Vol. 62, Issue 4, 836-846, October 2002

Molecular Basis of Pimarane Compounds as Novel Activators of Large-Conductance Ca²⁺-Activated K⁺ Channel -Subunit

Yuji Imaizumi, Kazuho Sakamoto, Aki Yamada, Aya Hotta, Susumu Ohya, Katsuhiko Muraki, Masanobu Uchiyama, and Tomohiko Ohwada

Departments of Molecular and Cellular Pharmacology (Y.J., K.S., A.Y., A.H., S.O., K.M.), and Organic and Medicinal Chemistry (M.U., T.O.), Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan; and Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (M.U., T.O.)

Effects of pimaric acid (PiMA) and eight closely related compounds on large-conductance K⁺ (BK) channels were examined using human embryonic kidney (HEK) 293 cells, in which either the  subunit of BK channel (HEKBK) or both  and 1 (HEKBK1) subunits were heterologously expressed. Effects of these compounds (10 µM) on the membrane potential of HEKBK1 were monitored by use of DiBAC₄(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid, and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BK1 opening under whole-cell voltage clamp showed that these six compounds activated BK1 in a very similar concentration range (1-10 µM); in contrast, abietic acid, sclareol, and methyl pimarate had no effect. PiMA did not affect the charybdotoxin-induced block of macroscopic BK1 current. Single channel recordings of BK1 in inside-out patches showed that 10 µM PiMA did not change channel conductance but significantly increased its open probability as a result of increase in sensitivity to Ca²⁺ and voltage. Because coexpression of the 1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BK subunit. PiMA was selective to BK over cloned small and intermediate Ca²⁺ activated K⁺ channels. In conclusion, PiMA (>1 µM) increases Ca²⁺ and voltage-sensitivity of BK when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BK activation.