Federico Sesti

MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia

A novel potassium channel gene has been cloned, characterized, and associated with cardiac arrhythmia. The gene encodes MinK-related peptide 1 (MiRP1), a small integral membrane subunit that assembles with HERG, a pore-forming protein, to alter its function. Unlike channels formed only with HERG, mixed complexes resemble native cardiac IKr channels in their gating, unitary conductance, regulation by potassium, and distinctive biphasic inhibition by the class III antiarrhythmic E-4031. Three missense mutations associated with long QT syndrome and ventricular fibrillation are identified in the gene for MiRP1. Mutants form channels that open slowly and close rapidly, thereby diminishing potassium currents. One variant, associated with clarithromycin-induced arrhythmia, increases channel blockade by the antibiotic. A mechanism for acquired arrhythmia is revealed: genetically based reduction in potassium currents that remains clinically silent until combined with additional stressors.

Single-walled Carbon Nanotubes Are a New Class of Ion Channel Blockers

Here we identify a novel class of biological membrane ion channel blockers called single-walled carbon nanotubes (SWNTs). SWNTs with diameter distributions peaked at ∼0.9 and 1.3 nm, C60 fullerenes, multi wall nanotubes (MWNTs), and hyperfullerenes (nano-“onions”) were synthesized by several techniques and applied to diverse channel types heterologously expressed in mammalian cells. External as-fabricated and purified SWNTs blocked K+ channel subunits in a dose-dependent manner. Blockage was dependent on the shape and dimensions of the nanoparticles used and did not require any electrochemical interaction. SWNTs were more effective than the spherical fullerenes and, for both, diameter was the determining factor. These findings postulate new uses for SWNTs in biological applications and provide unexpected insights into the current view of mechanisms governing the interaction of ion channels with blocking molecules.

Oxidation of potassium channels by ROS: a general mechanism of aging and neurodegeneration?

A wealth of evidence underscores the tight link between oxidative stress, neurodegeneration and aging. When the level of excess reactive oxygen species (ROS) increases in the cell, a phenomenon characteristic of aging, DNA is damaged, proteins are oxidized, lipids are degraded and more ROS are produced, all culminating in significant cell injury. Recently we showed that in the nematode, Caenorhabditis elegans, oxidation of K(+) channels by ROS is a major mechanism underlying the loss of neuronal function. The C. elegans results support an argument that K(+) channels controlling neuronal excitability and survival might provide a common, functionally important substrate for ROS in aging mammals. Here we discuss the implications that oxidation of K(+) channels by ROS might have for the mammalian brain during normal aging, as well as in neurodegenerative diseases such as Alzheimers and Parkinsons. We argue that oxidation of K(+) channels by ROS is a common theme in the aging brain and suggest directions for future experimentation.

A Molecular Target for Viral Killer Toxin: TOK1 Potassium Channels

Killer strains of S. cerevisiae harbor double-stranded RNA viruses and secrete protein toxins that kill virus-free cells. The K1 killer toxin acts on sensitive yeast cells to perturb potassium homeostasis and cause cell death. Here, the toxin is shown to activate the plasma membrane potassium channel of S. cerevisiae, TOK1. Genetic deletion of TOK1 confers toxin resistance; overexpression increases susceptibility. Cells expressing TOK1 exhibit toxin-induced potassium flux; those without the gene do not. K1 toxin acts in the absence of other viral or yeast products: toxin synthesized from a cDNA increases open probability of single TOK1 channels (via reversible destabilization of closed states) whether channels are studied in yeast cells or X. laevis oocytes.

The multi-ion nature of the cGMP-gated channel from vertebrate rods.

1. Native cGMP‐gated channels were studied in rod outer segments of the larval tiger salamander, Ambystoma tigrinum. The alpha‐subunit of the cGMP‐gated channel, here referred to as the wild type (WT), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside‐out configuration and were activated by the addition of 100 or 500 microM cGMP. The current carried by monovalent cations was measured under voltage‐clamp conditions. 2. In the presence of 110 mM Na+ in the extracellular medium and different amounts of Na+ in the intracellular medium, the I‐V relations of the native channel could be described by a single‐site model with a profile of Gibbs free energy with two barriers and a well. A similar result was obtained in the presence of 110 mM Li+ in the extracellular medium and different amounts of Li+ in the intracellular medium. The well depth was 1.4RT (where R is the gas constant and T is the absolute temperature) for both Li+ and Na+. 3. The I‐V relations of the native channel in the presence of 110 mM Na+ on one side of the membrane and 110 mM Li+ on the other side could not be described by the same single‐site model with identical values of barriers and well obtained in the presence of Li+ or Na+ alone: the well for Li+ had to be at least 4RT. 4. In the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane, an anomalous mole fraction effect was observed both in the native and the WT channel. No anomalous behaviour was seen in the presence of Li(+)‐Na+ and Li(+)‐NH4+ mixtures. 5. The anomalous mole fraction effect with mixtures of Li+ and Cs+ was not observed in the channel where glutamate 363 was mutated to a glutamine (E363Q) or an asparagine (E363N). When glutamate 363 was mutated to an aspartate (E363D), the anomalous mole fraction effect with mixtures of Li+ and Cs+ was still observed, although significantly reduced. 6. When lysine 346, arginine 369, aspartate 370 and glutamate 372 were neutralized by mutation to glutamine, the ion permeation through the mutant channels and the WT channel had largely similar properties.(ABSTRACT TRUNCATED AT 400 WORDS)

