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•Human SGLT1 is a monosaccharide transporter for glucose and galactose.•Mutations resulted in the loss of glucose transport or the gain of fructose transport.•Two mutants showed ...substrate selectivity for vibrio SGLT and human SGLT4, respectively.•Modeling analyses provided understanding for the binding properties of monosaccharides.•This study proposed the structural basis for monosaccharide selection of SGLT family.
Sodium-glucose cotransporters (SGLTs) are responsible for sugar absorption in small intestine and renal tubule epithelial cells. These proteins have attracted clinical attention as a cause of malabsorption and as a target for diabetes drugs. Each SGLT isoform has strict selectivity for its monosaccharide substrate. Few studies have attempted to elucidate the structural basis of sugar selectivity by allowing generating SGLT mutants that bind substrates not normally transported or by reproducing the substrate specificity of other isoforms. In this study, we built a structural homology model for the substrate binding states of human SGLT1 (hSGLT1), which primarily transports glucose and galactose. We also performed electrophysiological analysis of hSGLT1 using various natural sugars and mutants. By mutating the K321 residue, which forms hydrophilic interactions in the sugar binding pocket, we induced mannose and allose transport. We also changed the glucose/galactose transport ratio, which reproduces the substrate specificity of the prokaryotic galactose transporter. By adding mutations one-by-one to the residues in the binding pocket, we were able to reproduce the substrate specificity of SGLT4, which transports fructose. This suggests that fructose, which exhibits various structures in equilibrium, binds to SGLT in a pyranose conformation. These results reveal one state of the structural basis that determines selective transport by SGLT. These findings will be useful for predicting the substrates of other glucose transporters and to design effective inhibitors.
Hv1 is a voltage-gated proton-selective channel that plays critical parts in host defense, sperm motility, and cancer progression. Hv1 contains a conserved voltage-sensor domain (VSD) that is shared ...by a large family of voltage-gated ion channels, but it lacks a pore domain. Voltage sensitivity and proton conductivity are conferred by a unitary VSD that consists of four transmembrane helices. The architecture of Hv1 differs from that of cation channels that form a pore in the center among multiple subunits (as in most cation channels) or homologous repeats (as in voltage-gated sodium and calcium channels). Hv1 forms a dimer in which a cytoplasmic coiled coil underpins the two protomers and forms a single, long helix that is contiguous with S4, the transmembrane voltage-sensing segment. The closed-state structure of Hv1 was recently solved using X-ray crystallography. In this article, we discuss the gating mechanism of Hv1 and focus on cooperativity within dimers and their sensitivity to metal ions.
In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. ...VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca
-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca
-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.
Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate ...the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K+ channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.
Hv1/VSOP is a dimeric voltage-gated H(+) channel in which the gating of one subunit is reportedly coupled to that of the other subunit within the dimer. The molecular basis for dimer formation and ...intersubunit coupling, however, remains unknown. Here we show that the carboxy terminus ends downstream of the S4 voltage-sensor helix twist in a dimer coiled-coil architecture, which mediates cooperative gating. We also show that the temperature-dependent activation of H(+) current through Hv1/VSOP is regulated by thermostability of the coiled-coil domain, and that this regulation is altered by mutation of the linker between S4 and the coiled-coil. Cooperative gating within the dimer is also dependent on the linker structure, which circular dichroism spectrum analysis suggests is α-helical. Our results indicate that the cytoplasmic coiled-coil strands form continuous α-helices with S4 and mediate cooperative gating to adjust the range of temperatures over which Hv1/VSOP operates.
Transient receptor potential (TRP) channels comprise a large family of tetrameric cation-selective ion channels that respond to diverse forms of sensory input. Earlier studies showed that members of ...the TRPM subclass possess a self-assembling tetrameric C-terminal cytoplasmic coiled-coil domain that underlies channel assembly and trafficking. Here, we present the high-resolution crystal structure of the coiled-coil domain of the channel enzyme TRPM7. The crystal structure, together with biochemical experiments, reveals an unexpected four-stranded antiparallel coiled-coil architecture that bears unique features relative to other antiparallel coiled-coils. Structural analysis indicates that a limited set of interactions encode assembly specificity determinants and uncovers a previously unnoticed segregation of TRPM assembly domains into two families that correspond with the phylogenetic divisions seen for the complete subunits. Together, the data provide a framework for understanding the mechanism of TRPM channel assembly and highlight the diversity of forms found in the coiled-coil fold.
P2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel ...gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.
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•The ATP- and Zn2+-bound structure of an invertebrate P2X receptor is determined•Two distinct metal binding sites in the extracellular region are identified•The first site, located at the trimer interface, contributes to Zn2+ potentiation•The second site is coupled with ATP binding for regulation by Mg2+
Kasuya et al. report the structure of an invertebrate P2X receptor in the presence of ATP and Zn2+ ion, revealing two distinct metal binding sites. The first site, located at the trimer interface, is responsible for Zn2+ potentiation, whereas the second site is coupled with ATP binding for regulation by Mg2+.
Phosphoinositides (PIP n s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X 2 channels also are regulated by PIP n s, and investigated the structural ...background and the unique features of this regulation. We initially used two-electrode
voltage clamp to analyse the electrophysiological properties of P2X 2 channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization.
K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic
C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability
of the channel to N -methyl- d -glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated
by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region,
we then analysed their binding of the P2X 2 cytoplasmic domain to anionic lipids using PIP n s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound
to PIPs and PIP 2 s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that
fusion proteins comprising the proximal C-terminal region of P2X 2 with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound
PIP n s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the
proximal region of the P2X 2 cytoplasmic C-terminal domain.
The transient receptor potential vanilloid 2 (TRPV2) ion channel is activated by a chemical ligand (2-aminoethoxydiphenyl borate; 2-APB), noxious heat and mechanical stimulation. In a heterologous ...mammalian cell expression system, the oxidant chloramine T (ChT) sensitizes TRPV2 activation in response to 2-APB and heat by oxidation of methionine residues at positions 528 and 607 in rat TRPV2. Here, we used a Xenopus oocyte expression system to determine whether ChT-mediated oxidation can also sensitize TRPV2 to mechanical stimulation. In this system, we confirmed that ChT sensitized TRPV2 activation in response to 2-APB and heat, but we detected no sensitization to mechanical stimulation. This result suggests that the activation mechanism of TRPV2 by a chemical ligand and heat differs from that for mechanical stimulation. Further, we demonstrated that two-electrode voltage clamp recording in the Xenopus oocyte expression system is an excellent format for high throughput analysis of oxidization of redox-sensitive TRP channels.
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