Ion channel genes and human neurological disease: Recent progress, prospects, and challenges

What do epilepsy, migraine headache, deafness, episodic ataxia, periodic paralysis, malignant hyperthermia, and generalized myotonia have in common? These human neurological disorders can be caused by mutations in genes for ion channels. Many of the channel diseases are “paroxysmal disorders” whose principal symptoms occur intermittently in individuals who otherwise may be healthy and active. Some of the ion channels that cause human neurological disease are old acquaintances previously cloned and extensively studied by channel specialists. In other cases, however, disease-gene hunts have led the way to the identification of new channel genes. Progress in the study of ion channels has made it possible to analyze the effects of human neurological disease-causing channel mutations at the level of the single channel, the subcellular domain, the neuronal network, and the behaving organism.

An ATP-activated, ligand-gated ion channel on a cholinergic presynaptic nerve terminal.

ATP has recently been identified as a fast neurotransmitter in both the central and peripheral nervous systems. Several studies have suggested that ATP can also affect the release of classical neurotransmitters, including acetylcholine with which it is co-released. We have searched for ATP receptors on a cholinergic presynaptic nerve terminal using the calyx-type synapse of the chicken ciliary ganglion. ATP was pulsed onto the terminals under voltage clamp and induced a short latency cation current that exhibited inward rectification and marked desensitization. This current was not seen with adenosine but was mimicked by several sterically restricted ATP analogs and was blocked by suramin. ATP-activated single ion channels exhibited prominent flickering and had a conductance of approximately 17 pS. Our results demonstrate a ligand-gated P2X-like purinergic receptor on a cholinergic presynaptic nerve terminal.

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

Mechanisms of ion channel activation in primary cultured and clonal cell lines

The effects of hypotonic shock upon membrane C1 permeability of ROS 17/2.8 osteoblast-like cells was investigated using the patch-clamp technique. Hypotonic shock produced cell swelling that was accompanied by large amplitude, outwardly rectifying, currents that were active across the entire physiological range of membrane potentials (-80 to +100 mV). At strong depolarisations (> +50 mV) the currents exhibited time-dependent inactivation that followed a monoexponential time course. The currents were anion selective and exhibited a selectivity sequence of SCN- > I > Br- > Cl- > F- > gluconate. Current activation was unaffected by inhibitors of protein kinase (A (H-89) and tyrosine kinase (tyrphostin A25), and could not be mimicked by elevation of intracellular Ca2+ or activation of protein kinase C. Similarly, disruption of actin filaments by dihydrocytochalsin B, or generation of membrane tension by dipyridamole failed to elicit significant increases in cell chloride permeability. The mechanism of current activation is as yet undetermined. The currents were effectively inhibited by the chloride channel inhibitors NPPB and DIDS but resistant to DPC. A Cl- conductance with similar characteristics was found to be present in mouse primary cultured calvarial osteoblasts. The volume-sensitive Cl- current in ROS 17/2.8 cells was inhibited by arachidonic acid in two distinct phases. A rapid block that developed within 10 s, preceding a slower developing inhibitory phase that occurred approximately 90 s after onset of arachidonate superfusion. Arachidonic acid also induced kinetic modifications of the current which were evident as an acceleration of the time-dependent· inactivation exhibited at depolarised potentials. Inhibitors of cyclo-oxygenases, lipoxygenases and cytochrome P-4S0 were ineffectual against arachidonic acid's effects sugtgesting that arachidonic acid may elicit it's effects directly. Measurements of cell volume under hypotonic conditions showed that ROS 17/2,8 cells could effectively regulate their volume, However, effective inhibitors of the volume-sensitive CI" current drastically impaired this response suggesting that physiologically this current may have a vital role in cell volume regulation, In L6 skeletal myocytes, vasopressin was found to rapidiy hyperpolarise cells. This appears to occur as the result of activation of Ca2+ -sensitive K+ channels in a process dependent upon the presence of extracellular Ca2+.

Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch.

TRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of "forefront" signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.

'Neurons in Action' in Action - Educational settings for simulations and tutorials using NEURON

