Heme Is a Carbon Monoxide Receptor for Large-Conductance Ca ²⁺ -Activated K ⁺ Channels

Abstract

Abstract-Carbon monoxide (CO) is an endogenous paracrine and autocrine gaseous messenger that regulates physiological functions in a wide variety of tissues. CO induces vasodilation by activating arterial smooth muscle largeconductance Ca 2ϩ -activated potassium (BK Ca ) channels. However, the mechanism by which CO activates BK Ca channels remains unclear. Here, we tested the hypothesis that CO activates BK Ca channels by binding to channel-bound heme, a BK Ca channel inhibitor, and altering the interaction between heme and the conserved heme-binding domain (HBD) of the channel ␣ subunit C terminus. Data obtained using thin-layer chromatography, spectrophotometry, mass spectrometry (MS), and MS-MS indicate that CO modifies the binding of reduced heme to the ␣ subunit HBD. In contrast, CO does not alter the interaction between the HBD and oxidized heme (hemin), to which CO cannot bind. Consistent with these findings, electrophysiological measurements of native and cloned (cbv) cerebral artery smooth muscle BK Ca channels show that CO reverses BK Ca channel inhibition by heme but not by hemin. Site-directed mutagenesis of the cbv HBD from CKACH to CKASR abolished both heme-induced channel inhibition and CO-induced activation. Furthermore, on binding CO, heme switches from being a channel inhibitor to an activator. These findings indicate that reduced heme is a functional CO receptor for BK Ca channels, introduce a unique mechanism by which CO regulates the activity of a target protein, and reveal a novel process by which a gaseous messenger regulates ion channel activity. Key Words: vascular smooth muscle Ⅲ vasodilation Ⅲ potassium channels Ⅲ signal transduction L arge-conductance Ca 2ϩ -activated potassium (BK Ca ) channels regulate the physiological functions of many tissues, including smooth muscle, neuronal and endocrine cells. 1 BK Ca channels are typically composed of pore-forming ␣ subunits that are encoded by the Slo1 (or KCNMA1) gene, and accessory ␤ subunits that modulate channel gating. 2 In smooth muscle cells, BK Ca channels regulate cellular membrane potential and, thus, Ca 2ϩ entry through voltage-gated Ca 2ϩ channels, providing a mechanism to control contractility. 3 BK Ca channel activity is regulated by a variety of signaling molecules, including intracellular Ca 2ϩ ([Ca 2ϩ ] i ), protein kinases, 4 -6 tyrosine kinases, 7 cytochrome P-450 metabolites of arachidonic acid, 8 and heme. 9 BK Ca channels are also activated by physiologically relevant gases, including O 2 , CO, and NO. Although these gases can use cellular signaling pathways, O 2 , CO, and NO also activate BK Ca channels in cell-free membrane patches isolated from the intracellular milieu. 10 -12 Carbon monoxide is a physiological paracrine and autocrine messenger and neurotransmitter produced by heme oxygenase (HO) catalyzed metabolism of heme. [13][14][15] Heme is found in virtually all cell types, and many cell types contain HO-2, including arterial smooth muscle cells, endothelial cells, and neurons. CO regulates a variety o...

The RCK1 high-affinity Ca 2+ sensor confers carbon monoxide sensitivity to Slo1 BK channels

“…The modulatory effect of heme diminishes when the putative heme-binding sequence, 612 CKACH 616 , in the linker segment between the cytoplasmic RCK1 and RCK2 domains, is disrupted (19,21). This result is consistent with the idea that histidine is a common ligand for heme (25).…”

“…If CO stimulated the channel activity by interfering with the inhibitory effect of heme on the channel mediated by the sequence 612 CKACH 616 , mutation of this sequence should eliminate the stimulatory effect of CO. In the mutants H616R and C615S:H616R, both of which show diminished sensitivities to heme (21), application of CO robustly increased open probabilities (Fig. 1C).…”

“…Although increasing evidence suggests that the Slo1 BK channel is directly stimulated by CO without any requirement for a cGMP-dependent signaling pathway (8,10), how the allosteric gating mechanism of the Slo1 BK channel is directly altered by CO has not been clearly elucidated. The finding that heme is capable of acutely binding to the Slo1 BK channel and often inhibits the channel activity (19,20) prompted Jaggar et al (21) to examine whether the reversal of the inhibitory effect of heme by CO underlies the stimulatory action of CO on the Slo1 BK channel. Consistent with the widely held view that CO interacts only with reduced heme in hemoproteins (2,3), it was suggested that CO reverses the inhibitory effect of heme on the channel only under a reducing condition (21).…”

An oxygen-sensitive mechanism in regulation of epithelial sodium channel

“…Previous studies on the usage of haem have used 5-30 M haem in extracellular medium (16,17) and 1-200 nM haem in cytoplasmic medium (13,14,18) to interact with ion channels. The half-maximal inhibitory concentration (IC 50 ) of 23.3 nM reported here is consistent with these values and comparable with the dissociation constant values of other haemoproteins and haem-binding proteins (13,(19)(20)(21).…”

Glutamate-induced calcium signals stimulate CO production in piglet astrocytes

“…CO causes vasodilation by stimulating Ca 2ϩ sparks and large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channels and by increasing effective coupling between Ca 2ϩ sparks and BK Ca channels in arterial smooth muscle cells (17,18,25,38). Physiologically, CO is produced by heme oxygenase (HO)-catabolized breakdown of heme to CO, iron, and biliverdin (27).…”