Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach

Kodai Yajima; Takeshi Akiyoshi; Kazuho Sakamoto; Yoshiaki Suzuki; Takayuki Oka; Ayuko Imaoka; Hisao Yamamura; Junko Kurokawa; Hisakazu Ohtani

doi:10.1016/j.jphs.2023.08.008

Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach

J Pharmacol Sci. 2023 Nov;153(3):153-160. doi: 10.1016/j.jphs.2023.08.008. Epub 2023 Sep 1.

Authors

Kodai Yajima¹, Takeshi Akiyoshi², Kazuho Sakamoto³, Yoshiaki Suzuki⁴, Takayuki Oka⁵, Ayuko Imaoka⁶, Hisao Yamamura⁷, Junko Kurokawa⁸, Hisakazu Ohtani⁹

Affiliations

¹ Division of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30, Shibakoen Minato-ku, Tokyo, 105-8512, Japan. Electronic address: kodai.ss-kp8181@keio.jp.
² Division of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30, Shibakoen Minato-ku, Tokyo, 105-8512, Japan; Department of Clinical Pharmacy, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Electronic address: akiyoshi-tk@pha.keio.ac.jp.
³ Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka, 422-8526, Japan. Electronic address: kazuho@u-shizuoka-ken.ac.jp.
⁴ Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya, 467-8603, Japan. Electronic address: yoshisuz@phar.nagoya-cu.ac.jp.
⁵ Nanion Technologies Japan K.K., Tokyo Laboratory, Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-0056, Japan. Electronic address: Takayuki.Oka@nanion.jp.
⁶ Division of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30, Shibakoen Minato-ku, Tokyo, 105-8512, Japan. Electronic address: imaoka-ay@pha.keio.ac.jp.
⁷ Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya, 467-8603, Japan. Electronic address: yamamura@phar.nagoya-cu.ac.jp.
⁸ Department of Bio-Informational Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka, 422-8526, Japan. Electronic address: junkokuro@u-shizuoka-ken.ac.jp.
⁹ Division of Clinical Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30, Shibakoen Minato-ku, Tokyo, 105-8512, Japan; Department of Clinical Pharmacy, School of Medicine, Keio University, 35, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan; Department of Pharmacy, Keio University Hospital, 35, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan. Electronic address: ohtani-tky@umin.net.

PMID: 37770156
DOI: 10.1016/j.jphs.2023.08.008

Abstract

Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1. HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (Q_I). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (ρ) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CL_int) was calculated by dividing CL by Q_I·ρ. Q_I and ρ values were estimated to be 916 and 2.16, respectively, yielding CL_int of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters.

Keywords: Intrinsic activity; Patch clamp; Potassium channel; Targeted proteomics; Turnover rate.