Indirect (classical) and direct (novel) mechanisms of PIP₂ action on BK currents. Activated PLC via Gq, determined by either ligand binding to a Gq-coupled receptor (GPCR) or constitutive activity, cleaves PIP₂ into PIP₃, IP3, and DAG. IP3 releases Ca²⁺ from the sarcoplasmic reticulum (SR), raising Ca²⁺_i, whereas DAG activates PKC in the presence of Ca²⁺. BK channel modulation by PKC and Ca²⁺_i controls the degree of vascular myocyte contraction. Therefore, by generating DAG and IP3, PIP₂ indirectly modulates BK channels and, thus, vascular tone. We demonstrate that PIP₂ itself and other membrane phosphoinositides directly activate BK channels, that is, in the absence of cell integrity or organelles or in the continuous presence of cytosolic messengers. This activation involves the negative charges of the phosphoinositide headgroup and the sequence of positive residues RKK in the cbv1 S6–S7 cytosolic linker. However, PIP₂ activation is drastically and distinctly amplified by the channel accessory subunit of the β₁ type, which is abundantly expressed in smooth muscle. In intact myocytes, inhibition of PLC by U73122 increases paxilline-sensitive BK currents in the presence of a PI3 kinase blocker (wortmannin), PKC inhibitor (Ro 31-8220), and a selective blocker of the sarcoplasmic reticulum Ca²⁺-ATPase (thapsigargin). Under these conditions, PLC inhibition increases membrane PIP₂ (Narayanan et al., 1994), which leads to increased BK current in the intact cell (see main text).