beta 1-Subunits enhance the gating properties of large-conductance Ca2+-activated K+ channels (BKCa) formed by -subunits. In arterial vascular smooth muscle cells (VSMCs), 1-subunits are vital in coupling SR-generated Ca2+ sparks to BKCa activation, affecting contractility and blood pressure. Studies in cremaster and cerebral VSMCs show heterogeneity of BKCa activity due to apparent differences in the functional 1-subunit:-subunit ratio. To define these differences, studies were conducted at the single-channel level while siRNA was used to manipulate specific subunit expression. 1 modulation of the -subunit Ca2+ sensitivity was studied using patch-clamp techniques. BKCa channel normalized open probability (NPo) versus membrane potential (Vm) curves were more left-shifted in cerebral versus cremaster VSMCs as cytoplasmic Ca2+ was raised from 0.5 to 100 m. Calculated V1/2 values of channel activation decreased from 72.0 +/- 6.1 at 0.5 m Ca2+i to 89 +/- 9 mV at 100 m Ca2+i in cerebral compared with 101 +/- 10 to 63 +/- 7 mV in cremaster VSMCs. Cremaster BKCa channels thus demonstrated an approximate to 2.5-fold weaker apparent Ca2+ sensitivity such that at a value of Vm of 30 mV, a mean value of [Ca2+]i of 39 m was required to open half of the channels in cremaster versus 16 m[Ca2+]i in cerebral VSMCs. Further, shortened mean open and longer mean closed times were evident in BKCa channel events from cremaster VSMCs at either 30 or 30 mV at any given [Ca2+]. 1-Subunit-directed siRNA decreased both the apparent Ca2+ sensitivity of BKCa in cerebral VSMCs and the appearance of spontaneous transient outward currents. The data are consistent with a higher ratio of 1-subunit:-subunit of BKCa channels in cerebral compared with cremaster VSMCs. Functionally, this leads both to higher Ca2+ sensitivity and NPo for BKCa channels in the cerebral vasculature relative to that of skeletal muscle.