Mutant SOD1 protein increases Nav1.3 channel excitability.
Journal of biological physics
Thinking big with small molecules
JOURNAL OF CELL BIOLOGY
2015; 209 (1): 7-9
Amyotrophic lateral sclerosis (ALS) is a lethal paralytic disease caused by the degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) are present in ~20% of familial ALS and ~2% of all ALS cases. The most common SOD1 gene mutation in North America is a missense mutation substituting valine for alanine (A4V). In this study, we analyze sodium channel currents in oocytes expressing either wild-type or mutant (A4V) SOD1 protein. We demonstrate that the A4V mutation confers a propensity to hyperexcitability on a voltage-dependent sodium channel (Nav1.3) mediated by heightened total Na(+) conductance and a hyperpolarizing shift in the voltage dependence of Nav1.3 activation. To estimate the impact of these channel effects on excitability in an intact neuron, we simulated these changes in the program NEURON; this shows that the changes induced by mutant SOD1 increase the spontaneous firing frequency of the simulated neuron. These findings are consistent with the view that excessive excitability of neurons is one component in the pathogenesis of this disease.
View details for DOI 10.1007/s10867-016-9411-x
View details for PubMedID 27072680
NOVEL CHEMICAL TOOLS TO STUDY ION CHANNEL BIOLOGY
2015; 869: 77-100
Synthetic chemistry has enabled scientists to explore the frontiers of cell biology, limited only by the laws of chemical bonding and reactivity. As we investigate biological questions of increasing complexity, new chemical technologies can provide systems-level views of cellular function. Here we discuss some of the molecular probes that illustrate this shift from a "one compound, one gene" paradigm to a more integrated approach to cell biology.
View details for DOI 10.1083/jcb.201501084
View details for Web of Science ID 000352960200003
View details for PubMedID 25869661
Calmodulation meta-analysis: Predicting calmodulin binding via canonical motif clustering.
The Journal of general physiology
Ion channel complexes are challenging to study by traditional biochemical methods due to their membranous lipid environment and large size. Bioreactive tethers are specialized chemical probes that have been used in electrophysiological experiments to provide unique insight into ion channel structure and function. Because bioreactive tethers are small molecular probes, they can be used to manipulate ion channel function in heterologous expression systems, native cells and animal models. This chapter covers three classes of tethers: photoswitchable, molecular rulers, and chemically reactive. The modular nature of bioreactive tethers enables the facile synthesis of next generation reagents with enhanced functionalities to interrogate and control ion channels in novel and multifarious ways.
View details for DOI 10.1007/978-1-4939-2845-3_5
View details for Web of Science ID 000361794300005
View details for PubMedID 26381941
Structural insights into neuronal K+ channel-calmodulin complexes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (34): 13579-13583
The calcium-binding protein calmodulin (CaM) directly binds to membrane transport proteins to modulate their function in response to changes in intracellular calcium concentrations. Because CaM recognizes and binds to a wide variety of target sequences, identifying CaM-binding sites is difficult, requiring intensive sequence gazing and extensive biochemical analysis. Here, we describe a straightforward computational script that rapidly identifies canonical CaM-binding motifs within an amino acid sequence. Analysis of the target sequences from high resolution CaM-peptide structures using this script revealed that CaM often binds to sequences that have multiple overlapping canonical CaM-binding motifs. The addition of a positive charge discriminator to this meta-analysis resulted in a tool that identifies potential CaM-binding domains within a given sequence. To allow users to search for CaM-binding motifs within a protein of interest, perform the meta-analysis, and then compare the results to target peptide-CaM structures deposited in the Protein Data Bank, we created a website and online database. The availability of these tools and analyses will facilitate the design of CaM-related studies of ion channels and membrane transport proteins.
