Kathleen M. Giangiacomo

Electrostatic mutations in iberiotoxin as a unique tool for probing the electrostatic structure of the maxi-K channel outer vestibule.

Iberiotoxin (IbTX or alpha-KTx 1.3), a selective, high-affinity blocker of the large-conductance, calcium-activated (maxi-K) channel, exhibits a unique, asymmetric distribution of charge. To test how these charges control kinetics of IbTX binding, we generated five mutants at two positions, K27 and R34, that are highly conserved among other isotoxins. The dissociation and association rate constants, koff and kon, were determined from toxin-blocked and -unblocked durations of single maxi-K channels incorporated into planar lipid bilayers. Equilibrium dissociation constant (Kd) values were calculated from koff/kon. The IbTX mutants K27N, K27Q, and R34N caused large increases in Kd values compared to wild-type, suggesting that the IbTX interaction surface encompasses these residues. A well-established pore-blocking mechanism for IbTX predicts a voltage dependence of toxin-blocked times following occupancy of a potassium binding site in the channel pore. Time constants for block by K27R were approximately 5-fold slower at -20 mV versus +40 mV, while neutralization of K27 relieved the voltage dependence of block. This suggests that K27 in IbTX interacts with a potassium binding site in the pore. Neutralized mutants of K27 and R34, with zero net charge, displayed toxin association rate constants approximately 10-fold slower than wild-type. Association rates for R34N diminished approximately 19-fold when external potassium was increased from 30 to 300 mM. These findings suggest that simple net charge and diffusional processes do not control ingress of IbTX into the channel vestibule.

Glycine 30 in iberiotoxin is a critical determinant of its specificity for maxi-K versus KV channels

Iberiotoxin (IbTX) is a remarkably selective α‐K toxin peptide (α‐KTx) inhibitor of the maxi‐K channel. In contrast, the highly homologous charybdotoxin inhibits both the maxi‐K and KV1.3 channels with similar high affinity. The present study investigates the molecular basis for this specificity through mutagenesis of IbTX. The interactions of mutated peptides with maxi‐K and KV1.3 channels were monitored through dose‐dependent displacement of specifically bound iodinated α‐KTx peptides from membranes expressing these channels. Results of these studies suggest that the presence of a glycine at position 30 in IbTX is a major determinant of its specificity while the presence of four unique acidic residues in IbTX is not.

Novel α-KTx Sites in the BK Channel and Comparative Sequence Analysis Reveal Distinguishing Features of the BK and KV Channel Outer Pore

The α-KTx peptide toxins inhibit different types of potassium channels by occluding the outer channel pore composed of four identical α subunits. The large-conductance, calcium-activated (BK or Slo1) and voltage-dependent (KV) potassium channels differ in their specificity for the different α-KTx subfamilies. While many different α-KTx subfamilies of different sizes inhibit KV1 channels with high affinity, only one subfamily, α-KTx 1.x, inhibits BK channels with high affinity. Two solvent-exposed regions of the outer pore that influence α-KTx binding, the turret and loop, display high sequence variability among different potassium channels and may contribute to differences in α-KTx specificity. While these α-KTx domains have been studied in KV1 channels, little is known about the corresponding BK α-KTx domains. To define α-KTx sites in the BK outer pore, we examined the effect of 19 outer pore mutations on specific binding of 125I-labeled iberiotoxion (IbTX or α-KTx 1.3) and on their cell-surface expression. Similar to α-KTx sites in the Shaker KV1 loop, site-directed mutations in the BK loop disrupted specific IbTX binding. In contrast, mutations in the BK turret region revealed three novel α-KTx sites, Q267, N268, and L272, which are distinct from α-KTx sites in the KV1 turret. The BK turret region shows no sequence identity with KV1 and MthK turrets of known 3D structure. To define the BK turret, we used secondary structure prediction methods that incorporated information from sequence alignment of 30 different Slo1 and Slo3 turret sequences from 5 of the 7 major animal phyla representing 27 different species. Results of this analysis suggest that the BK turret contains 18 amino acids and is defined by a cluster of strictly conserved polar residues at the N-terminal side of the turret. Thus, the BK turret is predicted to have six more amino acids than the KV1 turret. Results of this work suggest that BK and KV1 outer pores have a similar α-KTx domain in the loop preceding the inner helix, but that the BK turret comprises a unique α-KTx interaction surface that likely contributes to the exclusive selectivity of BK channels for α-KTx1.x toxins.

