A facile approach for incorporating tyrosine esters to probe ion-binding sites and backbone hydrogen bonds.

Amide-to-ester substitutions are used to study the role of the amide bonds of the protein backbone in protein structure, function, and folding. An amber suppressor tRNA/synthetase pair has been reported for incorporation of p-hydroxy-phenyl-L-lactic acid (HPLA), thereby introducing ester substitution at tyrosine residues. However, the application of this approach was limited due to the low yields of the modified proteins and the high cost of HPLA. Here we report the in vivo generation of HPLA from the significantly cheaper phenyl-L-lactic acid. We also construct an optimized plasmid with the HPLA suppressor tRNA/synthetase pair that provides higher yields of the modified proteins. The combination of the new plasmid and the in-situ generation of HPLA provides a facile and economical approach for introducing tyrosine ester substitutions. We demonstrate the utility of this approach by introducing tyrosine ester substitutions into the K+ channel KcsA and the integral membrane enzyme GlpG. We introduce the tyrosine ester in the selectivity filter of the M96V mutant of the KcsA to probe the role of the second ion binding site in the conformation of the selectivity filter and the process of inactivation. We use tyrosine ester substitutions in GlpG to perturb backbone H-bonds to investigate the contribution of these H-bonds to membrane protein stability. We anticipate that the approach developed in this study will facilitate further investigations using tyrosine ester substitutions.

Structural basis for C-type inactivation in a Shaker family voltage-gated K+ channel.

C-type inactivation is a process by which ion flux through a voltage-gated K+ (Kv) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes has not been resolved. Here, we report the crystal structure of the Kv1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K+ occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.

Structures of Gating Intermediates in a K+ channel.

Regulation of ion conduction through the pore of a K+ channel takes place through the coordinated action of the activation gate at the bundle crossing of the inner helices and the inactivation gate located at the selectivity filter. The mechanism of allosteric coupling of these gates is of key interest. Here we report new insights into this allosteric coupling mechanism from studies on a W67F mutant of the KcsA channel. W67 is in the pore helix and is highly conserved in K+ channels. The KcsA W67F channel shows severely reduced inactivation and an enhanced rate of activation. We use continuous wave EPR spectroscopy to establish that the KcsA W67F channel shows an altered pH dependence of activation. Structural studies on the W67F channel provide the structures of two intermediate states: a pre- open state and a pre-inactivated state of the KcsA channel. These structures highlight key nodes in the allosteric pathway. The structure of the KcsA W67F channel with the activation gate open shows altered ion occupancy at the second ion binding site (S2) in the selectivity filter. This finding in combination with previous studies strongly support a requirement for ion occupancy at the S2 site for the channel to inactivate.

TRPV1, TRPA1, and TRPM8 are expressed in axon terminals in the cornea: TRPV1 axons contain CGRP and secretogranin II; TRPA1 axons contain secretogranin 3.

Purpose: The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea. Methods: Immunohistochemistry (IHC) was performed using wholemount and cryostat tissue preparations of mouse and monkey corneas. The expression patterns of TRPV1 and TRPA1 were determined using specific antisera, and further colocalization was performed with antibodies directed against calcitonin-related gene protein (CGRP), neurofilament protein NF200, and the secretogranins ScgII and SCG3. The expression of TRPM8 was determined using corneas from mice expressing EGFP under the direction of a TRPM8 promoter (TRPM8EGFP mice). Laser scanning confocal microscopy and image analysis were performed. Results: In the mouse cornea, TRPV1 and TRPM8 were expressed in distinct populations of small diameter C fibers extending to the corneal surface and ending either as simple or ramifying terminals, or in the case of TRPM8, as complex terminals. TRPA1 was expressed in large-diameter NF200-positive Aδ axons. TRPV1 and TRPA1 appeared to localize to separate intracellular vesicular structures and were primarily found in axons containing components of large dense vesicles with TRPV1 colocalizing with CGRP and ScgII, and TRPA1 colocalizing with SCG3. Monkey corneas showed similar colocalization of CGRP and TRPV1 on small-diameter axons extending to the epithelial surface. Conclusions: The mouse cornea is abundant in sensory neurons expressing TRPV1, TRPM8, and TRPA1, and provides an accessible tissue source for implementing a live tissue preparation useful for further exploration of the molecular mechanisms of hyperalgesia. This study showed that surprisingly, these TRP channels localize to separate neurons in the mouse cornea and likely have unique physiological functions. The similar TRPV1 expression pattern we observed in the mouse and monkey corneas suggests that mice provide a reasonable initial model for understanding the role of these ion channels in higher mammalian corneal physiology.

