Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K+ channels.

K(+) channels distinguish K(+) from Na(+) in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of C-type inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K(+) binding at the S2 site. We also show that an ester substitution at the S2 site in the KvAP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K(+) channels in K(+) compared with Rb(+) are due to different ion occupancies at the S2 site.

Semisynthetic K+ channels show that the constricted conformation of the selectivity filter is not the C-type inactivated state.

C-type inactivation of K(+) channels plays a key role in modulating cellular excitability. During C-type inactivation, the selectivity filter of a K(+) channel changes conformation from a conductive to a nonconductive state. Crystal structures of the KcsA channel determined at low K(+) or in the open state revealed a constricted conformation of the selectivity filter, which was proposed to represent the C-type inactivated state. However, structural studies on other K(+) channels do not support the constricted conformation as the C-type inactivated state. In this study, we address whether the constricted conformation of the selectivity filter is in fact the C-type inactivated state. The constricted conformation can be blocked by substituting the first conserved glycine in the selectivity filter with the unnatural amino acid d-Alanine. Protein semisynthesis was used to introduce d-Alanine into the selectivity filters of the KcsA channel and the voltage-gated K(+) channel KvAP. For semisynthesis of the KvAP channel, we developed a modular approach in which chemical synthesis is limited to the selectivity filter whereas the rest of the protein is obtained by recombinant means. Using the semisynthetic KcsA and KvAP channels, we show that blocking the constricted conformation of the selectivity filter does not prevent inactivation, which suggests that the constricted conformation is not the C-type inactivated state.

Engineering K+ channels using semisynthesis.

Potassium channels conduct K(+) ions selectively and at very high rates. Central to the function of K(+) channels is a structural unit called the selectivity filter. In the selectivity filter, a row of four K(+) binding sites are created using mainly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, site-directed mutagenesis is of limited utility in investigating the selectivity filter. In order to overcome this limitation, we have developed a semisynthetic approach, which permits the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that we have developed for the semisynthesis of the K(+) channel, KcsA. We anticipate that the protocols described in this chapter will also be applicable for the semisynthesis of other integral membrane proteins of interest.

Modular strategy for the semisynthesis of a K+ channel: investigating interactions of the pore helix.

Chemical synthesis is a powerful method for precise modification of the structural and electronic properties of proteins. The difficulties in the synthesis and purification of peptides containing transmembrane segments have presented obstacles to the chemical synthesis of integral membrane proteins. Here, we present a modular strategy for the semisynthesis of integral membrane proteins in which solid-phase peptide synthesis is limited to the region of interest, while the rest of the protein is obtained by recombinant means. This modular strategy considerably simplifies the synthesis and purification steps that have previously hindered the chemical synthesis of integral membrane proteins. We develop a SUMO fusion and proteolysis approach for obtaining the N-terminal cysteine containing membrane-spanning peptides required for the semisynthesis. We demonstrate the feasibility of the modular approach by the semisynthesis of full-length KcsA K(+) channels in which only regions of interest, such as the selectivity filter or the pore helix, are obtained by chemical synthesis. The modular approach is used to investigate the hydrogen bond interactions of a tryptophan residue in the pore helix, tryptophan 68, by substituting it with the isosteric analogue, beta-(3-benzothienyl)-l-alanine (3BT). A functional analysis of the 3BT mutant channels indicates that the K(+) conduction and selectivity of the 3BT mutant channels are similar to those of the wild type, but the mutant channels show a 3-fold increase in Rb(+) conduction. These results suggest that the hydrogen bond interactions of tryptophan 68 are essential for optimizing the selectivity filter for K(+) conduction over Rb(+) conduction.

Semisynthesis of K+ channels.

