Marcus D. Collins

Nanopore DNA sequencing with MspA

Nanopore sequencing has the potential to become a direct, fast, and inexpensive DNA sequencing technology. The simplest form of nanopore DNA sequencing utilizes the hypothesis that individual nucleotides of single-stranded DNA passing through a nanopore will uniquely modulate an ionic current flowing through the pore, allowing the record of the current to yield the DNA sequence. We demonstrate that the ionic current through the engineered Mycobacterium smegmatis porin A, MspA, has the ability to distinguish all four DNA nucleotides and resolve single-nucleotides in single-stranded DNA when double-stranded DNA temporarily holds the nucleotides in the pore constriction. Passing DNA with a series of double-stranded sections through MspA provides proof of principle of a simple DNA sequencing method using a nanopore. These findings highlight the importance of MspA in the future of nanopore sequencing.

Line Tensions, Correlation Lengths, and Critical Exponents in Lipid Membranes Near Critical Points

Membranes containing a wide variety of ternary mixtures of high chain-melting temperature lipids, low chain-melting temperature lipids, and cholesterol undergo lateral phase separation into coexisting liquid phases at a miscibility transition. When membranes are prepared from a ternary lipid mixture at a critical composition, they pass through a miscibility critical point at the transition temperature. Since the critical temperature is typically on the order of room temperature, membranes provide an unusual opportunity in which to perform a quantitative study of biophysical systems that exhibit critical phenomena in the two-dimensional Ising universality class. As a critical point is approached from either high or low temperature, the scale of fluctuations in lipid composition, set by the correlation length, diverges. In addition, as a critical point is approached from low temperature, the line tension between coexisting phases decreases to zero. Here we quantitatively evaluate the temperature dependence of line tension between liquid domains and of fluctuation correlation lengths in lipid membranes to extract a critical exponent, nu. We obtain nu = 1.2 +/- 0.2, consistent with the Ising model prediction nu = 1. We also evaluate the probability distributions of pixel intensities in fluorescence images of membranes. From the temperature dependence of these distributions above the critical temperature, we extract an independent critical exponent of beta = 0.124 +/- 0.03, which is consistent with the Ising prediction of beta = 1/8.

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal–Mueller planar bilayers in which one leaflets composition would phase-separate in a symmetric bilayer and the others would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions.

High-Pressure Protein Crystallography and NMR to Explore Protein Conformations

High-pressure methods for solving protein structures by X-ray crystallography and NMR are maturing. These techniques are beginning to impact our understanding of thermodynamic and structural features that define not only the proteins native conformation, but also the higher free energy conformations. The ability of high-pressure methods to visualize these mostly unexplored conformations provides new insight into protein function and dynamics. In this review, we begin with a historical discussion of high-pressure structural studies, with an eye toward early results that paved the way to mapping the multiple conformations of proteins. This is followed by an examination of several recent studies that emphasize different strengths and uses of high-pressure structural studies, ranging from basic thermodynamics to the suggestion of high-pressure structural methods as a tool for protein engineering.

Regulation of TRPV1 ion channel by phosphoinositide (4,5)-bisphosphate: the role of membrane asymmetry.

Background: Whether phosphoinositide 4,5-bisphosphate (PI(4,5)P2) activates or inhibits TRPV1 is controversial. Results: PI(4,5)P2 in the intracellular leaflet activates TRPV1, whereas PI(4,5)P2 in the extracellular leaflet inhibits TRPV1. Conclusion: Inhibition by PI(4,5)P2 in the extracellular leaflet may explain previous findings that TRPV1 reconstituted into PI(4,5)P2-containing liposomes is inhibited. Significance: PI(4,5)P2 in the physiologically relevant leaflet (the intracellular leaflet) of the membrane activates TRPV1. Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.

Regulation of TRPV1 by Phosphoinositide (4,5)-bisphosphate: Role of Membrane Asymmetry

Background: Whether phosphoinositide 4,5-bisphosphate (PI(4,5)P2) activates or inhibits TRPV1 is controversial. Results: PI(4,5)P2 in the intracellular leaflet activates TRPV1, whereas PI(4,5)P2 in the extracellular leaflet inhibits TRPV1. Conclusion: Inhibition by PI(4,5)P2 in the extracellular leaflet may explain previous findings that TRPV1 reconstituted into PI(4,5)P2-containing liposomes is inhibited. Significance: PI(4,5)P2 in the physiologically relevant leaflet (the intracellular leaflet) of the membrane activates TRPV1. Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P2) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P2 in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P2 was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P2 in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P2 in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P2 whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P2 must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P2 may resolve the long standing controversy about whether PI(4,5)P2 is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.

Phosphoinositides bind to a selective site in the proximal C-terminal region to regulate TRPV1.

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment. Electroformation is an efficient mean to produce giant liposomes >10 μm in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory. We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

PI(4,5)P2 Regulation of TRPV1 Reconstituted in Model Lipid Membranes

TRPV1 channels are non-selective cation channels activated by capsaicin, protons, and heat. We have previously shown that TRPV1 activation is potentiated in vivo by PI(4,5)P2. We are now investigating the mechanism of PI(4,5)P2 potentiation using TRPV1 channels reconstituted in synthetic lipid vesicles studied with patch-clamp electrophysiology. We will present our results exploring the effects of natural PIP2 extracts and di-C16 PI(4,5)P2 incorporated into asolectin giant unilamellar vesicles (GUVs) and synthetic lipid GUVs. We will compare the effects of these non-soluble forms of PIP2 to the effects of the more water soluble di-C8 PIP2 forms used in many cellular electrophysiology experiments.

Patch Clamped Giant Unilamellar Vesicles Containing Reconstituted Ion Channels

There is increasing interest in how the chemical and physical properties of lipid membranes affect the function of mammalian ion channels. As evidenced by recent work on KvAP and Kv1.2, as well as studies of the physical nature of patches themselves, these effects are both intriguing and difficult to study. For the TRP family of ion channels these questions are especially important as membrane lipids, such as phosphoinositide (4,5) bisphosphate (PIP2), play an active role in regulating their functional properties. Although much work has been done to address the mechanism by which lipids regulate TRP channels in intact cells and excised patches, the physical interactions that govern channel regulation are still unknown. We report here on our efforts to establish high resolution control over membrane chemical composition and physical properties. We find that giant unilammelar vesicles can be synthesized with desired lipid compositions and subsequently studied using patch-clamp techniques. Reconstitution of functional TRP channels into these synthetic lipid membranes would provide a well-controlled experimental paradigm for studying the function and mechanism of channel-lipid interactions.