Structures of TRPM5 channel elucidate mechanism of activation and inhibition

The Ca2+-activated TRPM5 channel plays an essential role in the perception of sweet, bitter, and umami stimuli in type II taste cells and in insulin secretion by pancreatic beta cells. Interestingly, the voltage dependence of TRPM5 in taste bud cells depends on the intracellular Ca2+ concentration, yet the mechanism remains elusive. Here we report cryo-electron microscopy structures of the zebrafish TRPM5 in an apo closed state, a Ca2+-bound open state, and an antagonist-bound inhibited state, at resolutions up to 2.3 Angstrom. We defined two novel ligand binding sites: a Ca2+ binding site (CaICD) in the intracellular domain (ICD), and an antagonist binding site in the transmembrane domain (TMD) for a drug (NDNA) that regulates insulin and GLP-1 release. The CaICD site is unique to TRPM5 and has two roles: shifting the voltage dependence toward negative membrane potential, and promoting Ca2+ binding to the CaTMD site that is conserved throughout Ca2+-sensitive TRPM channels. Replacing glutamate 337 in the CaICD site with an alanine not only abolished Ca2+ binding to CaICD but also reduced Ca2+ binding affinity to CaTMD, suggesting a cooperativity between the two sites. We have defined mechanisms underlying channel activation and inhibition. Conformational changes initialized from both Ca2+ sites, 70 Angstrom apart, are propagated to the ICD-TMD interface and cooperatively open the ion-conducting pore. The antagonist NDNA wedges into the space between the S1-S4 domain and pore domain, stabilizing the TMD in an apo-like closed state. Our results lay the foundation for understanding the voltage-dependent TRPM channels and developing new therapeutic agents to treat metabolic disorders.

Effect of Membrane Potential on Entry of Lactoferricin B-Derived 6-Residue Antimicrobial Peptide into Single Escherichia coli Cells and Lipid Vesicles

ABSTRACT An antimicrobial peptide (AMP) derived from lactoferricin B, LfcinB(4–9) (RRWQWR), and lissamine rhodamine B red-labeled peptide [Rh-LfcinB(4–9)] exhibit strong antimicrobial activities, and they can enter Escherichia coli cells without damaging the cell membranes. Thus, these peptides are cell-penetrating peptide (CPP)-type AMPs. In this study, to elucidate the effect of the membrane potential (Δφ) on the action of the CPP-type AMP Rh-LfcinB(4–9), we investigated the interactions of Rh-LfcinB(4–9) with single E. coli cells and spheroplasts containing calcein in the cytosol using confocal laser scanning microscopy. At low peptide concentrations, Rh-LfcinB(4–9) entered the cytosol of single E. coli cells and spheroplasts without damaging the cell membranes, and the H+ ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) suppressed its entry. Studies using the time-kill method indicate that these low concentrations of peptide exhibit antimicrobial activity, but CCCP inhibits this activity. Next, we investigated the effect of Δφ on the interaction of Rh-LfcinB(4–9) with single giant unilamellar vesicles (GUVs) comprising E. coli polar lipid extracts and containing a fluorescent probe, Alexa Fluor 647 hydrazide. At low concentrations (0.2 to 0.5 μM), Rh-LfcinB(4–9) showed significant entry into the single GUV lumen without pore formation in the presence of Δφ. The fraction of entry of the peptide increased with increasing negative membrane potential, indicating that the rate of peptide entry into the GUV lumen increased with increasing negative membrane potential. These results indicate that Δφ enhances the entry of Rh-LfcinB(4–9) into single E. coli cells, spheroplasts, and GUVs and its antimicrobial activity. IMPORTANCE Bacterial cells have a membrane potential (Δφ), but the effect of Δφ on the action of cell-penetrating peptide-type antimicrobial peptides (AMPs) is not clear. Here, we investigated the effect of Δφ on the action of a fluorescent probe-labeled AMP derived from lactoferricin B, Rh-LfcinB(4–9). At low peptide concentrations, Rh-LfcinB(4–9) enters the cytosol of Escherichia coli cells and spheroplasts without damaging their cell membrane, but a protonophore suppresses this entry and its antimicrobial activity. The rate of entry of Rh-LfcinB(4–9) into the giant unilamellar vesicles (GUVs) comprising E. coli lipids without pore formation increases with increasing Δφ. These results indicate that Δφ enhances the antimicrobial activity of Rh-LfcinB(4–9) and, hence, LfcinB(4–9) by increasing the rate of their entry into the cytosol.

