Crystallographic Analyses of Ion Channels: Lessons and Challenges

Rees, Douglas C.; Chang, Geoffrey; Spencer, Robert H.

doi:10.1074/jbc.275.2.713

Cited by 45 publications

(31 citation statements)

References 49 publications

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“…Similarly, the discovery of high affinity and selective agents that can target mechanically gated channels will represent a major breakthrough for the field. The determination of the crystal structure of bacterial MS channels [7,21,139] has provided a rich environment for model building and testing, and a similar trajectory is predicted for the recently identified MS channel proteins in animal cells. A key question that these studies should answer is whether a unified set of principles can account for the stretch sensitivity of channels in both prokaryotes and eukaryotes [54,88].…”

Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Twenty odd years of stretch-sensitive channels

Hamill

2006

Pflugers Arch - Eur J Physiol

110

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After formation of the giga-seal, the membrane patch can be stimulated by hydrostatic or osmotic pressure gradients applied across the patch. This feature led to the discovery of stretch-sensitive or mechanosensitive (MS) channels, which are now known to be ubiquitously expressed in cells representative of all the living kingdoms. In addition to mechanosensation, MS channels have been implicated in many basic cell functions, including regulation of cell volume, shape, and motility. The successful cloning, overexpression, and crystallization of bacterial MS channel proteins combined with patch clamp and modeling studies have provided atomic insight into the working of these nanomachines. In particular, studies of MS channels have revealed new understanding of how the lipid bilayer modulates membrane protein function. Three major membrane protein families, transient receptor potential, 2 pore domain K + , and the epithelial Na + channels, have been shown to form MS channels in animal cells, and their polymodal activation embrace fields far beyond mechanosensitivity. The discovery of new drugs highly selective for MS channels ("mechanopharmaceutics") and the demonstration of MS channel involvement in several major human diseases ("mechanochannelopathies") provide added motivation for devising new techniques and approaches for studying MS channels.

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Section: Conclusion and Future Prospectsmentioning

confidence: 99%

Twenty odd years of stretch-sensitive channels

Hamill

2006

Pflugers Arch - Eur J Physiol

110

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“…Membrane proteins control a number of cellular signaling pathways and are targets of Ͼ50% of pharmaceutical drugs (11,12). Crystallization of membrane proteins remains challenging (13,14), because samples are often limited in quantity (15) and may be unstable over time. Also, an extensive range of crystallization conditions must be searched, including screening detergents used to solubilize membrane proteins (15)(16)(17).…”

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confidence: 99%

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Li¹,

Mustafi²,

Fu³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

226

259

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High-throughput screening and optimization experiments are critical to a number of fields, including chemistry and structural and molecular biology. The separation of these two steps may introduce false negatives and a time delay between initial screening and subsequent optimization. Although a hybrid method combining both steps may address these problems, miniaturization is required to minimize sample consumption. This article reports a ''hybrid'' droplet-based microfluidic approach that combines the steps of screening and optimization into one simple experiment and uses nanoliter-sized plugs to minimize sample consumption. Many distinct reagents were sequentially introduced as Ϸ140-nl plugs into a microfluidic device and combined with a substrate and a diluting buffer. Tests were conducted in Ϸ10-nl plugs containing different concentrations of a reagent. Methods were developed to form plugs of controlled concentrations, index concentrations, and incubate thousands of plugs inexpensively and without evaporation. To validate the hybrid method and demonstrate its applicability to challenging problems, crystallization of model membrane proteins and handling of solutions of detergents and viscous precipitants were demonstrated. By using 10 l of protein solution, Ϸ1,300 crystallization trials were set up within 20 min by one researcher. This method was compatible with growth, manipulation, and extraction of high-quality crystals of membrane proteins, demonstrated by obtaining high-resolution diffraction images and solving a crystal structure. This robust method requires inexpensive equipment and supplies, should be especially suitable for use in individual laboratories, and could find applications in a number of areas that require chemical, biochemical, and biological screening and optimization.droplets ͉ plugs ͉ protein structure ͉ high-throughput ͉ miniaturization T his work reports a ''hybrid'' microfluidic approach that uses nanoliter plugs to perform screening and optimization simultaneously in the same experiment. To validate this method using a challenging problem, we demonstrate its compatibility with crystallization of membrane proteins. Small-scale screening and optimization experiments are important for biological assays, chemical screening, and protein crystallization (1-3). Screening and optimization are usually carried out sequentially. In the case of protein crystallization, random sparse matrix screening initially identifies the precipitants that may lead to crystallization. Subsequent gradient optimization establishes concentrations of these precipitants that lead to diffractionquality crystals (4). Combining screening and optimization steps into a single hybrid experiment would eliminate the need to wait for the outcome of the initial screen before carrying out subsequent optimizations. Furthermore, a hybrid experiment would reduce the false negatives (5) associated with screens performed at a single concentration. The hybrid experiment could also be more conclusive, because a single batch of the s...