Neurons in Action (NIA1, 2000; NIA1.5, 2004; NIA2, 2007), a set of tutorials and linked simulations, is designed to acquaint students with neuronal physiology through interactive, virtual laboratory experiments. Here we explore the uses of NIA in lecture, both interactive and didactic, as well as in the undergraduate laboratory, in the graduate seminar course, and as an examination tool through homework and problem set assignments. NIA, made with the simulator NEURON (http://www.neuron.yale.edu/neuron/), displays voltages, currents, and conductances in a membrane patch or signals moving within the dendrites, soma and/or axon of a neuron. Customized simulations start with the plain lipid bilayer and progress through equilibrium potentials; currents through single Na and K channels; Na and Ca action potentials; voltage clamp of a patch or a whole neuron; voltage spread and propagation in axons, motoneurons and nerve terminals; synaptic excitation and inhibition; and advanced topics such as channel kinetics and coincidence detection. The user asks and answers "what if" questions by specifying neuronal parameters, ion concentrations, and temperature, and the experimental results are then plotted as conductances, currents, and voltage changes. Such exercises provide immediate confirmation or refutation of the student's ideas to guide their learning. The tutorials are hyperlinked to explanatory information and to original research papers. Although the NIA tutorials were designed as a sequence to empower a student with a working knowledge of fundamental neuronal principles, we find that faculty are using the individual tutorials in a variety of educational situations, some of which are described here. Here we offer ideas to colleagues using interactive software, whether NIA or another tool, for educating students of differing backgrounds in the subject of neurophysiology.

ModelDB in Computational Neuroscience Education - A research tool as interactive educational media

ModelDB's mission is to link computational models and publications, supporting the field of computational neuroscience (CNS) by making model source code readily available. It is continually expanding, and currently contains source code for more than 300 models that cover more than 41 topics. Investigators, educators, and students can use it to obtain working models that reproduce published results and can be modified to test for new domains of applicability. Users can browse ModelDB to survey the field of computational neuroscience, or pursue more focused explorations of specific topics. Here we describe tutorials and initial experiences with ModelDB as an interactive educational tool.

KINETICS OF ION TRANSFER ACROSS LIQUID LIQUID INTERFACE BY CHRONOPOTENTIOMETRY WITH LINEAR CURRENT SCANNING

The equation of the potential-current curve for the ion transfer across the liquid/liquid interface during the linear current scanning has been derived theoretically. A method to calculate the kinetics parameters for the ion transfer by the way of linear current scanning is presented. The transfer of TPAs~+ ions, which is a typical basic electrolyte ion usually used in liquld/liquid interface electrochemistry, was practically investigated at the water/nitrobenzene interface.

A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

Structural changes of tumor necrosis factor alpha associated with membrane insertion and channel formation.

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.

Variants in the human potassium channel gene (KCNN3) are associated with migraine in a high risk genetic isolate

The calcium-activated potassium ion channel gene (KCNN3) is located in the vicinity of the familial hemiplegic migraine type 2 locus on chromosome 1q21.3. This gene is expressed in the central nervous system and plays a role in neural excitability. Previous association studies have provided some, although not conclusive, evidence for involvement of this gene in migraine susceptibility. To elucidate KCNN3 involvement in migraine, we performed gene-wide SNP genotyping in a high-risk genetic isolate from Norfolk Island, a population descended from a small number of eighteenth century Isle of Man ‘Bounty Mutineer’ and Tahitian founders. Phenotype information was available for 377 individuals who are related through the single, well-defined Norfolk pedigree (96 were affected: 64 MA, 32 MO). A total of 85 SNPs spanning the KCNN3 gene were genotyped in a sub-sample of 285 related individuals (76 affected), all core members of the extensive Norfolk Island ‘Bounty Mutineer’ genealogy. All genotyping was performed using the Illumina BeadArray platform. The analysis was performed using the statistical program SOLAR v4.0.6 assuming an additive model of allelic effect adjusted for the effects of age and sex. Haplotype analysis was undertaken using the program HAPLOVIEW v4.0. A total of four intronic SNPs in the KCNN3 gene displayed significant association (P < 0.05) with migraine. Two SNPs, rs73532286 and rs6426929, separated by approximately 0.1 kb, displayed complete LD (r 2 = 1.00, D′ = 1.00, D′ 95% CI = 0.96–1.00). In all cases, the minor allele led to a decrease in migraine risk (beta coefficient = 0.286–0.315), suggesting that common gene variants confer an increased risk of migraine in the Norfolk pedigree. This effect may be explained by founder effect in this genetic isolate. This study provides evidence for association of variants in the KCNN3 ion channel gene with migraine susceptibility in the Norfolk genetic isolate with the rarer allelic variants conferring a possible protective role. This the first comprehensive analysis of this potential candidate gene in migraine and also the first study that has utilised the unique Norfolk Island large pedigree isolate to implicate a specific migraine gene. Studies of additional variants in KCNN3 in the Norfolk pedigree are now required (e.g. polyglutamine variants) and further analyses in other population data sets are required to clarify the association of the KCNN3 gene and migraine risk in the general outbred population.

Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3') polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO). In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090). Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05). The prevalence of the long CAG repeat (>19 repeats) did not reach statistical significance in migraineurs (P = 0.15), nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively), or between MA vs MO (P = 0.40). Conclusion This association study provides no evidence that length variations of the second polyglutamine array in the N-terminus of the KCNN3 channel exert an effect in the pathogenesis of migraine.