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Small Molecule, Permeable, Internally Redistributing for Inner Membrane Targeting Rhodamine Voltage Reporter

<p>Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨ_m) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨ_m indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨ_m dynamics. Here, we report the first example of a fluorescent ΔΨ_m reporter that does not rely on ΔΨ_m-dependent accumulation. We re-directed the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxy methyl (AM) ester. Once within mitochondria, esterases remove the AM-ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨ_m. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨ_m induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca²⁺, plasma membrane potential, and reversible ΔΨ_m dynamics.</p>

Imaging Reversible Mitochondrial Membrane Potential Dynamics with a Small Molecule, Permeable, Internally Redistributing for Inner Membrane Targeting Rhodamine Voltage Reporter

<p>Mitochondria are the site of aerobic respiration, producing ATP via oxidative phosphorylation as protons flow down their electrochemical gradient through ATP synthase. This negative membrane potential across the inner mitochondrial membrane (ΔΨ_m) represents a fundamental biophysical parameter central to cellular life. Traditional, electrode-based methods for recording membrane potential are impossible to implement on mitochondria within intact cells. Fluorescent ΔΨ_m indicators based on cationic, lipophilic dyes are a common alternative, but these indicators are complicated by concentration-dependent artifacts and the requirement to maintain dye in the extracellular solution to visualize reversible ΔΨ_m dynamics. Here, we report the first example of a fluorescent ΔΨ_m reporter that does not rely on ΔΨ_m-dependent accumulation. We re-directed the localization of a photoinduced electron transfer (PeT)-based indicator, Rhodamine Voltage Reporter (RhoVR), to mitochondria by masking the carboxylate of RhoVR 1 as an acetoxy methyl (AM) ester. Once within mitochondria, esterases remove the AM-ester, trapping RhoVR inside of the mitochondrial matrix, where it can incorporate within the inner membrane and reversibly report on changes in ΔΨ_m. We show that this Small molecule, Permeable, Internally Redistributing for Inner membrane Targeting Rhodamine Voltage reporter, or SPIRIT RhoVR, localizes to mitochondria across a number of different cell lines and responds reversibly to changes in ΔΨ_m induced by exceptionally low concentrations of the uncoupler FCCP without the need for exogenous pools of dye (unlike traditional, accumulation-based rhodamine esters). SPIRIT RhoVR is compatible with multi-color imaging, enabling simultaneous, real-time observation of cytosolic Ca²⁺, plasma membrane potential, and reversible ΔΨ_m dynamics.</p>

Conformational transitions of the sodium-dependent sugar transporter, vSGLT

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron–electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

Amadori-glycated phosphatidylethanolamine enhances the physical stability and selective targeting ability of liposomes

Liposomes consisting of 100% phosphatidylcholine exhibit poor membrane fusion, cellular uptake and selective targeting capacities. To overcome these limitations, we used Amadori-glycated phosphatidylethanolamine, which is universally present in animals and commonly consumed in foods. We found that liposomes containing Amadori-glycated phosphatidylethanolamine exhibited significantly reduced negative membrane potential and demonstrated high cellular uptake.

