scholarly journals Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels

2015 ◽  
Vol 146 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Yee Ling Lam ◽  
Weizhong Zeng ◽  
Mehabaw Getahun Derebe ◽  
Youxing Jiang
Keyword(s):  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
Structural Difference ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Studies ◽  
Cyclic Nucleotide Gated Channels ◽  
Cng Channel ◽  
High Resolution Structure ◽  
Calcium Permeability

Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca2+ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2+ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca2+ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca2+ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.

scholarly journals Cyclic Nucleotide–Gated Channels

1999 ◽  
Vol 114 (3) ◽  
pp. 377-392 ◽  
Author(s):  
Andrea Becchetti ◽  
Katia Gamel ◽  
Vincent Torre
Keyword(s):  
Plasma Membrane ◽  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
Xenopus Laevis Oocytes ◽  
Gating Currents ◽  
Α Subunit ◽  
Channel Pore ◽  
Cyclic Nucleotide Gated Channels ◽  
Internal Side ◽  
Inside Out

In voltage- and cyclic nucleotide–gated ion channels, the amino-acid loop that connects the S5 and S6 transmembrane domains, is a major component of the channel pore. It determines ion selectivity and participates in gating. In the α subunit of cyclic nucleotide–gated channels from bovine rod, the pore loop is formed by the residues R345–S371, here called R1-S27. These 24 residues were mutated one by one into a cysteine. Mutant channels were expressed in Xenopus laevis oocytes and currents were recorded from excised membrane patches. The accessibility of the substituted cysteines from both sides of the plasma membrane was tested with the thiol-specific reagents 2-aminoethyl methanethiosulfonate (MTSEA) and [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET). Residues V4C, T20C, and P22C were accessible to MTSET only from the external side of the plasma membrane, and to MTSEA from both sides of the plasma membrane. The effect of MTSEA applied to the inner side of T20C and P22C was prevented by adding 10 mM cysteine to the external side of the plasma membrane. W9C was accessible to MTSET from the internal side only. L7C residue was accessible to internal MTSET, but the inhibition was partial, ∼50% when the MTS compound was applied in the absence of cGMP and 25% when it was applied in the presence of cGMP, suggesting that this residue is not located inside the pore lumen and that it changes its position during gating. Currents from T15C and T16C mutants were rapidly potentiated by intracellular MTSET. In T16C, a slower partial inhibition took place after the initial potentiation. Current from I17C progressively decayed in inside-out patches. The rundown was accelerated by inwardly applied MTSET. The accessibility results of MTSET indicate a well-defined topology of the channel pore in which residues between L7 and I17 are inwardly accessible, residue G18 and E19 form the narrowest section of the pore, and T20, P21, P22 and V4 are outwardly accessible.

scholarly journals New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Gabriella Angiulli ◽  
Harveer Singh Dhupar ◽  
Hiroshi Suzuki ◽  
Irvinder Singh Wason ◽  
Franck Duong Van Hoa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Complexes ◽  
Structural Basis ◽  
New Approach ◽  
Functional Studies ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Membrane Protein Complexes ◽  
Membrane Protein Reconstitution

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present ‘on-gradient’ reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional ‘on-bead’ approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

scholarly journals Cyclic Nucleotide-gated Channels on the Flagellum Control Ca2+ Entry into Sperm

1998 ◽  
Vol 142 (2) ◽  
pp. 473-484 ◽  
Author(s):  
Burkhard Wiesner ◽  
Jocelyn Weiner ◽  
Ralf Middendorff ◽  
Volker Hagen ◽  
U. Benjamin Kaupp ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Photoreceptor Cells ◽  
Structural Basis ◽  
Α Subunit ◽  
Cyclic Nucleotide Gated Channels ◽  
Bending Waves ◽  
Mammalian Sperm ◽  
Vertebrate Photoreceptor ◽  
Α And Β Subunits ◽  
Principal Piece

