Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes.

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell's glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.

The Proton-Coupled Monocarboxylate Transporter Hermes Is Necessary for Autophagy during Cell Death.

Nutrient availability influences the production and degradation of materials that are required for cell growth and survival. Autophagy is a nutrient-regulated process that is used to degrade cytoplasmic materials and has been associated with human diseases. Solute transporters influence nutrient availability and sensing, yet we know little about how transporters influence autophagy. Here, we screen for solute transporters that are required for autophagy-dependent cell death and identify CG11665/hermes. We show that hermes is required for both autophagy during steroid-triggered salivary gland cell death and TNF-induced non-apoptotic eye cell death. hermes encodes a proton-coupled monocarboxylate transporter that preferentially transports pyruvate over lactate. mTOR signaling is elevated in hermes mutant cells, and decreased mTOR function suppresses the hermes salivary gland cell death phenotype. Hermes is most similar to human SLC16A11, a protein that was recently implicated in type 2 diabetes, thus providing a link between pyruvate, mTOR, autophagy, and possibly metabolic disorders.

Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor.

Potassium (K+) exits electrically excitable cells during normal and pathophysiological activity. Currently, K+-sensitive electrodes and electrical measurements are the primary tools to detect K+ fluxes. Here, we describe the synthesis of a near-IR, oxazine fluorescent K+ sensor (KNIR-1) with a dissociation constant suited for detecting changes in intracellular and extracellular K+ concentrations. KNIR-1 treatment of cells expressing voltage-gated K+ channels enabled the visualization of intracellular K+ depletion upon channel opening and restoration of cytoplasmic K+ after channel closing.

Fluorescent Visualization of Cellular Proton Fluxes.

Cells use plasma membrane proton fluxes to maintain cytoplasmic and extracellular pH and to mediate the co-transport of metabolites and ions. Because proton-coupled transport often involves movement of multiple substrates, traditional electrical measurements provide limited information about proton transport at the cell surface. Here we visualize voltage-dependent proton fluxes over the entire landscape of a cell by covalently attaching small-molecule fluorescent pH sensors to the cell's glycocalyx. We found that the extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. Given that any small-molecule fluorescent sensor can be covalently attached to a cell's glycocalyx, our approach is readily adaptable to visualize most electrogenic and non-electrogenic transport events at the plasma membrane.

Mutant SOD1 protein increases Nav1.3 channel excitability.

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.

Bioreactive Tethers.

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.

LQT1 mutations in KCNQ1 C-terminus assembly domain suppress IKs using different mechanisms.

AIMS: Long QT syndrome 1 (LQT1) mutations in KCNQ1 that decrease cardiac IKs (slowly activating delayed rectifier K(+) current) underlie ventricular arrhythmias and sudden death. LQT1 mutations may suppress IKs by preventing KCNQ1 assembly, disrupting surface trafficking, or inhibiting gating. We investigated mechanisms underlying how three LQT1 mutations in KCNQ1 C-terminus assembly domain (R555H/G589D/L619M) decrease IKs in heterologous cells and cardiomyocytes. METHODS AND RESULTS: In Chinese hamster ovary (CHO) cells, mutant KCNQ1 + KCNE1 channels either produced no currents (G589D/L619M) or displayed markedly reduced IKs with a right-shifted voltage-dependence of activation (R555H). When co-expressed with wild-type (wt) KCNQ1, the mutant KCNQ1s displayed varying intrinsic dominant-negative capacities that were affected by auxiliary KCNE1. All three mutant KCNQ1s assembled with wt KCNQ1 as determined by fluorescence resonance energy transfer (FRET). We developed an optical quantum dot labelling assay to measure channel surface density. G589D/R555H displayed substantial reductions in surface density, which were either partially (G589D) or fully (R555H) rescued by wt KCNQ1. Unexpectedly, L619M showed no trafficking defect. In adult rat cardiomyocytes, adenovirus-expressed homotetrameric G589D/L619M + KCNE1 channels yielded no currents, whereas R555H + KCNE1 produced diminished IKs with a right-shifted voltage-dependence of activation, mimicking observations in CHO cells. In contrast to heterologous cells, homotetrameric R555H channels showed no trafficking defect in cardiomyocytes. CONCLUSION: Distinct LQT1 mutations in KCNQ1 assembly domain decrease IKs using unique combinations of biophysical and trafficking mechanisms. Functional deficits in IKs observed in heterologous cells are mostly, but not completely, recapitulated in adult rat cardiomyocytes. A 'methodological chain' combining approaches in heterologous cells and cardiomyocytes provides mechanistic insights that may help advance personalized therapy for LQT1 mutations.