[27] Construction of the photosynthetic reaction center—mitochondrial ubiquinol—cytochrome-c oxidoreductase hybrid system

Publisher Summary This chapter focuses on the construction of the photosynthetic reaction center-mitochondrial ubiquinol-cytochrome-c oxidoreductase hybrid system. The photochemical reaction center (RC) converts light energy into reducing equivalents at its low potential end and oxidizing equivalents at its high potential end. The bc1complex consumes quinol and ferricytochrome c generated by flash-activated RC. The photochemical RC from the photosynthetic bacterium Rhodopseudomonas sphaeroides can be combined with mitochondrial ubiquinol-cytochrome-c1 oxidoreductase (bc1 complex) to create an efficient light-activated electron-transfer system. It is a hybrid system of membrane redox proteins. Construction of hybrid system requires light-stimulated electron transfer between redox centers in the RC and the bc1 complex through both the high-potential cytochrome c and the low-potential quinol ends. Physiologically significant construction exhibits the sensitivities to inhibitors. The purified hybrid system promises application with new physical techniques, such as direct electrical measurement and voltage control of electron-transfer reactions in protein multilayers on electrodes.

Probing the structure and function of potassium channels with α-K toxin blockers

This review examines recent work aimed at revealing the molecular structures of the potassium channel vestibules using the α-K toxin (α-KTx) peptide blockers from the venoms of scorpions. The three subfamilies of α-K toxins are discussed in terms of their specificity for voltage-gated potassium (Kv) channels and for the large-conductance calcium-activated (maxi-K) channel. Among the α-KTx subfamilies, the three-dimensional solution structures all share a common β sheet/helix motif. However, there are differences in the toxin electrostatic structures and subtle differences in their α-carbon backbone structures that may underlie potassium channel specificity. The binding of these α-KTxs to the extracellular potassium channel pore is modulated by electrostatic interactions and by the channel gating conformation. Thus, these toxins are exceptional sensors of the electrostatic environment of the channel vestibule. Changes in toxin binding free energy, resulting from site-specific toxin mutants, have revealed a low resolution image of the α-KTx receptor surface and consequently the K channel vestibule. Interactions between specific residues on the toxin and on the channel were revealed by applying the principle of additivity of binding free energy to identify pairwise toxin:channel contacts. These interactions provide structural information about amino acids that line the Shaker potassium channel pore.

Stigmatellin and Other Electron Transfer Inhibitors as Probes for the QB Binding Site in the Reaction Center of Photosynthetic Bacteria

Quinones are vital for energy transduction both in photosynthetic and respiratory electron transfer chains. in the cyclic electron transfer system of photosynthetic bacteria there are four unique ubiquinone (UQ10) binding sites (1). The primary electron acceptor, QA, in the reaction center protein complex (RC) is a tightly bound quinone which is capable of accepting only single electrons from the light-activated (BChl)2- The secondary electron acceptor in the RC, QB, acts as a two electron gate between QA (n=l) quinol oxidation of the RC and the n=2 redox components of the Qpool and ultimately , the ubiquinol oxidoreductase (bc 1 complex) . in the bc 1 complex, the Qz site is a discrete n=2 site, and the Qc site appears to be a two electron gate analogous to QB. in this paper we present a method for probing the properties of the QB site in isolated photosynthetic RCs from Rhodopseudomonas sphaeroides. A comparison of these results with known properities of other Q binding sites in both photosynthetic and respiratory electron transport systems will allow us to determine common structural elements of Q binding sites.

Purification and functional reconstitution of high-conductance calcium-activated potassium channel from smooth muscle.

Publisher Summary High-conductance Ca2+-activated K+ (maxi-K+) channels are activated by both membrane depolarization and binding of Ca 2+ to sites at the intracellular face of the channel. Maxi-K+ channels are present in both electrically excitable and nonexcitable cells, and display high conductance and selectivity for K+. These channels are involved in regulation of the excitation-contraction coupling process in smooth muscle, as well as in control of transmitter release from neuroendocrine tissues. The pharmacology of maxi-K+ channels has been developed during the last few years and efforts are continuing to identify novel and selective modulators of this channel family. This chapter discusses procedures developed to purify maxi-K+ channels from smooth muscle tissues, as well as the way purified preparation can be reconstituted into liposomes for determination of channel activity. The chapter also discusses ways of obtaining protein sequence information from components of the maxi-K+ channel preparation that are useful for obtaining full-length cDNA clones of these proteins.