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 α subunit of BK channel (HEKBKα) or both α and β1 (HEKBKαβ1) subunits were heterologously expressed. Effects of these compounds (10 μM) on the membrane potential of HEKBKαβ1 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 BKαβ1 opening under whole-cell voltage clamp showed that these six compounds activated BKαβ1 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 BKαβ1 current. Single channel recordings of BKαβ1 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 β1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BKα 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 BKα 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 BKα activation.

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.

In articular cartilage inflammation, histamine release from mast cells is a key event. It can enhance cytokine production and matrix synthesis and also promote cell proliferation by stimulating chondrocytes. In this study, the functional impact of Ca²⁺-activated K⁺(K_Ca) channels in the regulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in chondrocytes in response to histamine was examined using OUMS-27 cells, as a model of chondrocytes derived from human chondrosarcoma. Application of histamine induced a significant [Ca²⁺]_irise and also membrane hyperpolarization, and both effects were mediated by the stimulation of H₁receptors. The histamine-induced membrane hyperpolarization was attenuated to ∼50% by large-conductance K_Ca(BK) channel blockers, and further reduced by intermediate (IK) and small conductance K_Ca(SK) channel blockers. The tonic component of histamine-induced [Ca²⁺]_irise strongly depended on the presence of extracellular Ca²⁺([Ca²⁺]_o) and was markedly reduced by La³⁺or Gd³⁺but not by nifedipine. It was significantly attenuated by BK channel blockers, and further blocked by the cocktail of BK, IK, and SK channel blockers. The K_Cablocker cocktail also significantly reduced the store-operated Ca²⁺entry (SOCE), which was induced by Ca²⁺addition after store-depletion by thapsigargin in [Ca²⁺]_ofree solution. Our results demonstrate that the histamine-induced membrane hyperpolarization in chondrocytes due to K_Cachannel activation contributes to sustained Ca²⁺entry mainly through SOCE channels in OUMS-27 cells. Thus, K_Cachannels appear to play an important role in the positive feedback mechanism of [Ca²⁺]_iregulation in chondrocytes in the presence of articular cartilage inflammation.

Functional coupling between large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca²⁺ release through RyRs (Ca²⁺ sparks) and subsequent BK_Ca channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BK_Ca channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca²⁺ microdomain function. Approximately half of all Ca²⁺ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-β-cyclodextrin treatments significantly reduced coupling efficiency between Ca²⁺ sparks and BK_Ca channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BK_Ca channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca²⁺ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BK_Ca channels, converts local Ca²⁺ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.