「様式３」P-31

The gating mechanism of human voltage-gated proton
channel Hv1 studied by fluorescence technology［１］Organization
Project Leader：Shu Jie Li（Nankai University, China）
Representative at RIE：
（Affiliation）
Participants：Masahito Yamazaki(Research Institute of Electronics）［２］Research Progress（10.5 points）
In this project, we investigated the role of Nand C-terminal domain of human voltage-gatedproton channel Hv1 in the gating mechanism
using fluorescence technology. The plan in details
is as follows:(1) To determine the interaction between Nand C-terminal domains, the N-terminal domain
was cloned, expressed and purified. (2) The
interaction between N- and C-terminal domains
was detected by GST-pull down and ITC. (3) The
interaction between divalent metal ions, classicalinhibitors of Hv1, with the C-terminal domain of
Hv1 was studied by CD spectra and fluorescence
lifetime spectroscopy.
［３］Results（10.5 points）(３－１) Research results(1) The N-terminal domain of Hv1 was successfully
expressed and purified.
The N-terminus of Hv1 fused with GST wasexpressed in E. coli.. A specific band with atime-dependent manner at a molecular weightof about 38 kDa corresponding to the molecularweight of the protein fused with GST (38.2 kDa),
was detected by the coomassie-stained
SDS-PAGE and the anti-GST-HRP . The fusion
protein induced with 0.5 mM IPTG was mainly
expressed in including body, while reducing theconcentration of IPTG could increase the soluble
expression of the protein. Moreover, addition of0.5% (v/v) Triton X-100 in lysis buffer obviously
increased the content of the protein insupernatant. The GST-fused protein wassuccessfully isolated from the GST moiety by
preScission protease. The isolated N-terminus ofHv1 from GST was further purified with an ion
exchange column and a gel filtration column.
(2) The interaction between N- and C-terminal
domains was detected by GST-pull down and ITC
In GST pull-down experiments, we foundthat the N-terminal domain of Hv1 interacts with
the C-terminal domain at pH 6.0. However, theN–terminal domain didn’t interact with theC-terminal domain at pH 7.5. These results
indicated that N-terminal domain interacts withC-terminal domain, and the interaction ispH-dependent.
To further confirm the interaction between
the N- and C-terminal domain of Hv1, ITC
experiments were carried out at 25 oC. The
binding of N-terminal domain to C-terminal
domain was an endothermic heat reaction. Fits of
the heat data for the titration was carried out
using the model which entails a single binding
event. The association constant (Ka) was ( 2.51±0.59)×105 M-1 with a n value of 1.38 ± 0.03.
(3) Divalent metal ions induce a conformationalchange of the C-terminal domain.
The internal C-terminal domain of Hv1 forms aparallel, two-stranded coiled-coil architecture. The
CD spectra in the range of 195–250 nm at 25 oC
were used to monitor the effect of binding ofdivalent metal ions on secondary structure of theprotein. At pH 5, the spectra of the protein without
divalent metal ions showed a characteristic ofa-helical secondary structure, and the molar
ellipticities at 222 and 208 nm were about -33557.5
and -32614.5 degrees cm2 dmol-1, respectively. In
the presence of divalent metal ions (75 μM), themolar ellipticities at 222 and 208 nm were bothdecreased. At pH 7, the molar ellipticities at 222
and 208 nm were more obviously changed by thetitration of divalent metal ions. These results
indicated that the interaction between divalent
metal ions and the C-terminal domain induced an
obvious secondary structural change of the protein
at pH 7.

(4) Interaction of divalent metal ions with theC-terminal domain
To estimate the interaction between divalent
metal ions and the C-terminal domain, weperformed the fluorescence lifetime spectroscopy
experiments. There are two His residues in theC-terminal domain of Hv1, His244 and His266, bothof which localize in the accessible region. Moreover,there is no Trp or Tyr residue within the C-terminaldomain. Then either of Phe247 or Leu262, which is
not in ‘a’ or ‘d’ position of the heptad repeat pattern
but close to His244 or His266, was mutated to Trp
residue to generate the mutant F247W or L262W,respectively. The CD spectra of the two mutants
were the same as the wild type, suggesting that thetwo mutants remained the characteristic ofa-helical secondary structure.
The fluorescence lifetime of F247W upon
addition of divalent metal ions were employed toinvestigate the binding of divalent metal ions to thesite close to His244 (site 1). The addition of divalent
metal ions quenched the fluorescence life time ofF247W at pH 7, except for Zn2+ which enhanced thefluorescence life time of F247W. KSV were obtained
from the Stern–Volmer plots. KSV is equal to kqs0
and the fluorescence lifetime of the biopolymer s0 is
10-8 s, so the quenching constant kq can becalculated. However, the maximum scatter collisionquenching constant of various quenchers with thebiopolymer is 2.0 × 1010 lmol-1s-1, and the rate
constants of protein quenching procedure initiatedby divalent metal ions were much greater than thekq of the scatter procedure, so the quenching should
be initiated by the formation of complex rather thanthe dynamic collision. The dissociation constants(KD) were estimated from the slopes. The order ofthe binding strength between divalent metal ionsand the site close to His244 (site 1) from strong toweak is Co2+, Ca2+, Ni2+, Mg2+ and Mn2+.
(３－２) Ripple effects and further developments(1) To investigate the role of the interaction
between N- and C-terminal domains in thechannel gating, the dynamic interaction between
N- and C-terminal domains during the activationof Hv1 could be captured by Förster Resonance
Energy Transfer (FRET).
(2) To determine the interaction between theN-terminal domain and PKC, the full-length Hv1
was used to be co-transfected with PKC. The
yellow fluorescence protein was divided into twopart reasonably and fused to Hv1 or PKC,
respectively. The interaction could be detected bybimolecularfluorescencecomplementationtechnology.
(3) To investigate the role of the interaction
between N-terminal domain and PKC in Hv1
enhance mold gating, the dynamic interaction
between N-terminal domain and PKC during theactivation of Hv1 could be captured by Förster
Resonance Energy Transfer (FRET).
［４］Achievements (List of Publications)（10.5
points）（１）Q. Zhao, S.J. Li, et al., The voltage-gatedproton channel Hv1 is expressed in pancreatic
islet β-cells and regulates insulin eecretion，Biochem. Biophys. Res. Commun., 468 (2015)746-751.
（２）Q. Zhao, S.J. Li, et al., Proton pumpinhibitors have pH-dependent effects on thethermostability of the carboxyl-terminal domainof voltage-gated proton channel Hv1, underreview.