The ability to selectively conduct K(+) ions is central to the function of K(+) channels. Selection for K(+) and rejection of Na(+) takes place in a conserved structural element referred to as the selectivity filter. The selectivity filter consists of four K(+)-specific ion binding sites that are created using predominantly the backbone carbonyl oxygen atoms. Due to the involvement of the protein backbone, experimental manipulation of the ion binding sites in the selectivity filter is not possible using traditional site directed mutagenesis. The limited suitability of the site-directed mutagenesis for studies on the selectivity filter has motivated the development of a semisynthesis approach, which enables the use of chemical synthesis to manipulate the selectivity filter. In this chapter, we describe the protocols that are presently used in our laboratory for the semisynthesis of the bacterial K(+) channel, KcsA. We show the introduction of a spectroscopic probe into the KcsA channel using semisynthesis. We also review previous applications of semisynthesis in investigations of K(+) channels. While the protocols described in this chapter are for the KcsA K(+) channel, we anticipate that similar protocols will also be applicable for the semisynthesis of other integral membrane proteins.

Rectification properties and pH-dependent selectivity of meningococcal class 1 porin.

We studied the current rectification properties and selectivity of class 1 porin (PorA) from Neisseria meningitidis (strain H44/76 Delta 3 Delta 4) reconstituted in planar lipid membranes varying salt concentrations and pH. PorA channel shows voltage gating with a characteristic time remarkably longer than other porins. Its current-voltage asymmetry, evaluated as the current rectification ratio, changes nonmonotonically with salt concentration. Interestingly, it reaches its maximum value at physiological concentration. Porin selectivity, quantified by reversal potential measurements, is also significantly asymmetric. Depending on the direction of the salt gradient, the channel becomes more or less selective (10:1 vs. 5:1 Na(+)/Cl(-)). Besides, the reversal potential measurements suggest that porin inserts directionally following the concentration gradient. Measurements over a wide range of pH show that although PorA is strongly cation selective at pH >6, its selectivity gradually changes to anionic in an acidic medium (pH < 4). We show that a continuum electrodiffusion model quantitatively accounts for conductance and reversal potential measurements at positive and negative applied voltages.

New insights into the mechanism of permeation through large channels.

The mitochondrial channel, VDAC, regulates metabolite flux across the outer membrane. The open conformation has a higher conductance and anionic selectivity, whereas closed states prefer cations and exclude metabolites. In this study five mutations were introduced into mouse VDAC2 to neutralize the voltage sensor. Inserted into planar membranes, mutant channels lack voltage gating, have a lower conductance, demonstrate cationic selectivity, and, surprisingly, are still permeable to ATP. The estimated ATP flux through the mutant is comparable to that for wild-type VDAC2. The outer membranes of mitochondria containing the mutant are permeable to NADH and ADP/ATP. Both experiments support the counterintuitive conclusion that converting a channel from an anionic to a cationic preference does not substantially influence the flux of negatively charged metabolites. This finding supports our previous proposal that ATP translocation through VDAC is facilitated by a set of specific interactions between ATP and the channel wall.

The physiological properties of a novel family of VDAC-like proteins from Drosophila melanogaster.

VDAC, a major protein of the mitochondrial outer membrane, forms voltage-dependent, anion-selective channels permeable to most metabolites. Although multiple isoforms of VDAC have been found in different organisms, only one isoform (porin/DVDAC) has been previously reported for Drosophila melanogaster. We have examined the physiological properties of three other Drosophila proteins (CG17137, CG17139, and CG17140) whose primary sequences have significant homology to DVDAC. A comparison of their hydropathy profiles (beta-pattern) with known VDAC sequences indicates the same fundamental folding pattern but with major insertions and deletions. The ability of these proteins to form channels was tested on planar membranes and liposomes. Channel activity was observed with varying degrees of similarity to VDAC. Two of these proteins (CG17137 and CG17140) produced channels with anionic selectivity in the open state. Sometimes channels exhibited closure and voltage gating, but for CG17140 this occurred at much higher voltages than is typical for VDAC. CG17139 was not able to form channels. DVDAC and CG17137 were able to rescue the temperature-sensitive conditional-lethal phenotype of VDAC-deficient yeast, whereas CG17139 and CG17140 demonstrated no complementation. Similar structure and channel formation indicate that VDAC-like proteins are part of the larger VDAC family but the modifications are indicative of specialized functions.