Solution Structure of the HsapBK K⁺ Channel Voltage-Sensor Paddle Sequence^,

Abstract

Voltage-gated potassium channels open and close in response to changes in the membrane potential. In this study, we have determined the NMR solution structure of the putative S3b-S4 voltage-sensor paddle fragment, the part that moves to mediate voltage gating, of the HsapBK potassium channel in dodecylphosphocholine (DPC) micelles. This paper presents the first structure of the S3b-S4 fragment from a BK channel. Diffusion coefficients as determined from PFG NMR experiments showed that a well-defined complex between the peptide and DPC molecules was formed. The structure reveals a helix-turn-helix motif, which is in agreement with crystal structures of other voltage-gated potassium channels, thus indicating that it is feasible to study the isolated fragment. The paddle motifs generally contain several basic residues, implicated in the gating. The critical Arg residues in this structure all reside on the surface, which is in agreement with crystal structures of K(v) channels. Similarities in the structure of the S3b-S4 fragment in BK and K(v) channels as well as important differences are seen, which may be important for explaining the details in paddle movement within a bilayer.

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

“…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.…”

“…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] .…”

“…CD spectra of both KvAPp and HsapBKp in DPC micelles showed clear α-helical features, which were very similar to earlier observations in bicelles. 19 Addition of methanol did not affect the secondary structure significantly, suggesting that the methanol added to the LUV samples does not affect the secondary structure significantly either.…”

“…A third possibility is that the thickness of the lipid bilayer is significantly reduced by the paddle domain due to hydrophobic mismatch, which means that the movement needs not to be that large. 8,9,18 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.…”

Operation of the voltage sensor of a human voltage- and Ca 2+ -activated K + channel

“…Sequence homology (Fig. 1C), hydropathy analysis (12), and NMR spectroscopy (53) suggest that the extracellular linker between S3 and S4 in BK Ca is only three residues long. Thus, the voltage-evoked movements of S3, which bears the voltage-sensing D186 (44), could contribute to the fluorescence deflections reported from position 202.…”