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“…Concerning the MscL system, recent work has attempted to rationalize the extensive functional studies on Escherichia coli MscL (Eco-MscL) 1 in light of the crystal structure, which was obtained for the Mycobacterium tuberculosis homologue (Tb-MscL) (3)(4)(5)(6)(7). Additionally, several different models for channel opening have been proposed by considering E. coli gain of function (GOF) mutations in light of the M. tuberculosis crystal structure (1,3,8).…”

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confidence: 99%

Comparing and Contrasting Escherichia coliand Mycobacterium tuberculosis Mechanosensitive Channels (MscL)

Maurer¹,

Elmore²,

Lester³

et al. 2000

Journal of Biological Chemistry

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Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. The recent crystal structures of two bacterial ion channels, the KcsA potassium channel and the mechanosensitive channel MscL, provide unique opportunities to study ion channel structure-function relationships (1, 2). Concerning the MscL system, recent work has attempted to rationalize the extensive functional studies on Escherichia coli MscL (Eco-MscL) 1 (17). This difference may result from protein structural differences, a difference in interactions with lipids, or both.Sequence alignment is essential to map previously studied E. coli GOF mutations onto the M. tuberculosis MscL sequence. In this work we report an optimal sequence alignment of 35 MscL homologues and an analysis of regions of conservation and variability. Consistent with previous studies, we find greater conservation in the transmembrane regions than in the loop or intracellular regions. Interestingly, the various channels clearly fall into subfamilies based on sequence similarity, with Eco-MscL and Tb-MscL in different subfamilies.By using the optimal alignment, we have prepared Tb-MscL analogues of several critical Eco-MscL GOF mutations (Fig. 1A). Perhaps surprisingly, we find that several well established Eco-MscL GOF mutants do not translate to the Tb-MscL system. We also directly evaluate a predicted intersubunit hydrogen bond in the Tb-MscL crystal structure (Fig. 1B). Crosslinking studies establish that these residues are indeed close in the reconstituted channel and firmly establish the pentameric nature of the channel. Mutations of this pair generally lead to GOF mutants, suggesting an important functional role for this specific region of the channel. Interestingly, no analogous interaction is apparent in the Eco-MscL alignment. Our results indicate that the functional studies performed on the Eco-MscL channel may not map directly onto the Tb-MscL crystal structure. MATERIALS AND METHODSSequence Analysis-Multiple sequence alignments were obtained using alignment of multiple sequences (AMPS) (20,21), and consensus group analysis was performed using multiple EM for motif elicitation (MEME) (22,23). The alignment was broken into regions, extracellular

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Crystallographic Analyses of Ion Channels: Lessons and Challenges

Cited by 45 publications

References 49 publications

Twenty odd years of stretch-sensitive channels

Twenty odd years of stretch-sensitive channels

Nanoliter microfluidic hybrid method for simultaneous screening and optimization validated with crystallization of membrane proteins

Comparing and Contrasting Escherichia coliand Mycobacterium tuberculosis Mechanosensitive Channels (MscL)

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