Crina M Nimigean

Weill Cornell Medical College, New York City, New York, United States

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Publications (37)190.61 Total impact

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    ABSTRACT: To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl(+)) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H(+)). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H(+) gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H(+) affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels' lipid bilayer environment or other regulatory processes.
    Biophysical Journal 03/2014; 106(5):1070-1078. · 3.67 Impact Factor
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    ABSTRACT: Cyclic nucleotide-gated (CNG) and hyperpolarization-activated and cyclic nucleotide-gated (HCN) ion channels play crucial roles in signal transduction in eukaryotes. The molecular mechanism by which ligand binding leads to channel opening remains poorly understood, however, due in part to the lack of a robust method for preparing sufficient amounts of purified, stable protein required for structural and biochemical characterization. To overcome this limitation, we designed a stable, highly-expressed chimeric ion channel consisting of the transmembrane domains of the well-characterized potassium channel KcsA and the cyclic nucleotide-binding domains (CNBDs) of the prokaryotic cyclic nucleotide-modulated channel, MloK1. This chimera demonstrates KcsA-like, pH-sensitive activity, which is modulated by cAMP, reminiscent of the dual modulation in HCN channels that display voltage-dependent activity that is also modulated by cAMP. Using this chimeric construct, we were able to measure, for the first time, the binding thermodynamics of cAMP to an intact cyclic nucleotide-modulated ion channel using isothermal titration calorimetry. The energetics of ligand binding to channels reconstituted in lipid bilayers are substantially different than those observed in detergent micelles, suggesting that the conformation of the chimera's transmembrane domain is sensitive to its (lipid or lipid-mimetic) environment, and that ligand binding induces conformational changes in the transmembrane domain. Nevertheless, because cAMP on its own does not activate these chimeric channels, cAMP binding likely has a smaller energetic contribution to gating than proton binding suggesting that there is only a small difference in cAMP binding energy between the open and closed states of the channel.
    Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
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    ABSTRACT: Cyclic nucleotide-modulated ion channels are important for signal transduction and pacemaking in eukaryotes. The molecular determinants of ligand gating in these channels are still unknown, mainly because of a lack of direct structural information. Here we report ligand-induced conformational changes in full-length MloK1, a cyclic nucleotide-modulated potassium channel from the bacterium Mesorhizobium loti, analysed by electron crystallography and atomic force microscopy. Upon cAMP binding, the cyclic nucleotide-binding domains move vertically towards the membrane, and directly contact the S1-S4 voltage sensor domains. This is accompanied by a significant shift and tilt of the voltage sensor domain helices. In both states, the inner pore-lining helices are in an 'open' conformation. We propose a mechanism in which ligand binding can favour pore opening via a direct interaction between the cyclic nucleotide-binding domains and voltage sensors. This offers a simple mechanistic hypothesis for the coupling between ligand gating and voltage sensing in eukaryotic HCN channels.
    Nature Communications 01/2014; 5:3106. · 10.02 Impact Factor
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    ABSTRACT: The bacterial potassium channel KcsA is gated open by the binding of protons to amino acids on the intracellular side of the channel. We have identified, via channel mutagenesis and x-ray crystallography, two pH-sensing amino acids and a set of nearby residues involved in molecular interactions that influence gating. We found that the minimal mutation of one histidine (H25) and one glutamate (E118) near the cytoplasmic gate completely abolished pH-dependent gating. Mutation of nearby residues either alone or in pairs altered the channel's response to pH. In addition, mutations of certain pairs of residues dramatically increased the energy barriers between the closed and open states. We proposed a Monod-Wyman-Changeux model for proton binding and pH-dependent gating in KcsA, where H25 is a "strong" sensor displaying a large shift in pKa between closed and open states, and E118 is a "weak" pH sensor. Modifying model parameters that are involved in either the intrinsic gating equilibrium or the pKa values of the pH-sensing residues was sufficient to capture the effects of all mutations.
    The Journal of General Physiology 11/2013; · 4.73 Impact Factor
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    ABSTRACT: We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.
    Protein Expression and Purification 08/2013; · 1.43 Impact Factor
  • Biophysical Journal 01/2013; 104(2):542-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):373-. · 3.67 Impact Factor
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    David J Posson, Jason G McCoy, Crina M Nimigean
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    ABSTRACT: Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. Blockers bind in an aqueous cavity between two putative gates: an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location-an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.
    Nature Structural & Molecular Biology 12/2012; · 11.90 Impact Factor
  • David J. Posson, Crina Nimigean
    Biophysical Journal 01/2012; 102(3):690-. · 3.67 Impact Factor
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    Jason G McCoy, Crina M Nimigean
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    ABSTRACT: Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the "selectivity filter". The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. This article is part of a Special Issue entitled: Membrane protein structure and function.
