Transferring knowledge towards understanding the pore stabilizing variations in K(+) channels: pore stability in K(+) channels.

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K(+) channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K(+) channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K(+) channels.

Improved technique for reconstituting incredibly high and soluble amounts of tetrameric K⁺ channel in natural membranes.

The reconstitution of large amounts of integral proteins into lipid vesicles is largely prompted by the complexity of most biological membranes and protein stability. We optimized a particular system which maximized the incorporation efficiency of large soluble amounts of KcsA potassium channel in Escherichia coli membranes. The effects of two detergents, octylglucoside and 3-[(cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), on KcsA reconstitution were compared. Reconstitution efficiency was found to be incredibly high for CHAPS-treated proteoliposomes followed by dialysis at room temperature. This approach may allow more accurate investigation of integral membrane proteins in their natural membrane environment via biophysical or biochemical techniques.

The "flipped" state in E71A-K+-channel KcsA exclusively alters the channel gating properties by tetraethylammonium and phosphatidylglycerol.

Mutation E71A in the bacterial K(+)-channel KcsA has been shown to abolish the activation-coupled inactivation of KcsA via significant alterations of the peptide backbone in the vicinity of the selectivity filter. In the present study, we examined channel-blocking behavior of KcsA-E71A by tetraethylammonium (TEA) from both the extra- and the intracellular sides. First, we found that E71A is inserted either in cis or trans orientation in a planar lipid bilayer; however, it exhibits only one orientation in proteoliposomes as determined by extravesicular partial chymotrypsin digestion. Second, E71A exhibits a lower extracellular TEA affinity and is more sensitive to intracellular TEA compared to wild-type KcsA, which apparently has >50-fold higher affinity for extracellular TEA and approximately 2.5-fold lower affinity for intracellular TEA compared to E71A. In additional experiments, we investigated the influence of negatively charged phosphatidylglycerol (PG) on channel-gating properties in phosphatidylcholine lipid bilayers. It was found that high PG content decreases the single-channel conductance and increases the channel open time and open probability. Taken together, our data suggest that the "flipped" conformation of the selectivity filter present in E71A allows weaker extracellular and stronger intracellular TEA binding, whereas higher PG content decreases channel conductivity and stabilizes the channel open "flipped" state via electrostatic interaction in the proximity of the channel pore.

Sodium permeability and sensitivity induced by mutations in the selectivity filter of the KcsA channel towards Kir channels.

The bacterial potassium (K(+)) channel KcsA provides an attractive model system to study ion permeation behavior in a selective K(+)-channel. We changed residue at the N-terminal end of the selectivity filter of KcsA (T74V) to its counterpart in inwardly rectifying K(+)-channels (Kir). The tetramer was found to be stable as unmodified KcsA. Under symmetrical and asymmetrical conditions, Na(+) increased the inward current in the virtual absence of K(+) however outward currents were nearly abolished which could be recovered upon internal K(+) addition. Na(+) also drastically increased the channel open time either in the presence or virtual absence of K(+). Furthermore, the T74V mutation decreased the internal Ba(2+) affinity of the channel possibly by binding to a K(+) site in the pore. In additional experiments, another point mutation V76I in T74V mutant was carried out thus the selectivity filter resembled more the selectivity filter of Kir channels. The mutant tetramer was converted into monomers as determined by conventional gel electrophoresis. However, native like gel electrophoresis, Trp fluorescence and acrylamide quenching experiments indicated that this mutant still formed a tetramer and apparently adopted similar folding properties as unmodified KcsA. Single-channel experiments further demonstrated that the channel was selective for K(+) over Na(+) as Na(+) blocked channel currents. These data suggest that single point mutation T74V alters the selectivity filter and allows simultaneous occupancy and conduction of K(+) and Na(+) probably via ion-ion interaction in the pore. In contrast, both mutations (T74V and V76I) in the same molecule seem to reorganize the pore conformation which controls the overall stability of a selective K(+)-channel.

Mutations in the K(+)-channel KcsA toward Kir channels alter salt-induced clusterization and blockade by quaternary alkylammonium ions.

Protein aggregation is a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment, thus triggering many human diseases. We have shown that bacterial K(+)-channel KcsA, which acts as a representative model for ion channels, forms salt-induced large conductive complexes in a particular environment. In the present study, we investigated the effects of point mutations in the selectivity filter of KcsA on intrinsic stability, aggregation, and channel blocking behavior. First, we found that a low sodium chloride concentration in potassium-containing media induced fast transfer of single channels to a planar lipid bilayer. Second, increasing the sodium chloride concentration drastically increased the total channel current, indicating enhanced vesicle fusion and transfer of multiple channels to a planar lipid bilayer. However, such complexes exhibited high conductance as well as higher open probability compared to the unmodified KcsA behavior shown previously. Interestingly, the affinity of aggregated complexes for larger symmetric quaternary alkylammonium ions (QAs) was found to be much higher than that for tetraethylammonium, a classical blocker of the K(+) channel. Based on these findings, we propose that mutant channel complexes exhibit larger pore dimensions, thus resembling more the topological properties of voltage-gated and inwardly rectifying K(+) channels.