The middle X residue influences cotranslational N-glycosylation consensus site skipping.

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X ≠ P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B's role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.

Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering.

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.

Molecular determinants of co- and post-translational N-glycosylation of type I transmembrane peptides.

Type I transmembrane peptides acquire N-linked glycans during and after protein synthesis to facilitate anterograde trafficking through the secretory pathway. Mutations in N-glycosylation consensus sites (NXT and NXS, where X≠P) that alter the kinetics of the initial N-glycan attachment have been associated with cardiac arrhythmias; however, the molecular determinants that define co- and post-translational consensus sites in proteins are not known. In the present study, we identified co- and post-translational consensus sites in the KCNE family of K+ channel regulatory subunits to uncover three determinants that favour co-translational N-glycosylation kinetics of type I transmembrane peptides which lack a cleavable signal sequence: threonine-containing consensus sites (NXT), multiple N-terminal consensus sites and long C-termini. The identification of these three molecular determinants now makes it possible to predict co- and post-translational consensus sites in type I transmembrane peptides.

Chemical derivatization and purification of peptide-toxins for probing ion channel complexes.

Ion channels function as multi-protein complexes made up of ion-conducting α-subunits and regulatory ß-subunits. To detect, identify, and quantitate the regulatory ß-subunits in functioning K(+) channel complexes, we have chemically derivatized peptide-toxins that specifically react with strategically placed cysteine residues in the channel complex. Two protein labeling approaches have been developed to derivatize the peptide-toxin, charybdotoxin, with hydrophilic and hydrophobic bismaleimides, and other molecular probes. Using these cysteine-reactive peptide-toxins, we have specifically targeted KCNQ1-KCNE1 K(+) channel complexes expressed in both Xenopus oocytes and mammalian cells. The modular design of the reagents should permit this approach to be applied to the many ion channel complexes involved in electrical excitability as well as salt and water homoeostasis.

Structural insights into neuronal K+ channel-calmodulin complexes.

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.

The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3ß-dependent, protein kinase C (PKC)-mediated endocytic trafficking.

The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3ß and a novel potassium channel endocytic motif, because depleting either 14-3-3ß protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3ß-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs.

Xenopus laevis oocytes infected with multi-drug-resistant bacteria: implications for electrical recordings.

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.

Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease.

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel ß-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ∼20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.

O-glycosylation of the cardiac I(Ks) complex.

Post-translational modifications of the KCNQ1­KCNE1 (Kv7) K+ channel complex are vital for regulation of the cardiac IKs current and action potential duration. Here, we show the KCNE1 regulatory subunit is O-glycosylated with mucin-type glycans in vivo. As O-linked glycosylation sites are not recognizable by sequence gazing, we designed a novel set of glycosylation mutants and KCNE chimeras and analysed their glycan content using deglycosylation enzymes. Our results show that KCNE1 is exclusively O-glycosylated at Thr-7, which is also required for N-glycosylation at Asn-5. For wild type KCNE1, the overlapping N- and O-glycosylation sites are innocuous for subunit biogenesis; however, mutation of Thr-7 to a non-hydroxylated residue yielded mostly unglycosylated protein and a small fraction of mono-N-glycosylated protein. The compounded hypoglycosylation was equally deleterious for KCNQ1­KCNE1 cell surface expression, demonstrating that KCNE1 O-glycosylation is a post-translational modification that is integral for the proper biogenesis and anterograde trafficking of the cardiac IKs complex. The enzymatic assays and panel of glycosylation mutants used here will be valuable for identifying the different KCNE1 glycoforms in native cells and determining the roles N- and O-glycosylation play in KCNQ1­KCNE1 function and localization in cardiomyocytes,