H þ currents when expressed in HEK-293 cells. The data indicate that H þ transfer is unlikely to be mediated by H þ shuttling through pairs of nearly acidic side chains, but are consistent with the hypothesis that 'aqueous' or water-wire proton transfer is the primary mechanism of H þ conduction in Hv1. Different mutant combinations produce similar defects in pH-dependent gating, suggesting that interactions among networked side chains confer emergent biophysical properties in Hv1. Experimental data were used to build a refined activatedstate Hv1 VS model structure (Hv1 J) that was subjected to molecular dynamics simulation. Although Hv1 J is similar overall to Hv1 B (Ramsey, et al., 2010), differences in the positions of specific side chains suggest a channel-opening conformational rearrangement that follows S4 movement, as predicted from gating currents (De La Rosa, et al., 2016).

Herein, we report the synthesis and mass spectrometry studies of several N-alkylbenzenesulfonamides structurally related to sulfanilic acid. The compounds were synthesized using a modified Schotten-Baumann reaction coupled with Meisenheimer arylation. Sequential mass spectrometry by negative mode electrospray ionization (ESI(-)-MS/MS) showed the formation of sulfoxylate anion (m/z 65) observed in the mass spectrum of p-chloro-N-alkylbenzenesulfonamides. Investigation of the unexpected loss of two water molecules, as observed by electron ionization mass spectrometry (EI-MS) analysis of p-(N-alkyl)lactam sulfonamides, led to the proposal of corresponding fragmentation pathways. These compounds showed loss of neutral iminosulfane dioxide molecule (M-79) with formation of ions observed at m/z 344 and 377. These ions were formed by rearrangement on ESI(+)-MS/MS analysis. Some of the molecules showed antagonistic activity against Kv3.1 voltage-gated potassium channels.

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assess the significance of toxin residues. Here, we show that geneticallyencoded, membrane-tethered toxins (T-toxins) allow rapid screening of the residues to determine the mechanistic basis for toxin-ion channel interaction, including kinetics parameters of interaction, through study of two channels, KcsA and Kv1.3. First, the structure of the sea anemone type I (SAK1) toxin HmK is determined by NMR. Then, T-HmK residues are scanned by point mutation to identify the seven residues in close contact with the KcsA pore. T-HmK-Lys 22 is shown to interact with K þ ions traversing the permeation pathway from the cytoplasm conferring voltage-dependence to the toxin offrate, a classic mechanism we observe as well for HmK peptide with both KcsA and K V 1.3 channels. In contrast, two related SAK1 toxins, Hui1 and ShK, block KcsA and K V 1.3, respectively, via an arginine rather than the canonical lysine, when tethered as well as free peptides. Our study changes a long-held conclusion about the voltage-dependent mechanism of ShK-Kv1.3 interaction, and demonstrates that there are two orientations for SAK1 toxins in K þ channel pores.

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