sites: the olfactory epithelium (OE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). While the OE can detect volatile chemicals released from numerous sources, the VNO appears to be specialized to detect pheromones that are emitted by other animals and that convey information of behavioral or physiological importance. The mechanisms underlying sensory transduction in the OE have been well studied and a number of components of the transduction cascade have been cloned. Here, we investigated sensory transduction in the VNO by asking whether VNO neurons express molecules that have been implicated in sensory transduction in the OE. Using in situ hybridization and Northern blot analyses, we found that most of the olfactory transduction components examined, including the guanine nucleotide binding protein alpha subunit (G-alpha-olf), adenylyl cyclase type III, and an olfactory cyclic nucleotide-gated (CNG) channel subunit (oCNC1), are not expressed by VNO sensory neurons. In contrast, VNO neurons do express a second olfactory CNG channel subunit (oCNC2). These results indicate that VNO sensory transduction is distinct from that in the OE but raise the possibility that, like OE sensory transduction, sensory transduction in the VNO might involve cyclic nucleotide-gated ion channels.

Primary structure and functional expression of a cGMP-gated potassium channel.

Cyclic nucleotides modulate potassium (K) channel activity in many cells and are thought to act indirectly by inducing channel protein phosphorylation. Herein we report the isolation from rabbit of a gene encoding a K channel (Kcn1) that is specifically activated by cGMP and not by cAMP. Analysis of the deduced amino acid sequence (725 amino acids) indicates that, in addition to a core region that is highly homologous to Shaker K channels, Kcn1 also contains a cysteine-rich region similar to that of ligand-gated ion channels and a cyclic nucleotide-binding region. Northern blot analysis detects gene expression in kidney, aorta, and brain. Kcn1 represents a class of K channels that may be specifically regulated by cGMP and could play an important role in mediating the effects of substances, such as nitric oxide, that increase intracellular cGMP.

Nramp defines a family of membrane proteins.

Nramp (natural resistance-associated macrophage protein) is a newly identified family of integral membrane proteins whose biochemical function is unknown. We report on the identification of Nramp homologs from the fly Drosophila melanogaster, the plant Oryza sativa, and the yeast Saccharomyces cerevisiae. Optimal alignment of protein sequences required insertion of very few gaps and revealed remarkable sequence identity of 28% (yeast), 40% (plant), and 55% (fly) with the mammalian proteins (46%, 58%, and 73% similarity), as well as a common predicted transmembrane topology. This family is defined by a highly conserved hydrophobic core encoding 10 transmembrane segments. Other features of this hydrophobic core include several invariant charged residues, helical periodicity of sequence conservation suggesting conserved and nonconserved faces for several transmembrane helices, a consensus transport signature on the intracytoplasmic face of the membrane, and structural determinants previously described in ion channels. These characteristics suggest that the Nramp polypeptides form part of a group of transporters or channels that act on as yet unidentified substrates.