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 alpha subunit of BK channel (HEKBKalpha) or both alpha and beta1 (HEKBKalphabeta1) subunits were heterologously expressed. Effects of these compounds (10 microM) on the membrane potential of HEKBKalphabeta1 were monitored by use of DiBAC(4)(3), a voltage-sensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid, and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BKalphabeta1 opening under whole-cell voltage clamp showed that these six compounds activated BKalphabeta1 in a very similar concentration range (1-10 microM); in contrast, abietic acid, sclareol, and methyl pimarate had no effect. PiMA did not affect the charybdotoxin-induced block of macroscopic BKalphabeta1 current. Single channel recordings of BKalphabeta1 in inside-out patches showed that 10 microM PiMA did not change channel conductance but significantly increased its open probability as a result of increase in sensitivity to Ca(2+) and voltage. Because coexpression of the beta1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKalpha subunit. PiMA was selective to BK over cloned small and intermediate Ca(2+) activated K(+) channels. In conclusion, PiMA (>1 microM) increases Ca(2+) and voltage-sensitivity of BKalpha 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 BKalpha activation.