Single Streptomyces lividans K+ Channels

100

Water molecules are constrained to move with K ؉ ions through the narrow part of the Streptomyces lividans K ؉ channel because of the single-file nature of transport. In the presence of an osmotic gradient, a water molecule requires <10 ps to cross the purified protein reconstituted into planar bilayers. Rinsing K ؉ out of the channel, water may be 1,000 times faster than the fastest experimentally observed K ؉ ion and 20 times faster than the onedimensional bulk diffusion of water. Both the anomalously high water mobility and its inhibition observed at high K ؉ concentrations are consistent with the view that liquid-vapor oscillations occur because of geometrical confinements of water in the selectivity filter. These oscillations, where the chain of molecules imbedded in the channel (the ''liquid'') cooperatively exits the channel, leaving behind a near vacuum (the ''vapor''), thus far have only been discovered in hydrophobic nanopores by molecular dynamics simulations

Section: Resultsmentioning

confidence: 99%

Beyond the diffusion limit: Water flow through the empty bacterial potassium channel

Saparov

Pohl

2004

100

“…Recently, the molecular determinants of inactivation in KcsA have been demonstrated to occur at the selectivity filter (47). In these channels, however, the activation gate that opens and closes the permeation pathway in response to voltage or pH has been established to be at the intracellular end of the last transmembrane segment (10)(11)(12)(13)(14)(15)(16)(48)(49)(50). In stark contrast, our findings point to the selectivity filter itself acting as the primary gate in CNG channels, which might be a mechanism occurring in other cation channels.…”

Section: Discussionmentioning

confidence: 99%

Gating at the selectivity filter in cyclic nucleotide-gated channels

Contreras

Srikumar²,

Holmgren³

2008

By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (KV) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.cGMP ͉ ion channel ͉ signal transduction C yclic nucleotide-gated (CNG) channels sense variations in the intracellular concentration of cyclic nucleotides that occur in response to visual or olfactory stimuli, therefore playing essential roles in the transduction of visual and olfactory information (1, 2). In many ways, CNG channels are similar to voltage-activated potassium (K V ) channels. They coassemble as tetramers of homologous subunits (3-6), each containing six transmembrane segments (TM), a positively charged TM4 and a reentry P region between TM5 and TM6, suggesting that CNG channels belong to the same superfamily of voltage-activated cation channels (7). The main difference is that CNG channels are only weakly voltage-dependent. Instead, they open and close the pore in response to changes in the intracellular concentrations of cGMP or cAMP, a property conferred by the presence of a cyclic nucleotide binding domain at the C terminus of each subunit (8, 9).Our understanding of how CNG channels open and close their pore in response to cyclic nucleotide binding is much less refined than our understanding of how K V channels gate in response to voltage. A large body of evidence, using a variety of approaches, has established that K V channels open and close their permeation pathway at the intracellular end of the pore (10-17). Attempts to extend those ideas to CNG channels have encountered some resistance. For example, studies using intracellularly applied molecules that block the permeation pathway of CNG channels, such as divalent ions (18), tetracaine (19,20), or quaternary ammonium ions (21), have shown that blockade is not state-dependent, as if these molecules can access the pore in both open and closed channels, which is in stark contrast with the blockade properties observed in K V channels (10,11,14,(22)(23)(24). In addition, experiments examining the state dependence of cysteine modification by intracellular application of methanethiosulfonate (MTS) reagents have failed to show dramatic differences between open and closed states in the inner-vestibule region (21,25,26), results that are inconsistent with an intracellular gate in TM6, as shown in K V channels (12,15).Se...

“…The MthK pore and NaKN⌬19 constructs were purified as described previously (18,21). The proteins were reconstituted in lipid vesicles composed of a 3:1 ratio of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine and 1-palmitoyl-2-oleoyl-phosphatidylglycerol at a protein/lipid ratio of 6 and 10 g/mg, respectively, as described previously (31), with the following modifications: 10 mM DM was used to solubilize the lipid and dialysis [against a reconstitution buffer of 10 mM Hepes (pH 7.4), 400 mM KCl, and 4 mM N-methyl-Dglucamine] was used to slowly remove the detergent from the detergent/lipid/protein mixture. The reconstituted liposome samples were kept at Ϫ80°C in 100-l aliquots.…”

Section: Methodsmentioning

confidence: 99%

Structural insight into Ca ²⁺ specificity in tetrameric cation channels

Alam

Shi

Jiang

2007

Apparent blockage of monovalent cation currents by the permeating blocker Ca 2؉ is a physiologically essential phenomenon relevant to cyclic nucleotide-gated (CNG) channels. The recently determined crystal structure of a bacterial homolog of CNG channel pores, the NaK channel, revealed a Ca 2؉ binding site at the extracellular entrance to the selectivity filter. This site is not formed by the side-chain carboxylate groups from the conserved acidic residue, Asp-66 in NaK, conventionally thought to directly chelate Ca 2؉ in CNG channels, but rather by the backbone carbonyl groups of residue Gly-67. Here we present a detailed structural analysis of the NaK channel with a focus on Ca 2؉ permeability and blockage. Our results confirm that the Asp-66 residue, although not involved in direct chelation of Ca 2؉ , plays an essential role in external Ca 2؉ binding. Furthermore, we give evidence for the presence of a second Ca 2؉ binding site within the NaK selectivity filter where monovalent cations also bind, providing a structural basis for Ca 2؉ permeation through the NaK pore. Compared with other Ca 2؉ -binding proteins, both sites in NaK present a novel mode of Ca 2؉ chelation, using only backbone carbonyl oxygen atoms from residues in the selectivity filter. The external site is under indirect control by an acidic residue (Asp-66), making it Ca 2؉ -specific. These findings give us a glimpse of the possible underlying mechanisms allowing Ca 2؉ to act both as a permeating ion and blocker of CNG channels and raise the possibility of a similar chemistry governing Ca 2؉ chelation in Ca 2؉ channels.calcium blockage ͉ cyclic nucleotide-gated channel pore ͉ NaK channel ͉ nonselective cation channel