Valeria Vásquez

Molecular driving forces determining potassium channel slow inactivation

K+ channels undergo a time-dependent slow inactivation process that plays a key role in modulating cellular excitability. Here we show that in the prokaryotic proton-gated K+ channel KcsA, the number and strength of hydrogen bonds between residues in the selectivity filter and its adjacent pore helix determine the rate and extent of C-type inactivation. Upon channel activation, the interaction between residues at positions Glu71 and Asp80 promotes filter constriction parallel to the permeation pathway, which affects K+-binding sites and presumably abrogates ion conduction. Coupling between these two positions results in a quantitative correlation between their interaction strength and the stability of the inactivated state. Engineering of these interactions in the eukaryotic voltage-dependent K+ channel Kv1.2 suggests that a similar mechanistic principle applies to other K+ channels. These observations provide a plausible physical framework for understanding C-type inactivation in K+ channels.

Crystal structure of full-length KcsA in its closed conformation

KcsA is a proton-activated, voltage-modulated K+ channel that has served as the archetype pore domain in the Kv channel superfamily. Here, we have used synthetic antigen-binding fragments (Fabs) as crystallographic chaperones to determine the structure of full-length KcsA at 3.8 Å, as well as that of its isolated C-terminal domain at 2.6 Å. The structure of the full-length KcsA–Fab complex reveals a well-defined, 4-helix bundle that projects ≈70 Å toward the cytoplasm. This bundle promotes a ≈15° bending in the inner bundle gate, tightening its diameter and shifting the narrowest point 2 turns of helix below. Functional analysis of the full-length KcsA–Fab complex suggests that the C-terminal bundle remains whole during gating. We suggest that this structure likely represents the physiologically relevant closed conformation of KcsA.

A structural mechanism for MscS gating in lipid bilayers.

The mechanosensitive channel of small conductance (MscS) is a key determinant in the prokaryotic response to osmotic challenges. We determined the structural rearrangements associated with MscS activation in membranes, using functorial measurements, electron paramagnetic resonance spectroscopy, and computational analyses. MscS was trapped in its open conformation after the transbilayer pressure profile was modified through the asymmetric incorporation of lysophospholipids. The transition from the closed to the open state is accompanied by the downward tilting of the transmembrane TM1-TM2 hairpin and by the expansion, tilt, and rotation of the TM3 helices. These movements expand the permeation pathway, leading to an increase in accessibility to water around TM3. Our open MscS model is compatible with single-channel conductance measurements and supports the notion that helix tilting is associated with efficient pore widening in mechanosensitive channels.

Three Dimensional Architecture of Membrane-Embedded MscS in the Closed Conformation

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH(2)-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.

Studying mechanosensitive ion channels using liposomes

Mechanosensitive (MS) ion channels are the primary molecular transducers of mechanical force into electrical and/or chemical intracellular signals in living cells. They have been implicated in innumerable mechanosensory physiological processes including touch and pain sensation, hearing, blood pressure control, micturition, cell volume regulation, tissue growth, or cellular turgor control. Much of what we know about the basic physical principles underlying the conversion of mechanical force acting upon membranes of living cells into conformational changes of MS channels comes from studies of MS channels reconstituted into artificial liposomes. Using bacterial MS channels as a model, we have shown by reconstituting these channels into liposomes that there is a close relationship between the physico-chemical properties of the lipid bilayer and structural dynamics bringing about the function of these channels.

Omega-3 Fatty Acids Modulate TRPV4 Function through Plasma Membrane Remodeling

Dietary consumption of ω-3 polyunsaturated fatty acids (PUFAs), present in fish oils, is known to improve the vascular response, but their molecular targets remain largely unknown. Activation of the TRPV4 channel has been implicated in endothelium-dependent vasorelaxation. Here, we studied the contribution of ω-3 PUFAs to TRPV4 function by precisely manipulating the fatty acid content in Caenorhabditis elegans. By genetically depriving the worms of PUFAs, we determined that the metabolism of ω-3 fatty acids is required for TRPV4 activity. Functional, lipid metabolome, and biophysical analyses demonstrated that ω-3 PUFAs enhance TRPV4 function in human endothelial cells and support the hypothesis that lipid metabolism and membrane remodeling regulate cell reactivity. We propose a model whereby the eicosanoids epoxide group location increases membrane fluidity and influences the endothelial cell response by increasing TRPV4 channel activity. ω-3 PUFA-like molecules might be viable antihypertensive agents for targeting TRPV4 to reduce systemic blood pressure.

Sensory Biology: It Takes Piezo2 to Tango

A trio of papers has resolved an outstanding controversy regarding the function of Merkel cells and their afferent nerve fiber partners. Merkel cells sense mechanical stimuli (through Piezo2), fire action potentials, and are sufficient to activate downstream sensory neurons.

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

Arachidonic Acid is Essential for Normal Touch Sensation

Touch, proprioception, and blood pressure regulation rely on mechanoreceptor neurons and mechano-electrical transduction (MeT) channels to convert mechanical cues into electrical signals. The protein partners that form native MeT channels are known only for a small group of mechanoreceptor neurons, including the touch receptor neurons responsible for gentle body touch in C. elegans. Such MeT channels are formed from at least four membrane proteins: two amiloride-sensitive sodium channel proteins (MEC-4 and MEC-10) and two putative lipid binding protein subunits (MEC-2 and MEC-6). It remains unknown how touch activates MeT channels, but interactions between the channel and the lipid membrane are likely to be essential. To learn more about interactions between MeT channels and specific lipids, we utilized C. elegans strains deficient in the synthesis of long-chain, poly-unsaturated fatty acids (PUFAs) and identified arachidonic acid (AA) as the sole PUFA required for full touch sensitivity. Touch receptor neurons retain normal morphology in PUFA-deficient mutants and normal touch sensitivity can be restored by exogenous PUFAs. We will present evidence garnered from behavioral and optogenetic studies suggesting that long-chain PUFAs are needed to activate native MeT channels, but dispensable for subsequent events linking sensory signaling in touch receptor neurons to behavior. Our data suggest that AA and its non-metabolizable analog ETYA directly modulate the activity of the MeT channel.

A Functional Analysis of NaK at the Single Channel Level

NaK is a non-selective monovalent cation channel from Bacillus cereus. Despite being unable to discriminate between Na+ and K+, NaK shows high sequence similarity to other K+ channels. Based on recently solved crystal structures in the closed and putatively open state, NaK exhibits an overall architecture similar to that found in the pore domain of tetrameric K+ channels. Rb+ influx studies suggest the channel conducts cations, however net flux is unusually low for a channel. The absence of electrophysiological data from NaK precludes significant understanding of its functional behavior. Using a random mutagenesis approach together with a K+ transport based screen, we have identified gain-of-function mutants in an attempt to develop a system for electrophysiological studies. One of these purified and reconstituted mutants was further studied by liposome patch-clamp. The channel displays non-selective conductances at 25 and 91 pS and is characterized by a low probability spiking behavior. In addition, we show that NaK undergoes a voltage-dependent inactivation process, which is functionally similar to that seen in K+ channels. This inactivation may contribute to the low flux of Rb+ through NaK. Our functional characterization, along with the known crystal structures, now allows us to use NaK as a model system to further investigate structure-function correlations in non-selective channels and related selectivity filters.