Hugo McGuire

Fluorescence detection of the movement of single KcsA subunits reveals cooperativity

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.

Automating single subunit counting of membrane proteins in mammalian cells.

Background: Although powerful, single subunit counting is time-consuming, prone to user bias, and largely restricted to Xenopus expression. Results: PIF is an automated analysis program that identifies subunit stoichiometry of any fluorescently tagged membrane protein from TIRF recordings. Conclusion: PIF is accurate to more than 90% even in noisy data typical for mammalian expression system. Significance: The PIF approach is generalizable to any membrane protein and TIRF microscope. Elucidating subunit stoichiometry of neurotransmitter receptors is preferably carried out in a mammalian expression system where the rules of native protein assembly are strictly obeyed. Although successful in Xenopus oocytes, single subunit counting, manually counting photobleaching steps of GFP-tagged subunits, has been hindered in mammalian cells by high background fluorescence, poor control of expression, and low GFP maturation efficiency. Here, we present a fully automated single-molecule fluorescence counting method that separates tagged proteins on the plasma membrane from background fluorescence and contaminant proteins in the cytosol or the endoplasmic reticulum and determines the protein stoichiometry. Lower GFP maturation rates observed in cells cultured at 37 °C were partly offset using a monomeric version of superfolder GFP. We were able to correctly identify the stoichiometry of GluK2 and α1 glycine receptors. Our approach permits the elucidation of stoichiometry for a wide variety of plasma membrane proteins in mammalian cells with any commercially available TIRF microscope.

Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization

Background: The stoichiometry of pore-forming toxins is frequently unknown because crystal structures do not reflect the active conformations. Results: We used single subunit counting on fluorescently labeled Cry1Aa toxin of Bacillus thuringiensis to follow its oligomerization process. Conclusion: We determined that the final architecture of the pores is tetrameric. Significance: The stochastic analysis introduced permits the application of single subunit counting to dynamic processes such as oligomerization. Pore-forming toxins constitute a class of potent virulence factors that attack their host membrane in a two- or three-step mechanism. After binding to the membrane, often aided by specific receptors, they form pores in the membrane. Pore formation either unfolds a cytolytic activity in itself or provides a pathway to introduce enzymes into the cells that act upon intracellular proteins. The elucidation of the pore-forming mechanism of many of these toxins represents a major research challenge. As the toxins often refold after entering the membrane, their structure in the membrane is unknown, and key questions such as the stoichiometry of individual pores and their mechanism of oligomerization remain unanswered. In this study, we used single subunit counting based on fluorescence spectroscopy to explore the oligomerization process of the Cry1Aa toxin of Bacillus thuringiensis. Purified Cry1Aa toxin molecules labeled at different positions in the pore-forming domain were inserted into supported lipid bilayers, and the photobleaching steps of single fluorophores in the fluorescence time traces were counted to determine the number of subunits of each oligomer. We found that toxin oligomerization is a highly dynamic process that occurs in the membrane and that tetramers represent the final form of the toxins in a lipid bilayer environment.

A limited 4 Å radial displacement of the S4-S5 linker is sufficient for internal gate closing in Kv channels.

Background: For Kv channels, only crystal structures for the open state are available. Results: Using LRET, we determined the movement of the S4-S5 linker during gating in KvAP channels. Conclusion: A small displacement of the S6 by only 4 Å is sufficient for closing of the Kv channels. Significance: We provide the first Kv channel closed state model based on cytosolic restraints. Voltage-gated ion channels are responsible for the generation of action potentials in our nervous system. Conformational rearrangements in their voltage sensor domains in response to changes of the membrane potential control pore opening and thus ion conduction. Crystal structures of the open channel in combination with a wealth of biophysical data and molecular dynamics simulations led to a consensus on the voltage sensor movement. However, the coupling between voltage sensor movement and pore opening, the electromechanical coupling, occurs at the cytosolic face of the channel, from where no structural information is available yet. In particular, the question how far the cytosolic pore gate has to close to prevent ion conduction remains controversial. In cells, spectroscopic methods are hindered because labeling of internal sites remains difficult, whereas liposomes or detergent solutions containing purified ion channels lack voltage control. Here, to overcome these problems, we controlled the state of the channel by varying the lipid environment. This way, we directly measured the position of the S4-S5 linker in both the open and the closed state of a prokaryotic Kv channel (KvAP) in a lipid environment using Lanthanide-based resonance energy transfer. We were able to reconstruct the movement of the covalent link between the voltage sensor and the pore domain and used this information as restraints for molecular dynamics simulations of the closed state structure. We found that a small decrease of the pore radius of about 3–4 Å is sufficient to prevent ion permeation through the pore.

Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation

Most ligand- and voltage-gated ion channels assemble as signaling complexes consisting of pore-forming and auxiliary subunits. In the mammalian brain, AMPA-type ionotropic glutamate receptors (AMPARs) coassemble with several families of auxiliary subunits that regulate channel gating as well as ion channel block and permeation. Previous work has shown that auxiliary proteins stargazin (or &ggr;2) and cornichon-3 (CNIH-3) attenuate the cytoplasmic polyamine channel block of AMPARs, although the underlying mechanism has yet to be established. Here, we show that &ggr;2 and CNIH-3 relieve channel block by enhancing the rate of blocker permeation. Surprisingly, the relative permeability of the polyamine spermine (Spm) through the pore of the AMPAR-&ggr;2 or -CNIH-3 complexes is considerably more than AMPARs expressed alone. Spm permeability is comparable to that of Na+ for the GluA2-&ggr;2 complex and four times greater than Na+ with GluA2 + CNIH-3. A modified model of permeant channel block fully accounts for both the voltage- and time-dependent nature of Spm block. Estimates of block rate constants reveal that auxiliary subunits do not attenuate block by shifting the location of the block site within the membrane electric field, and they do not affect the blocker’s ability to reach it. Instead, &ggr;2 and CNIH-3 relieve channel block by facilitating the blocker’s exit rates from the open channel. From a physiological perspective, the relief of channel block exerted by &ggr;2 and CNIH-3 ensures that there is unfettered signaling by AMPARs at glutamatergic synapses. Moreover, the pronounced ability of AMPARs to transport polyamines may have an unexpected role in regulating cellular polyamine levels.

Full-length Cellular Beta-secretase Has a Trimeric Subunit Stoichiometry, and Its Sulfur-rich Transmembrane Interaction Site Modulates Cytosolic Copper Compartmentalization

The β-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aβ peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.

A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells

Determining the composition and stoichiometry of membrane-bound proteins has been a perennial problem that has plagued biology for a long time. The most recurring issue is that composition and subunit stoichiometry is commonly inferred from bulk biochemical assays that can only shed light on the “averaged” makeup of the protein complex. However, recent studies have been able to circumvent this issue by studying the stoichiometry of individual protein complexes. The most common approach has been to express GFP-tagged subunits in Xenopus laevis oocytes and then manually count the number of photobleaching steps to report mature protein stoichiometry. Although valuable, an important drawback of this technique is that the strict rules of mammalian protein assembly are not always adhered to in this surrogate expression system. Furthermore, manual counting of bleaching steps is subject to user bias and places practical limits on the amount of data that can be analyzed. In this chapter, we provide a step-by-step account of how we adapted the subunit counting method for mammalian cells to study the composition and stoichiometry of ionotropic glutamate receptors. Using custom-made software, we have automated the entire counting process so that it is much less time consuming and no longer subject to user bias. Given its universality, this methodological approach permits the elucidation of subunit number and stoichiometry for a wide variety of plasma-membrane-bound proteins in mammalian cells.