TRPV1, TRPA1, and TRPM8 are expressed in axon terminals in the cornea: TRPV1 axons contain CGRP and secretogranin II; TRPA1 axons contain secretogranin 3.

Purpose: The cornea is highly enriched in sensory neurons expressing the thermal TRP channels TRPV1, TRPA1, and TRPM8, and is an accessible tissue for study and experimental manipulation. The aim of this work was to provide a concise characterization of the expression patterns of various TRP channels and vesicular proteins in the mammalian cornea. Methods: Immunohistochemistry (IHC) was performed using wholemount and cryostat tissue preparations of mouse and monkey corneas. The expression patterns of TRPV1 and TRPA1 were determined using specific antisera, and further colocalization was performed with antibodies directed against calcitonin-related gene protein (CGRP), neurofilament protein NF200, and the secretogranins ScgII and SCG3. The expression of TRPM8 was determined using corneas from mice expressing EGFP under the direction of a TRPM8 promoter (TRPM8EGFP mice). Laser scanning confocal microscopy and image analysis were performed. Results: In the mouse cornea, TRPV1 and TRPM8 were expressed in distinct populations of small diameter C fibers extending to the corneal surface and ending either as simple or ramifying terminals, or in the case of TRPM8, as complex terminals. TRPA1 was expressed in large-diameter NF200-positive Aδ axons. TRPV1 and TRPA1 appeared to localize to separate intracellular vesicular structures and were primarily found in axons containing components of large dense vesicles with TRPV1 colocalizing with CGRP and ScgII, and TRPA1 colocalizing with SCG3. Monkey corneas showed similar colocalization of CGRP and TRPV1 on small-diameter axons extending to the epithelial surface. Conclusions: The mouse cornea is abundant in sensory neurons expressing TRPV1, TRPM8, and TRPA1, and provides an accessible tissue source for implementing a live tissue preparation useful for further exploration of the molecular mechanisms of hyperalgesia. This study showed that surprisingly, these TRP channels localize to separate neurons in the mouse cornea and likely have unique physiological functions. The similar TRPV1 expression pattern we observed in the mouse and monkey corneas suggests that mice provide a reasonable initial model for understanding the role of these ion channels in higher mammalian corneal physiology.

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor.

Membrane proteins are universal signal decoders. The helical transmembrane segments of these proteins play central roles in sensory transduction, yet the mechanistic contributions of secondary structure remain unresolved. To investigate the role of main-chain hydrogen bonding on transmembrane function, we encoded amide-to-ester substitutions at sites throughout the S4 voltage-sensing segment of Shaker potassium channels, a region that undergoes rapid, voltage-driven movement during channel gating. Functional measurements of ester-harboring channels highlight a transitional region between α-helical and 310 segments where hydrogen bond removal is particularly disruptive to voltage-gating. Simulations of an active voltage sensor reveal that this region features a dynamic hydrogen bonding pattern and that its helical structure is reliant upon amide support. Overall, the data highlight the specialized role of main-chain chemistry in the mechanism of voltage-sensing; other catalytic transmembrane segments may enlist similar strategies in signal transduction mechanisms.

A facile approach for the in vitro assembly of multimeric membrane transport proteins.

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.

Patch-Clamp Recordings of the KcsA K+ Channel in Unilamellar Blisters.

Patch-clamp electrophysiology is the standard technique used for the high-resolution functional measurements on ion channels. While studies using patch clamp are commonly carried out following ion channel expression in a heterologous system such as Xenopus oocytes or tissue culture cells, these studies can also be carried out using ion channels reconstituted into lipid vesicles. In this chapter, we describe the methodology for reconstituting ion channels into liposomes and the procedure for the generation of unilamellar blisters from these liposomes that are suitable for patch clamp. Here, we focus on the bacterial K+ channel KcsA, although the methodologies described in this chapter should be applicable for the functional analysis of other ion channels.

