An engineered scorpion toxin analogue with improved Kv1.3 selectivity displays reduced conformational flexibility

Abstract

The voltage-gated Kv1.3 K+ channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.

Probing pattern and dynamics of disulfide bridges using synthesis and NMR of an ion channel blocker peptide toxin with multiple diselenide bonds

“…75% of the peak current (Fig. 1A), which is comparable to the inhibition of Kv1.3 with the double mutated peptide [N17A/F32T]-AnTx at identical concentration (89% inhibition in the latter case,26Fig. 1C).…”

“…For the preparation of the anuroctoxin derived peptides, both the Boc and Fmoc chemistry were used previously with success 26. Since the stability of several Boc compatible Se protecting groups is significantly lower than the corresponding sulfur protecting groups ( e.g.…”

“…Improving selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3 opens the door to the toxin's therapeutic use. In line with these, we have constructed a double substituted analog of AnTx, [N17A/F32T]-AnTx, which is characterized by comparable Kv1.3 affinity to the wild-type peptide but shows a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2 and other ion channels tested (Kv1.1 and KCa3.1) 26. Thus, [N17A/F32T]-AnTx may serve as an excellent template for a novel therapeutically applicable peptide, and, interestingly, understanding the structural features of this peptide may lead to a better understanding of the nature of the selectivity of scorpion toxins between Kv1.3 and Kv1.2.…”

Kv1.3: a multifunctional channel with many pathological implications

“…Other potential Kv1.3 blockers for autoimmune diseases include BmKTX-D33H, an analog of Buthusmartensi's kaliotoxin (BmKTX) [20] and (N17A/F32T)-AnTx, an analog of the scorpion toxin anuroctoxin [21]. The use of these compounds is successful because Kv1.3 is so dominant in T EM lymphocytes that the concentration needed for the inhibition of this channel in these cells has no effect on leukocyte antimicrobial capabilities [5].…”

The Role of Proton Transport in Gating Current in a Voltage Gated Ion Channel, as Shown by Quantum Calculations

“…TOXINS: A number of toxins, e.g. particular toxins from specific snails (151)(152)(153), scorpions (154)(155)(156)(157)(158), and spiders (159)(160)(161), interact with voltage gated ion channels, sometimes with an extracellular loop of the VSD, or else possibly near the membrane interface. Suggestions of how these interactions with channels are relevant to gating have been proposed.…”