Carbon monoxide activates KCachannels in newborn arteriole smooth muscle cells by increasing apparent Ca2+sensitivity of α-subunits

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Abstract
Carbon monoxide (CO) is a gaseous vasodilator produced by many cell types, including endothelial and smooth muscle cells. The goal of the present study was to investigate signaling mechanisms responsible for CO activation of large-conductance Ca2+-activated K+ (KCa) channels in newborn porcine cerebral arteriole smooth muscle cells. In intact cells at 0 mV, CO (3 μM) or CO released from dimanganese decacarbonyl (10 μM), a novel light-activated CO donor, increased KCa channel activity 4.9- or 3.5-fold, respectively. KCa channel activation by CO was not blocked by 1-H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (25 μM), a soluble guanylyl cyclase inhibitor. In inside-out patches at 0 mV, CO shifted the Ca2+ concentration-response curve for KCa channels leftward and decreased the apparent dissociation constant for Ca2+ from 31 to 24 μM. Western blotting data suggested that the low Ca2+ sensitivity of newborn KCa channels may be due to a reduced β-subunit-to-α-subunit ratio. CO activation of KCa channels was Ca2+ dependent. CO increased open probability 3.7-fold with 10 μM free Ca2+ at the cytosolic membrane surface but only 1.1-fold with 300 nM Ca2+. CO left shifted the current-voltage relationship of cslo-α currents expressed in HEK-293 cells, increasing currents 2.2-fold at +50 mV. In summary, data suggest that in newborn arteriole smooth muscle cells, CO activates low-affinity KCa channels via a direct effect on the α-subunit that increases apparent Ca2+ sensitivity. The optimal tuning by CO of the micromolar Ca2+ sensitivity of KCa channels will lead to preferential activation by signaling modalities, such as Ca2+ sparks, which elevate the subsarcolemmal Ca2+ concentration within this range.