Probing the Effects of Gating on the Ion Occupancy of the K+ Channel Selectivity Filter Using Two-Dimensional Infrared Spectroscopy.

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.

Instantaneous ion configurations in the K+ ion channel selectivity filter revealed by 2D IR spectroscopy.

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein. We demonstrate the feasibility of using this approach for the K(+) channels KcsA and MVP and the amino acid transporter LeuT. We determine the crystal structure of the KcsA channel obtained by CFPS and in vitro folding to show the structural similarity to the cellular expressed KcsA channel and to establish the feasibility of using this two-step approach for membrane protein production for structural studies. Our studies show that the correct folding of these membrane proteins with complex topologies can take place in vitro without the involvement of the cellular machinery for membrane protein biogenesis. This indicates that the folding instructions for these complex membrane proteins are contained entirely within the protein sequence.

Individual Ion Binding Sites in the K(+) Channel Play Distinct Roles in C-type Inactivation and in Recovery from Inactivation.

The selectivity filter of K(+) channels contains four ion binding sites (S1-S4) and serves dual functions of discriminating K(+) from Na(+) and acting as a gate during C-type inactivation. C-type inactivation is modulated by ion binding to the selectivity filter sites, but the underlying mechanism is not known. Here we evaluate how the ion binding sites in the selectivity filter of the KcsA channel participate in C-type inactivation and in recovery from inactivation. We use unnatural amide-to-ester substitutions in the protein backbone to manipulate the S1-S3 sites and a side-chain substitution to perturb the S4 site. We develop an improved semisynthetic approach for generating these amide-to-ester substitutions in the selectivity filter. Our combined electrophysiological and X-ray crystallographic analysis of the selectivity filter mutants show that the ion binding sites play specific roles during inactivation and provide insights into the structural changes at the selectivity filter during C-type inactivation.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation. Together, these regions determine when the channel conducts ions. The K(+) channel from Streptomyces lividians (KcsA) undergoes an inactivation process that is functionally similar to KV channels, which has led to its use as a practical system to study inactivation. Crystal structures of KcsA channels with an open intracellular gate revealed a selectivity filter in a constricted conformation similar to the structure observed in closed KcsA containing only Na(+) or low [K(+)]. However, recent work using a semisynthetic channel that is unable to adopt a constricted filter but inactivates like WT channels challenges this idea. In this study, we measured the equilibrium ion-binding properties of channels with conductive, inactivated, and constricted filters using isothermal titration calorimetry (ITC). EPR spectroscopy was used to determine the state of the intracellular gate of the channel, which we found can depend on the presence or absence of a lipid bilayer. Overall, we discovered that K(+) ion binding to channels with an inactivated or conductive selectivity filter is different from K(+) ion binding to channels with a constricted filter, suggesting that the structures of these channels are different.

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels.

K(+) channels distinguish K(+) from Na(+) in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K(+) binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site.

Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.

C-type inactivation of K(+) channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K(+) channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K(+) or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K(+) channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d-Alanine. Protein semisynthesis was used to introduce d-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K(+) channel KvAP. For semisynthesis of the KvAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and KvAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.

Surprises from an unusual CLC homolog.

The chloride channel (CLC) family is distinctive in that some members are Cl(-) ion channels and others are Cl(-)/H(+) antiporters. The molecular mechanism that couples H(+) and Cl(-) transport in the antiporters remains unknown. Our characterization of a novel bacterial homolog from Citrobacter koseri, CLC-ck2, has yielded surprising discoveries about the requirements for both Cl(-) and H(+) transport in CLC proteins. First, even though CLC-ck2 lacks conserved amino acids near the Cl(-)-binding sites that are part of the CLC selectivity signature sequence, this protein catalyzes Cl(-) transport, albeit slowly. Ion selectivity in CLC-ck2 is similar to that in CLC-ec1, except that SO(4)(2-) strongly competes with Cl(-) uptake through CLC-ck2 but has no effect on CLC-ec1. Second, and even more surprisingly, CLC-ck2 is a Cl(-)/H(+) antiporter, even though it contains an isoleucine at the Glu(in) position that was previously thought to be a critical part of the H(+) pathway. CLC-ck2 is the first known antiporter that contains a nonpolar residue at this position. Introduction of a glutamate at the Glu(in) site in CLC-ck2 does not increase H(+) flux. Like other CLC antiporters, mutation of the external glutamate gate (Glu(ex)) in CLC-ck2 prevents H(+) flux. Hence, Glu(ex), but not Glu(in), is critical for H(+) permeation in CLC proteins.

