Controlling the level of potassium in cells is critical to cellular function, and therefore all cells have membrane-embedded proteins that act as gatekeepers for selectively guiding potassium ions in and out of the cell. These potassium channels are potential drug targets since cellular activity can be limited and even shut down entirely by blocking the potassium conduction pathway. The bacterial potassium channel KcsA serves as a paradigm to study the structure and function of mammalian channels. Clearly, understanding the process by which toxins bind to this channel can be helpful to pharmaceutical companies interested in designing channel-inhibiting molecules.
One example of such molecules are toxins, small proteins naturally found in scorpion, spider, snail and snake venom, and even in Caribbean sea anemones, that bind to mammalian channels and in this fashion paralyze and eventually kill their prey. However, up until five years ago no toxins were known to inhibit the KcsA channel. That changed when researchers from the University of California Irvine reported their discovery of a family of KcsA-blocking toxins that blocks this channel.
Now, in a study just published in the journal Science Advances (Tethered peptide neurotoxins display two blocking mechanisms in the K+ channel pore as do their untethered analogs), the researchers, co-led by Prof. Steve A.N. Goldstein of UC Irvine, and Prof. Jordan H. Chill of the Department of Chemistry at Bar-Ilan University, report on a quicker and more cost-effective way to test a multitude of toxin-channel combinations in order to investigate how such toxins recognize various channels. This was accomplished using toxins tethered to the cell membrane. Channels, looking like goblets with their opening defining the extracellular ‘turret’ region of the channel while the stems represent a membrane-spanning ion conduction pathway, were expressed and embedded in a cell membrane. Meanwhile, a toxin (like an olive or a cherry that can ‘block’ the opening to the stem) is tethered by a flexible linker to the membrane close to the channel. However, rather than chemically synthesizing the toxins – a time-consuming and expensive proposition – the Goldstein team hijacked the cell machinery to synthesize and display various toxins on the cell surface, giving this approach the name ‘zombie-scanning’. To prove the merits of this new approach, several variants of a toxin from the Heterotactica magnifica sea anemone (hence the name HmK – K is the periodic table symbol for potassium) were assayed for their ability to ‘fit’ into the channel and block it. Single amino-acid changes (amino acids are the building blocks of proteins) along the HmK sequence were introduced, and the effect of each on channel blocking noted, thus pinpointing amino-acids with significant contributions to the toxin-channel interaction.
Unfortunately, at this point HmK was only known as a schematic oblate object without structural detail. To establish the structural basis for the results of the tethered-toxin experiment, Prof. Jordan Chill and Dr. Netanel Mendelman, of Bar-Ilan University, used nuclear magnetic resonance (NMR) to elucidate the three-dimensional structure of the toxin for the first time. “We employed sophisticated NMR experiments to measure hundreds of distances between atom pairs, and these distance constraints eventually defined the position of each atom in the toxin, finally arriving at the full structure which was not known to date,” says Prof. Chill. Mapping upon this structure of the residues shown by the zombie-scanning method to contribute most to binding immediately revealed the exact location of the toxin binding surface.
And herein lay a surprise. Toxins of this sea-anemone family have two immutable positively charged residues, one of which inserts into the potassium-conducting pore, masquerades as a potassium ion, and thus blocks the channel. Curiously, findings of the Goldstein team showed that HmK inserted the opposite residue in comparison to toxins studied earlier by the two groups. “We assumed HmK would conform to the same binding scheme, but the toxin proved us wrong,” says Prof. Chill. “Based on the toxin structure, the two binding modes we observed seem to involve a ‘flipping’ of the toxin or some rearrangement of its atomic structure.” The realization that toxins can block the channel in two different ‘poses’ (spatial orientations of the toxin in relation to the channel) has interesting implications, immediately raising the question of what determines the chosen pose, and how is this pose related to the selectivity profile of channels. This query is the motivating factor behind current research in the framework of the collaboration. Prof. Chill summarizes, “the NMR-derived structural map of HmK greatly enhanced the impact of this study by providing a more detailed view of the toxin and how it recognizes the opening to a potassium channel.”
This study was funded in part by grants from the National Institutes of Health and the US-Israel Binational Science Foundation.