    Biochimica et Biophysica Acta 09/2011; 1818(2):272-85. · 4.66 Impact Factor
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    Crina M Nimigean, Toby W Allen
    The Journal of General Physiology 05/2011; 137(5):405-13. · 4.73 Impact Factor
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    ABSTRACT: Structures of the prokaryotic K(+) channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K(+) channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K(+) selectivity. In E71A channels, Na(+) permeates at higher rates as seen with and flux measurements and analysis of intracellular Na(+) block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K(+), but a "flipped" conformation, that is also observed in high K(+), high Na(+), and even Na(+) only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K(+) selectivity. We propose a molecular mechanism by which inactivation and K(+) selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.
    Proceedings of the National Academy of Sciences 03/2011; 108(13):5272-7. · 9.81 Impact Factor
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    Sourabh Banerjee, Crina M Nimigean
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    ABSTRACT: Discoidal lipoproteins are a novel class of nanoparticles for studying membrane proteins (MPs) in a soluble, native lipid environment, using assays that have not been traditionally applied to transmembrane proteins. Here, we report the successful delivery of an ion channel from these particles, called nanoscale apolipoprotein-bound bilayers (NABBs), to a distinct, continuous lipid bilayer that will allow both ensemble assays, made possible by the soluble NABB platform, and single-molecule assays, to be performed from the same biochemical preparation. We optimized the incorporation and verified the homogeneity of NABBs containing a prototypical potassium channel, KcsA. We also evaluated the transfer of KcsA from the NABBs to lipid bilayers using single-channel electrophysiology and found that the functional properties of the channel remained intact. NABBs containing KcsA were stable, homogeneous, and able to spontaneously deliver the channel to black lipid membranes without measurably affecting the electrical properties of the bilayer. Our results are the first to demonstrate the transfer of a MP from NABBs to a different lipid bilayer without involving vesicle fusion.
    The Journal of General Physiology 02/2011; 137(2):217-23. · 4.73 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Sourabh Banerjee, Crina M. Nimigean
    Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
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    Mikio Tanabe, Crina M Nimigean, T M Iverson
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    ABSTRACT: PorB is the second most prevalent outer membrane protein in Neisseria meningitidis. PorB is required for neisserial pathogenesis and can elicit a Toll-like receptor mediated host immune response. Here, the x-ray crystal structure of PorB has been determined to 2.3 A resolution. Structural analysis and cocrystallization studies identify three putative solute translocation pathways through the channel pore: One pathway transports anions nonselectively, one transports cations nonselectively, and one facilitates the specific uptake of sugars. During infection, PorB likely binds host mitochondrial ATP, and cocrystallization with the ATP analog AMP-PNP suggests that binding of nucleotides regulates these translocation pathways both by partial occlusion of the pore and by restricting the motion of a putative voltage gating loop. PorB is located on the surface of N. meningitidis and can be recognized by receptors of the host innate immune system. Features of PorB suggest that Toll-like receptor mediated recognition outer membrane proteins may be initiated with a nonspecific electrostatic attraction.
    Proceedings of the National Academy of Sciences 03/2010; 107(15):6811-6. · 9.81 Impact Factor
  • David J. Posson, Crina M. Nimigean
    Biophysical Journal 01/2010; 98. · 3.67 Impact Factor
  • Ameer N. Thompson, Crina M. Nimigean
    Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
  • Sourabh Banerjee, Crina M. Nimigean
    Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: Potassium channels allow K(+) ions to diffuse through their pores while preventing smaller Na(+) ions from permeating. Discrimination between these similar, abundant ions enables these proteins to control electrical and chemical activity in all organisms. Selection occurs at the narrow selectivity filter containing structurally identified K(+) binding sites. Selectivity is thought to arise because smaller ions such as Na(+) do not bind to these K(+) sites in a thermodynamically favorable way. Using the model K(+) channel KcsA, we examined how intracellular Na(+) and Li(+) interact with the pore and the permeant ions using electrophysiology, molecular dynamics simulations and X-ray crystallography. Our results suggest that these small cations have a separate binding site within the K(+) selectivity filter. We propose that selective permeation from the intracellular side primarily results from a large energy barrier blocking filter entry for Na(+) and Li(+) in the presence of K(+), not from a difference of binding affinity between ions.
    Nature Structural & Molecular Biology 11/2009; 16(12):1317-24. · 11.90 Impact Factor

Publication Stats

626 Citations
190.61 Total Impact Points

Institutions

  • 2008–2014
    • Weill Cornell Medical College
      • Department of Anesthesiology
      New York City, New York, United States
  • 2011
    • Washington University in St. Louis
      • Department of Cell Biology and Physiology
      Saint Louis, MO, United States
    • Cornell University
      • Department of Anesthesiology
      Ithaca, NY, United States
  • 2006–2007
    • University of California, Davis
      • • Department of Molecular and Cellular Biology
      • • Department of Physiology and Membrane Biology
      Davis, CA, United States
  • 2002–2004
    • Brandeis University
      • Department of Biochemistry
      Waltham, MA, United States
  • 1999–2000
    • University of Miami Miller School of Medicine
      • Department of Physiology and Biophysics
      Miami, FL, United States