A designed inhibitor of a CLC antiporter blocks function through a unique binding mode.

The lack of small-molecule inhibitors for anion-selective transporters and channels has impeded our understanding of the complex mechanisms that underlie ion passage. The ubiquitous CLC "Chloride Channel" family represents a unique target for biophysical and biochemical studies because its distinctive protein fold supports both passive chloride channels and secondary-active chloride-proton transporters. Here, we describe the synthesis and characterization of a specific small-molecule inhibitor directed against a CLC antiporter (ClC-ec1). This compound, 4,4'-octanamidostilbene-2,2'-disulfonate (OADS), inhibits ClC-ec1 with low micromolar affinity and has no specific effect on a CLC channel (ClC-1). Inhibition of ClC-ec1 occurs by binding to two distinct intracellular sites. The location of these sites and the lipid dependence of inhibition suggest potential mechanisms of action. This compound will empower research to elucidate differences between antiporter and channel mechanisms and to develop treatments for CLC-mediated disorders.

Measurement of k(on) without a rapid-mixing device.

We have recently designed a biochemistry laboratory experiment for the purpose of providing students an advanced experience with enzyme kinetics and the kinetics of binding. Bestatin, a well-known and commercially available general protease inhibitor, is a slow-binding inhibitor of aminopeptidase isolated from Aeromonas proteolytica. The binding is on a timescale slow enough for measurement without the use of a rapid-mixing device. Aminopeptidase inhibition is detected via a standard colorimetric assay with an inexpensive commercially available substrate. The binding of bestatin follows first order binding kinetics with a rate constant k(on) of 59 ± 5 M(-1) s(-1) . This aminopeptidase is well characterized with several crystal structures and a published K(i) , which students can then use to calculate the value for k(off) .

Discovery of potent CLC chloride channel inhibitors.

Anion-transport proteins are central to all of physiology, for processes ranging from regulating bone-density, muscle excitability, and blood pressure, to facilitating extreme-acid survival of pathogenic bacteria. 4,4-Diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) has been used as an anion-transport inhibitor for decades. In this study, we demonstrate that polythiourea products derived from DIDS hydrolysis inhibit three different CLC chloride-transport proteins, ClC-ec1, ClC-0, and ClC-Ka, more effectively than DIDS itself. The structures of the five major products were determined by NMR spectroscopy, mass spectrometry, and chemical synthesis. These compounds bind directly to the CLC proteins, as evidenced by the fact that inhibition of ClC-0 occurs only from the intracellular side and inhibition of ClC-Ka is prevented by the point mutation N68D. These polythioureas are the highest affinity inhibitors known for the CLCs and provide a new class of chemical probes for dissecting the molecular mechanisms of chloride transport.

The CLC 'chloride channel' family: revelations from prokaryotes.

Members of the CLC 'chloride channel' family play vital roles in a wide variety of physiological settings. Research on prokaryotic CLC homologues provided long-anticipated high-resolution structures as well as the unexpected discovery that some CLCs are not chloride channels, but rather are proton-chloride antiporters. Hence, CLCs encompass two functional classes of transport proteins once thought to be fundamentally different from one another. In this review, we discuss the structural features and molecular mechanisms of CLC channels and antiporters. We focus on ClC-0, the most thoroughly studied CLC channel, and ClC-ec1, the prokaryotic antiporter of known structure. We highlight some striking similarities between these CLCs and discuss compelling questions that remain to be addressed. Prokaryotic CLCs will undoubtedly continue to shed light upon this understudied family of proteins.