Portability of paddle motif function and pharmacology in voltage sensors

Abstract

Voltage-sensing domains enable membrane proteins to sense and react to changes in membrane voltage. Although identifiable S1-S4 voltage-sensing domains are found in an array of conventional ion channels and in other membrane proteins that lack pore domains, the extent to which their voltage sensing mechanisms are conserved is unknown. Here we show that the voltage-sensor paddle, a motif composed of S3b and S4 helices, can drive channel opening with membrane depolarization when transplanted from an archaebacterial voltage-activated potassium (Kv) channel (KvAP) or voltagesensing domain proteins (Hv1 and Ci-VSP) into eukaryotic Kv channels. Tarantula toxins that partition into membranes can interact with these paddle motifs at the protein-lipid interface and similarly perturb voltage sensor activation in both ion channels and voltage-sensing domain proteins. Our results show that paddle motifs are modular, that their functions are conserved in voltage sensors, and that they move in the relatively unconstrained environment of the lipid membrane. The widespread targeting of voltage-sensor paddles by toxins demonstrates that this modular structural motif is an important pharmacological target.Ion channels that open and close in response to changes in membrane voltage have a modular architecture, with a central pore domain that determines ion selectivity, and four surrounding voltage sensing domains that move in response to changes in membrane voltage to drive opening of the pore 1-5 (Fig 1a). Although X-ray structures have now been solved for two voltage-activated potassium (Kv) channels 1, 6-9 , the structural basis of voltage sensing remains controversial 10-12 . A seminal observation in the X-ray structures of the KvAP channel, an archaebacterial Kv channel from Aeropyrum pernix, was that the S3b helix and the charge-bearing S4 helix within the voltage-sensing domain form a helix-turn-helix structure, termed the paddle motif 1, 8, 9 . Studies on KvAP 1, 9, 13-16 suggest that this voltage-sensor paddle is buried in the membrane and that it moves at the protein-lipid interface, which contrasts with models for eukaryotic Kv channels where the S4 helix is protected from membrane lipids by other regions of the protein 10-12, 17-21 . Voltage-sensing domains have also recently been described in voltage-sensing proteins that lack associated pore domains 5, 22, 23 . In Ci-VSP the voltage-sensing domain is coupled to a phosphatase domain and in Hv1 the voltage-sensing domain itself is thought to function as a proton channel. Here we explore whether the mechanisms of voltage-sensing are conserved between the distantly related eukaryotic and Chimeras between Kv channelsWe began by generating chimeras between the archaebacterial KvAP channel 24 and the eukaryotic Kv2.1 channel from rat brain 25 to define the interchangeable regions. Transfer of the KvAP pore domain into Kv2.1 results in channels that open in response to membrane depolarization (Fig 1a ; Supplementary Fig 1 and Table 1), so long as the S4-S5 linker hel...

Potassium Ion Channels: Could They Have Evolved from Viruses?

“…Only small portions of the two domains, e.g. the "paddle" motif of the VS domain and the P loop with most of the transmembrane domains of the pore, could be exchanged between K + channels (Lu et al, 2001;Alabi et al, 2007). These results led to the conclusion that membrane modules have coevolved so intimately in Kv proteins that it is impossible to exchange them entirely without losing the function of the chimeric channels.…”

Functional diversity of potassium channel voltage-sensing domains

“…[9][10][11] In fact, VSDs exist as independent gene products, coupled to other kinds of proteins, as demonstrated by the voltage-sensitive phosphatases 12 and can even mediate proton transport, as shown in the voltage-sensitive proton channels. 13,14 As diverse as the functions of VSDs may be, they all work by converting electrostatic energy into conformation changes and it seems that the basic mechanism of voltage sensing is the same.…”

Functionality of the voltage-gated proton channel truncated in S4

“…S4 of both mVSOP and Hv1 contain three arginines (R201, R204, and R207 in mVSOP, which are equivalent to R205, R208, and R211 in Hv1) that correspond to positively charged amino acids in S4 of voltagegated ion channels. Moreover, the area called the "paddle region," which is comprised of parts of S3 and S4 in Hv1, can replace the corresponding region in voltage-gated potassium channels without affecting voltage-dependent ion channel activity, indicating that a paddle motif-like structure is conserved among Hv and voltagegated potassium channels (20).Earlier studies by ourselves and others showed that Hv channels form dimers via their cytoplasmic regions (21−23), although monomeric Hv channels lacking their cytoplasmic regions remain functional and exhibit robust Hv channel activity (21,22). This motivated us to further define the minimum unit for Hv channel activity.…”