Protein ligands for studying ion channel proteins

Protein toxins from venomous organisms have provided remarkable insights into the structure and mechanism of ion channel proteins (Kalia et al., 2015). One classic example is the work of Rod MacKinnon, Chris Miller, and their colleagues on the mechanism by which high-conductance calcium-activated potassium channels (or BK channels for Big K conductance) are inhibited by charybdotoxin (Anderson et al., 1988; MacKinnon and Miller, 1988)—a small protein toxin found in the venom of the Israeli deathstalker scorpion, Leiurus quinquestriatus (Miller et al., 1985). They imagined at the time that the high single-channel conductance of BK might reflect a whirlpool-like structure, and lore has it that a young Gary Yellen made the winning suggestion to name the toxin after Charybdis, the daughter of Poseidon who was turned into a whirlpool-producing sea monster by Zeus. In this issue of The Journal of General Physiology, Daniel Turman and Randy Stockbridge revisit these timeless experiments during their investigation of the mechanism by which monobodies inhibit Fluc fluoride channels. Like MacKinnon, Miller, and colleagues, they find that channel inhibition occurs via a pore block mechanism. In their pioneering experiments, MacKinnon and Miller used powerful single-channel recordings to show that charybdotoxin binds to BK with bimolecular kinetics expected for a 1:1 complex. Moreover, even though the toxin binds to the extracellular side of the BK channel, they discovered that the toxin unbound more rapidly when they increased the concentration of internal potassium ions or depolarized the voltage across the membrane. Remarkably, this voltage-dependent toxin knock-off vanished when potassium ions were replaced with impermeant ions such as sodium, suggesting that the influence of membrane voltage was to increase potassium occupancy of an ion-binding site near the external side of the pore where the toxin binds. They reasoned that such observations could be understood if charybdotoxin inhibited the channel by physically blocking the pore, as illustrated in the cartoon depicting this bold conclusion from their 1988 JGP paper (MacKinnon and Miller, 1988), reproduced here in Fig. 1 A. This now famous cartoon also speculates that the toxin positions a basic residue at the external end of the permeation pathway, enabling the toxin to sense ion occupancy of the pore. With such pore-blocking scorpion toxins in hand, MacKinnon and Miller localized the pore-forming region within the Shaker potassium channel (MacKinnon and Miller, 1989), MacKinnon demonstrated that this potassium channel is a tetramer (MacKinnon, 1991), and together with Rick Aldrich and Alice Lee, they revealed that one N-terminal inactivation particle was sufficient to inactivate the Shaker channel (MacKinnon et al., 1993). The structural depiction of the toxin inserting a basic residue into the pore gained further traction when Chul-Seung Park and Miller succeeded in producing charybdotoxin recombinantly and showing that mutation of K27 on the toxin greatly reduced the ability of internal potassium or membrane voltage to destabilize the toxin–channel complex (Park and Miller, 1992). Recently, Anirban Banerjee and MacKinnon obtained an even clearer picture when they solved the x-ray structure of charybdotoxin bound to a voltage-activated potassium channel known as the Kv1.2/2.1 paddle chimera and showed that K27 is indeed positioned near the ion selectivity filter within the outer pore of the channel (Fig. 1 B; Banerjee et al., 2013). In the work described in this issue, Turman and Stockbridge (2017) hark back to the classical studies on charybdotoxin block of potassium channels by demonstrating that a synthetic monobody inhibits the Fluc family of fluoride channels through a remarkably similar mechanism. Flucs are an interesting and recently discovered family of fluoride channels that bacteria, archaea, single-celled eukaryotes, and plants use to rid themselves of toxic fluoride ions found ubiquitously in nature (Baker et al., 2012; Stockbridge et al., 2013; Ji et al., 2014). Fluc subunits come in two varieties, those encoded by a single subunit containing four predicted T he J o u rn al o f G e ne ra l P hy si o lo g y