A Potassium Channel-MiRP Complex Controls Neurosensory Function in Caenorhabditis elegans

MinK-related peptides (MiRPs) are single transmembrane proteins that associate with mammalian voltage-gated K+ subunits. Here we report the cloning and functional characterization of a MiRP β-subunit, MPS-1, and of a voltage-gated pore-forming potassium subunit, KVS-1, from the nematodeCaenorhabditis elegans. mps-1 is expressed in chemosensory and mechanosensory neurons and co-localizes withkvs-1 in a subset of these. Inactivation of eithermps-1 or kvs-1 by RNA interference (RNAi) causes partially overlapping neuronal defects and results in broad-spectrum neuronal dysfunction, including defective chemotaxis, disrupted mechanotransduction, and impaired locomotion. Inactivation of one subunit by RNAi dramatically suppresses the expression of the partner subunit only in cells where the two proteins co-localize. Co-expression of MPS-1 and KVS-1 in mammalian cells gives rise to a potassium current distinct from the KVS-1 current. Taken together these data indicate that potassium currents constitute a basic determinant for C. elegans neuronal function and unravel a unifying principle of evolutionary significance: that potassium channels in various organisms use MiRPs to generate uniqueness of function with rich variation in the details.

Oxidation of a potassium channel causes progressive sensory function loss during aging

Potassium channels are key regulators of neuronal excitability. Here we show that oxidation of the K+ channel KVS-1 during aging causes sensory function loss in Caenorhabditis elegans and that protection of this channel from oxidation preserves neuronal function. Chemotaxis, a function controlled by KVS-1, was significantly impaired in worms exposed to oxidizing agents, but only moderately affected in worms harboring an oxidation-resistant KVS-1 mutant (C113S). In aging C113S transgenic worms, the effects of free radical accumulation were significantly attenuated compared to those in wild type. Electrophysiological analyses showed that both reactive oxygen species (ROS) accumulation during aging and acute exposure to oxidizing agents acted primarily to alter the excitability of the neurons that mediate chemotaxis. Together, these findings establish a pivotal role for ROS-mediated oxidation of voltage-gated K+ channels in sensorial decline during aging in invertebrates.

Toxicity induced enhanced extracellular matrix production in osteoblastic cells cultured on single-walled carbon nanotube networks

A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix.

Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel.

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homolo-gous channels in mammals and plants are expected to function similarly.

Toxic Role of K+ Channel Oxidation in Mammalian Brain

Potassium (K+) channels are essential to neuronal signaling and survival. Here we show that these proteins are targets of reactive oxygen species in mammalian brain and that their oxidation contributes to neuropathy. Thus, the KCNB1 (Kv2.1) channel, which is abundantly expressed in cortex and hippocampus, formed oligomers upon exposure to oxidizing agents. These oligomers were ∼10-fold more abundant in the brain of old than young mice. Oxidant-induced oligomerization of wild-type KCNB1 enhanced apoptosis in neuronal cells subject to oxidative insults. Consequently, a KCNB1 variant resistant to oxidation, obtained by mutating a conserved cysteine to alanine, (C73A), was neuroprotective. The fact that oxidation of KCNB1 is toxic, argues that this mechanism may contribute to neuropathy in conditions characterized by high levels of oxidative stress, such as Alzheimers disease (AD). Accordingly, oxidation of KCNB1 channels was exacerbated in the brain of a triple transgenic mouse model of AD (3xTg-AD). The C73A variant protected neuronal cells from apoptosis induced by incubation with β-amyloid peptide (Aβ1–42). In an invertebrate model (Caenorhabditis elegans) that mimics aspects of AD, a C73A-KCNB1 homolog (C113S-KVS-1) protected specific neurons from apoptotic death induced by ectopic expression of human Aβ1–42. Together, these data underscore a novel mechanism of toxicity in neurodegenerative disease.