Changing Val-76 towards Kir channels drastically influences the folding and gating properties of the bacterial potassium channel KcsA.

All K(+)-channels are stabilized by K(+)-ions in the selectivity filter. However, they differ from each other with regard to their selectivity filter. In this study, we changed specific residue Val-76 in the selectivity filter of KcsA to its counterpart Ile in inwardly rectifying K(+)-channels (Kir). The tetramer was exclusively converted into monomers as determined by conventional gel electrophoresis. However, by perfluoro-octanoic acid (PFO) gel electrophoresis mutant channel was mostly detected as tetramer. Tryptophan fluorescence and acrylamide quenching experiments demonstrated significant alteration in channel folding properties via increase in hydrophilicity of local environment. Furthermore, in planar lipid bilayer experiments V76I exhibited drastically lower conductance and decreased channel open time as compared to the unmodified KcsA. These studies suggest that V76I might contribute to determine the stabilizing, folding and channel gating properties in a selective K(+)-channel.

Dissimilarity in the channel intrinsic stability among the bacterial KcsA and the inwardly rectifying potassium channel ROMK1.

In this study, we compared the channel intrinsic stability of the bacterial K(+)-channel KcsA and the inwardly rectifying potassium channel (Kir) ROMK1. ROMK1 was successfully cloned, expressed and purified from Saccharomyces cerevisae. By conventional gel electrophoresis, ROMK1 was detected in monomeric form running exclusively at approximately 45 kDa either in its oxidized or reduced form. By perfluoro-octanoic acid (PFO)-PAGE, the reduced ROMK1 was identified as tetrameric as well as oligomeric complex. However, in its oxidized form ROMK1 was exclusively detected in oligomeric form thus indicating the role of intrinsic cysteine residues and formation of disulfide bonds in stabilizing the oligomeric ROMK1. On the other hand, KcsA purified from E. coli was detected as an extremely stable tetramer both in its oxidized or reduced forms as determined by conventional or PFO-PAGE. Furthermore, in planar lipid bilayer ROMK1 exhibited prominent inward rectification, low single channel conductance and high channel open probability as compared to the KcsA channel which showed typically slight outward rectification and low open probability under similar conditions. Our experiments clearly indicate that KcsA and ROMK1 channels differ with regard to their intrinsic stability which might be related to their structural and functional differences.

Effects of sodium chloride on membrane fusion and on the formation of aggregates of potassium channel KcsA in Escherichia coli membrane.

In the current study, we found that NaCl induces fast vesicle fusion and aggregation of the bacterial potassium channel KcsA in Escherichia coli (E. coli) membranes. Conventional gel electrophoresis and tryptophan fluorescence experiments were performed to detect NaCl-induced aggregation or supramolecular complexes. Interestingly, in a planar lipid bilayer, increasing NaCl concentration dramatically increased the total internal current indicating enhanced vesicle fusion and transfer of high number of channels to the lipid bilayer. Such channels appeared in clusters of variable size which exhibited higher conductivity, increased open probability and efficient blocking by K(+)-channel blocker tetraethylammonium (TEA). Furthermore, NaCl-induced KcsA aggregation was found to be specific for E. coli membrane as compared to the artificial membrane system. Our preliminary data suggest the role of NaCl in the formation of KcsA aggregates as well as in the enhancement of membrane fusion capability in E. coli membranes.

The first ankyrin-like repeat is the minimum indispensable key structure for functional assembly of homo- and heteromeric TRPC4/TRPC5 channels.

The closely related TRPC4 and TRPC5 proteins, members of the canonical transient receptor potential (TRPC) family, assemble into either homo- or heterotetrameric, non-selective cation-channels. To elucidate domains that mediate channel complex formation, we evaluated dominant negative effects of N- or C-terminal TRPC4/5 fragments on respective currents of full-length proteins overexpressed in HEK293 cells with whole-cell electrophysiology. Confocal Förster Resonance Energy Transfer (FRET) measurements enabled to probe the interaction potential of these CFP/YFP-labelled fragments in vivo. Only N-terminal fragments that included the first ankyrin-like repeat potently down-regulated TRPC4/TRPC5 currents, while fragments including either the second ankyrin-like repeat and the coiled-coil domain or the C-terminus remained ineffective. Total internal reflection fluorescence (TIRF) microscopy data suggested that the dominant negative N-terminal fragments led to a predominantly intracellular localisation of coexpressed TRPC5 proteins. FRET measurements clearly revealed that only fragments including the first ankyrin-like repeat were able to multimerise. Moreover a TRPC5 mutant that lacked the first ankyrin-like repeat was unable to homo-multimerise, failed to interact with wild-type TRPC5 and resulted in non-functional channels.