Cyclic nucleotide-gated (CNG) channels are key elements of cGMP- and cAMP-signaling pathways in vertebrate photoreceptor cells and in olfactory sensory neurons, respectively. These channels form heterooligomeric complexes composed of at least two distinct subunits (α and β). The α subunit of cone photoreceptors is also present in mammalian sperm. Here we identify one short and several long less abundant transcripts of β subunits in testis. The α and β subunits are expressed in a characteristic temporal and spatial pattern in sperm and precursor cells. In mature sperm, the α subunit is observed along the entire flagellum, whereas the short β subunit is restricted to the principal piece of the flagellum. These findings suggest that different forms of CNG channels coexist in the flagellum. Confocal microscopy in conjunction with the Ca2+ indicator Fluo-3 shows that the CNG channels serve as a Ca2+ entry pathway that responds more sensitively to cGMP than to cAMP. Assuming that CNG channel subtypes differ in their Ca2+ permeability, dissimilar localization of α and β subunits may give rise to a pattern of Ca2+ microdomains along the flagellum, thereby providing the structural basis for control of flagellar bending waves.

scholarly journals Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

2015 ◽  
Vol 112 (50) ◽  
pp. 15366-15371 ◽  
Author(s):  
Joshua B. Brettmann ◽  
Darya Urusova ◽  
Marco Tonelli ◽  
Jonathan R. Silva ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Coupling ◽  
Dynamic Coupling ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Dependent Inactivation ◽  
Ion Binding Sites ◽  
Essential Connection

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

scholarly journals Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 729-739 ◽  
Author(s):  
Yu-Yang Zhang ◽  
Janice L. Robertson ◽  
Daniel A. Gray ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Single Amino Acid ◽  
Structural Basis ◽  
K Channels ◽  
Chord Conductance ◽  
In Series ◽  
Carboxy Terminal

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at Vm = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba2+, suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl+:K+, accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K+ transport.

scholarly journals K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

2020 ◽  
Author(s):  
Marco Lolicato ◽  
Andrew M. Natale ◽  
Fayal Abderemane-Ali ◽  
David Crottès ◽  
Sara Capponi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Anomalous Scattering ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Ion Binding Sites ◽  
Physical And Chemical

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

scholarly journals Structural insights into the ion selectivity of the MgtE channel for Mg2+ over Ca2+

2021 ◽  
Author(s):  
Xinyu Teng ◽  
Danqi Sheng ◽  
Jin Wang ◽  
Ye Yu ◽  
Motoyuki Hattori
Keyword(s):  
Metal Binding ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Structural Basis ◽  
Recognition Mechanism ◽  
High Resolution Structure ◽  
Moderate Resolution ◽  
Tm Domain ◽  
Functional Analyses ◽  
Computational Analyses

MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 angstrom resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ ~500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 angstrom resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.

scholarly journals Global alignment and assessment of TRP channel transmembrane domain structures to explore functional mechanisms

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Katherine E Huffer ◽  
Antoniya A Aleksandrova ◽  
Andrés Jara-Oseguera ◽  
Lucy R Forrest ◽  
Kenton J Swartz
Keyword(s):  
Transmembrane Domain ◽  
Ion Selectivity ◽  
Structural Data ◽  
Trp Channel ◽  
Structural Basis ◽  
Global Alignment ◽  
Functional Studies ◽  
Domain Structures ◽  
Mechanistic Basis ◽  
Channel Structures

The recent proliferation of published TRP channel structures provides a foundation for understanding the diverse functional properties of this important family of ion channel proteins. To facilitate mechanistic investigations, we constructed a structure-based alignment of the transmembrane domains of 120 TRP channel structures. Comparison of structures determined in the absence or presence of activating stimuli reveals similar constrictions in the central ion permeation pathway near the intracellular end of the S6 helices, pointing to a conserved cytoplasmic gate and suggesting that most available structures represent non-conducting states. Comparison of the ion selectivity filters toward the extracellular end of the pore supports existing hypotheses for mechanisms of ion selectivity. Also conserved to varying extents are hot spots for interactions with hydrophobic ligands, lipids and ions, as well as discrete alterations in helix conformations. This analysis therefore provides a framework for investigating the structural basis of TRP channel gating mechanisms and pharmacology, and, despite the large number of structures included, reveals the need for additional structural data and for more functional studies to establish the mechanistic basis of TRP channel function.

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