View details for DOI 10.1085/jgp.201311140
View details for PubMedID 24935744
Xenopus laevis oocytes infected with multi-drug-resistant bacteria: implications for electrical recordings
JOURNAL OF GENERAL PHYSIOLOGY
2011; 138 (2): 271-277
Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that directly binds to and modulates a wide variety of ion channels. Despite the large repository of high-resolution structures of CaM bound to peptide fragments derived from ion channels, there is no structural information about CaM bound to a fully folded ion channel at the plasma membrane. To determine the location of CaM docked to a functioning KCNQ K(+) channel, we developed an intracellular tethered blocker approach to measure distances between CaM residues and the ion-conducting pathway. Combining these distance restraints with structural bioinformatics, we generated an archetypal quaternary structural model of an ion channel-CaM complex in the open state. These models place CaM close to the cytoplasmic gate, where it is well positioned to modulate channel function.
View details for DOI 10.1073/pnas.1207606109
View details for Web of Science ID 000308085200033
View details for PubMedID 22869708
Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes
2009; 4 (1)
The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug-resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3-4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug-resistant bacteria.
View details for DOI 10.1085/jgp.201110661
View details for Web of Science ID 000293122500011
View details for PubMedID 21788613
Preligand assembly domain-mediated ligand-independent association between TRAIL receptor 4 (TR4) and TR2 regulates TRAIL-induced apoptosis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (50): 18099-18104
KCNQ1 voltage-gated K(+) channels (Kv7.1) associate with the family of five KCNE peptides to form complexes with diverse gating properties and pharmacological sensitivities. The varied gating properties of the different KCNQ1-KCNE complexes enables the same K(+) channel to function in both excitable and non excitable tissues. Small molecule activators would be valuable tools for dissecting the gating mechanisms of KCNQ1-KCNE complexes; however, there are very few known activators of KCNQ1 channels and most are ineffective on the physiologically relevant KCNQ1-KCNE complexes. Here we show that a simple boronic acid, phenylboronic acid (PBA), activates KCNQ1/KCNE1 complexes co-expressed in Xenopus oocytes at millimolar concentrations. PBA shifts the voltage sensitivity of KCNQ1 channel complexes to favor the open state at negative potentials. Analysis of different-sized charge carriers revealed that PBA also targets the permeation pathway of KCNQ1 channels. Activation by the boronic acid moiety has some specificity for the Kv7 family members (KCNQ1, KCNQ2/3, and KCNQ4) since PBA does not activate Shaker or hERG channels. Furthermore, the commercial availability of numerous PBA derivatives provides a large class of compounds to investigate the gating mechanisms of KCNQ1-KCNE complexes.
View details for DOI 10.1371/journal.pone.0004236
View details for Web of Science ID 000265482100008
View details for PubMedID 19156197
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a cytokine with potential therapeutic value against cancers because of its selective cytotoxicity to many transformed, but not normal, cells. The "decoy receptors" TRAIL-R3 (TR3) and TRAIL-R4 (TR4) were believed to negatively regulate TRAIL-induced cytotoxicity by competing for ligand binding with TRAIL-R1 (TR1) and TRAIL-R2 (TR2). Here, we show that inhibition of TRAIL-induced apoptosis by TR4 critically depends on its association with TR2 via the NH(2)-terminal preligand assembly domain overlapping the first partial cysteine-rich domain of both receptors. By contrast, ligand binding by TR4 is dispensable for its apoptosis inhibitory function, thereby excluding the possibility that TR4 was a "decoy" to inhibit apoptosis by binding up TRAIL. In primary CD8(+) T cells, which express only TR2 and TR4 and are resistant to TRAIL-induced apoptosis, stimulation with phorbol myristate acetate abrogated the ligand-independent interaction between TR2 and TR4 and enhanced their sensitivity to TRAIL-induced apoptosis. Hence, whereas most TNF receptors normally form only homotrimeric complexes, the preligand assembly domains in TR2 and TR4 permit mixed complex formation as a means to regulate apoptosis induction. We propose that TR4 is a "regulatory" rather than "decoy" receptor that inhibits apoptosis signaling by TRAIL through this previously uncharacterized ligand-independent mechanism.
View details for DOI 10.1073/pnas.0507329102
View details for Web of Science ID 000234010500040
View details for PubMedID 16319225