Poster: Membrane Transport

Abs # 1198: Topogenesis of the voltage sensor in Arabidopsis K channel, KAT1

Presenter:	Uozumi, Nobuyuki , uozumi@agr.nagoya-u.ac.jp
Authors	Uozumi, Nobuyuki  (A) (B)  Sato, Yoko  (B)   Sakaguchi, Masao  (C)   Goshima, Shinobu  (A)   Nakamura, Tatsunosuke  (D)
Affiliations:	(A): Bioscience Center, Nagoya University (B): Graduate School of Bioagricultural Sciences, Nagoya University (C): Department of Molecular Biology, Graduate School of Medical Science, Kyushu University (D): Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences
Web Site:	http://www.agr.nagoya-u.ac.jp/~donew/uozumiG/index.html

KAT1 is a well characterized plant hyperpolarization-activated K channel. Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes. The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3. The process involved in the integration of the protein into the membrane remains to be elucidated. In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane. A D95V mutation in S2 showed less S3-S4 integration into the membrane, while a D105V mutation allowed S4 to be released into the ER lumen. These results indicate that D95 assists in the membrane insertion of S3-S4 and that D105 helps in preventing S4 from being releasing into the ER lumen. The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen. A series of constructs containing different C-terminal truncations of S4 showed that R174 was required for correct integration of S3 and S4 into the membrane. Interactions between D105 and R171 and between negative residues in S2 or S3 and R174 may be formed transiently during membrane integration. These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane. Uozumi et al., PNAS 95, 9773-9778 (1998); Sato et al. PNAS 99, 60-65 (2002); Sato et al. JBC, in press; (2003)