Membrane-Perturbing Properties of Two Arg-Rich Paddle Domains from Voltage-Gated Sensors in the KvAP and HsapBK K⁺ Channels

Abstract

Voltage-gated K + channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains,KvAPp from the K v channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy.We show that both paddle domains induce calcein leakage in large unilamellar vesicles and we suggest that this leakage represent a general thinning of the bilayer making movement of the whole paddle domain plausible. That HsapBKp induces more leakage than KvAPp, may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic/hydrophobic interface and disturb tight packing. In magnetically aligned bicelles KvAPp increases the order along the whole acyl chain, while HsapBKp affects the morphology, also indicating thatKvAPp adapts more to the lipid environment. Relaxation NMR measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is wellstructured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility for HsapBKp to undergo structural transitions is also shown by relaxation NMR, which may be of importance for the gating mechanism. The aim of present study was to investigate how paddle domains from two different channels, the K v channel KvAP (from Aeropyrum pernix, GI:14601099) 2,3,6 and the BK channel HsapBK (from Homo sapiens, GI:2570854) 19 interact with membranes, to be able to draw general conclusions about the mechanism of gating and to be able to compare paddle domains from two different subtypes. We have previously determined the high-resolution NMR structure of the paddle-domain from HsapBK (HsapBKp, corresponding to HsapBK(233-260)) in DPC micelles. 19 The solution structure revealed a helix-turn-helix structure similar to the structure of the corresponding sequence in KvAP as determined by X-ray crystallography. 2,3,19 In the present study the interactions between the paddle domains from KvAP (KvAPp, corresponding to KvAP(112-146)) and from HsapBK (HsapBKp) and lipids were studied by fluorescence, circular dichroism (CD) and NMR spectroscopy. To investigate bilayer perturbation induced by the paddle domains, leakage of the fluorophore calcein from large unilamellar vesicles (LUVs) was monitored by fluorescence spectroscopy [20][21][22][23] . Circular dichroism was used to examine the lipiddependent structure induction, while deuterium NMR spectra of acyl-chain deuterated DMPC and DMPG in magnetically aligned bicelles were acquired to investigate the effects of the paddle domains on bilayer order and integrity. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (...

A surface plasmon resonance approach to monitor toxin interactions with an isolated voltage-gated sodium channel paddle motif

“…CD measurements reveal that the peptide folds and mainly adopts an α-helical conformation (∼53%; Fig. 3 A ), an observation that fits well with an earlier report about the NMR solution structure of an isolated paddle motif from the HsapBK K v channel ( Unnerståle et al, 2009 , 2012 ). To serve as a control for toxins interacting exclusively with the VSD IV paddle motif, we also synthesized the previously defined VSD II paddle motif of rNa v 1.2a, including a V843A gain-of-function mutation and a C-terminal K-linked biotin group ( Bosmans et al, 2008 ; Zhang et al, 2011 ).…”

Purification and Structural Study of the Voltage-Sensor Domain of the Human KCNQ1 Potassium Ion Channel

“…This is reasonable in light of the higher polarity of S4 relative to the polarity of the other TM segments. Finally, it is believed that the membrane is thinned in the vicinity of the S4 transmembrane segment, such that water can permeate into what would normally be the plane of the membrane to hydrate the multiply charged residues of this segment, − an effect that may also apply to micellar conditions and that would also be expected to contribute to enhanced H–D exchange rates.…”

The Magic of Bicelles Lights Up Membrane Protein Structure

“…The interaction of two different Arg-rich paddle domains of voltage gated K+ channels with the lipid bilayer have been characterized in oriented bicelles. 266 The myristoylated 14-residue peptide Cat14 from the catalytic unit of cAMP-dependent protein kinase A was incorporated in q = 3.5 bicelles to study interaction with lipids by 2 H NMR. 267 A myristoylated N-terminal 14-residue peptide from pp60 ν-src was studied in neutral and acidic bicelles.…”

“…In addition, this study used isotropic bicelles to characterize membrane binding affinity of hERG. The interaction of two different Arg-rich paddle domains of voltage gated K+ channels with the lipid bilayer have been characterized in oriented bicelles . The myristoylated 14-residue peptide Cat14 from the catalytic unit of cAMP-dependent protein kinase A was incorporated in q = 3.5 bicelles to study interaction with lipids by 2 H NMR .…”