Quantum Calculations Of A Large Section Of The Voltage Sensing Domain Of The Kv1.2 Channel Show That Proton Transfer, Not S4 Motion, Provides The Gating Current

Quantum calculations on much of the voltage sensing domain (VSD) of the Kv1.2 potassium channel (pdb: 3Lut) have been carried out on a 904 atom subset of the VSD, plus 24 water molecules (total, 976 atoms). Those side chains that point away from the center of the VSD were truncated; in all calculations, S1,S2,S3 end atoms were fixed; in some calculations, S4 end atoms were also fixed, while in other calculations they were free. After optimization at Hartree-Fock level, single point calculations of energy were carried out using DFT (B3LYP/6-31G**), allowing accurate energies of different cases to be compared. Open conformations (i.e., zero or positive membrane potentials) are consistent with the known X-ray structure of the open state when the salt bridges in the VSD are not ionized (H+ on the acid), whether S4 end atoms were fixed or free (closer fixed than free). Based on these calculations, the backbone of the S4 segment, free or not, moves no more than 2.5 Å upon switching from positive to negative membrane potential, and the movement is in the wrong direction for closing the channel. This leaves H+ motion as the principal component of gating current. Groups of 3-5 side chains are important for proton transport, based on the calculations. Our calculations point to a pair of steps in which a proton transfers from a tyrosine, Y266, through arginine (R300), to a glutamate (E183); this would account for approximately 20-25% of the gating charge. The calculated charges on each arginine and glutamate are appreciably less than one. Groupings of five amino acids appear to exchange a proton; the group is bounded by the conserved aromatic F233. Dipole rotations appear to also contribute. Alternate interpretations of experiments usually understood in terms of the standard model are shown to be plausible.

The expression, regulation, and function of Kir4.1 (Kcnj10) in the mammalian kidney

Kir4.1 is an inwardly rectifying potassium (K+) channel and is expressed in the brain, inner ear, and kidney. In the kidney, Kir4.1 is expressed in the basolateral membrane of the late thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule (CNT)/cortical collecting duct (CCD). It plays a role in K+ recycling across the basolateral membrane in corresponding nephron segments and in generating negative membrane potential. The renal phenotypes of the loss-function mutations of Kir4.1 include mild salt wasting, hypomagnesemia, hypokalemia, and metabolic alkalosis, suggesting that the disruption of Kir4.1 mainly impairs the transport in the DCT. Patch-clamp experiments and immunostaining demonstrate that Kir4.1 plays a predominant role in determining the basolateral K+ conductance in the DCT. However, the function of Kir4.1 in the TAL and CNT/CCD is not essential, because K+ channels other than Kir4.1 are also expressed. The downregulation of Kir4.1 in the DCT reduced basolateral chloride (Cl−) conductance, suppressed the expression of ste20 proline-alanine-rich kinase (SPAK), and decreased Na-Cl cotransporter (NCC) expression and activity. This suggests that Kir4.1 regulates NCC expression by the modulation of the Cl−-sensitive with-no-lysine kinase–SPAK pathway.

Biochemical Characterization of the C4-Dicarboxylate Transporter DctA from Bacillus subtilis

ABSTRACT Bacterial secondary transporters of the DctA family mediate ion-coupled uptake of C4-dicarboxylates. Here, we have expressed the DctA homologue from Bacillus subtilis in the Gram-positive bacterium Lactococcus lactis. Transport of dicarboxylates in vitro in isolated membrane vesicles was assayed. We determined the substrate specificity, the type of cotransported ions, the electrogenic nature of transport, and the pH and temperature dependence patterns. DctA was found to catalyze proton-coupled symport of the four C4-dicarboxylates from the Krebs cycle (succinate, fumurate, malate, and oxaloacetate) but not of other mono- and dicarboxylates. Because (i) succinate-proton symport was electrogenic (stimulated by an internal negative membrane potential) and (ii) the divalent anionic form of succinate was recognized by DctA, at least three protons must be cotransported with succinate. The results were interpreted in the light of the crystal structure of the homologous aspartate transporter GltPh from Pyrococcus horikoshii.

Amphiphilic Blockers Punch through a Mutant CLC-0 Pore

Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl−, the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than −80 mV, a phenomenon similar to the blocker “punch-through” shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at −80 mV in symmetrical 140 mM Cl−, and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl− reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential. That the shift of the CPA-dissociation rate follows the Cl− gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl− reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.