Large-conductance (BK type) Ca(2+)-dependent K(+) channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing. BK channels are activated by membrane depolarization and intracellular Ca(2+) and Mg(2+) (refs 6-10). The energy provided by voltage, Ca(2+) and Mg(2+) binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways. Here we report a molecular investigation of a Mg(2+)-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg(2+)-binding site in the RCK/Rossman fold domain -- an intracellular structural motif that immediately follows the activation gate S6 helix -- is responsible for Mg(2+)-dependent activation. Mutations that impair or abolish Mg(2+) sensitivity do not affect Ca(2+) sensitivity, and vice versa. These results indicate distinct structural pathways for Mg(2+)- and Ca(2+)-dependent activation and suggest a possible mechanism for the coupling between Mg(2+) binding and channel opening.

Summary Ca2+-activated BK channels modulate neuronal activities including spike frequency adaptation and synaptic transmission. Previous studies found that Ca2+ binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca2+ binding opens the gate over this distance remains unknown. By studying an Asp to Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca2+ binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca2+ sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca2+ binding to channel opening.

The S4 transmembrane segment is the primary voltage sensor in voltage-dependent ion channels. Its movement in response to changes in membrane potential leads to the opening of the activation gate, which is formed by a separate structural component, the S6 segment. Here we show in voltage-, Ca 2؉ -, and Mg 2؉ -dependent, large conductance K ؉ channels that the S4 segment participates not only in voltage-but also Mg 2؉ -dependent activation. Mutations in S4 and the S4-S5 linker alter voltagedependent activation and have little or no effect on activation by micromolar Ca 2؉ . However, a subset of these mutations in the C-terminal half of S4 and in the S4-S5 linker either reduce or abolish the Mg 2؉ sensitivity of channel gating. Cysteine residues substituted into positions R210 and R213, marking the boundary between S4 mutations that alter Mg 2؉ sensitivity and those that do not, are accessible to a modifying reagent [sodium (2-sulfonatoethyl)methane-thiosulfonate] (MTSES) from the extracellular and intracellular side of the membrane, respectively, at ؊80 mV. This implies that interactions between S4 and a cytoplasmic domain may be involved in Mg 2؉ -dependent activation. These results indicate that the voltage sensor is critical for Mg 2؉ -dependent activation and the coupling between the voltage sensor and channel gate is a converging point for voltage-and Mg 2؉ -dependent activation pathways.

Intracellular Ca2+ release events ('Ca 2+ sparks') and transient activation of large-conductance Ca 2+ -activated potassium (BK) channels represent an important vasodilator pathway in the cerebral vasculature. Considering the frequent occurrence of cerebral artery constriction after subarachnoid hemorrhage (SAH), our objective was to determine whether Ca 2+ spark and BK channel activity were reduced in cerebral artery myocytes from SAH model rabbits. Using laser scanning confocal microscopy, we observed B50% reduction in Ca 2+ spark activity, reflecting a decrease in the number of functional Ca 2+ spark discharge sites. Patch-clamp electrophysiology showed a similar reduction in Ca 2+ spark-induced transient BK currents, without change in BK channel density or single-channel properties. Consistent with a reduction in active Ca 2+ spark sites, quantitative real-time PCR and western blotting revealed decreased expression of ryanodine receptor type 2 (RyR-2) and increased expression of the RyR-2-stabilizing protein, FKBP12.6, in the cerebral arteries from SAH animals. Furthermore, inhibitors of Ca 2+ sparks (ryanodine) or BK channels (paxilline) constricted arteries from control, but not from SAH animals. This study shows that SAH-induced decreased subcellular Ca 2+ signaling events disable BK channel activity, leading to cerebral artery constriction. This phenomenon may contribute to decreased cerebral blood flow and poor outcome after aneurysmal SAH. Keywords: cerebral aneurysm; FKBP12.6; potassium channels; ryanodine receptors; vascular smooth muscle; vasospasm IntroductionCerebral aneurysm rupture and the ensuing subarachnoid hemorrhage (SAH) has an enormous impact on individuals and society, with 30-day mortality rates approaching 50% and the majority of survivors left with moderate-to-severe disability (Hop et al, 1997). For decades, 'angiographically defined' cerebral vasospasm of conduit arteries ( > 1 mm in diameter) has been considered to be the major contributor to death and disability in SAH patients surviving the initial intracranial bleed. However, recent evidence indicates that factors other than large-artery vasospasm contribute to SAH-induced pathologies (Macdonald et al, 2007). Additional factors contributing to the deleterious consequences of aneurysmal SAH may include global transient ischemia, early brain injury, disruption of the bloodbrain barrier, and activation of inflammatory pathways (Ostrowski et al, 2006;Prunell et al, 2005). It has now been realized that SAH may also impact small-diameter arteries and arterioles, i.e., those involved in the autoregulation of cerebral blood flow (Hattingen et al, 2008;Ishiguro et al, 2002;Ohkuma et al, 2000).In resistance arteries from healthy animals, vasoconstrictor stimuli such as physiologic increases in intravascular pressure lead to smooth muscle membrane potential depolarization, increased voltage-dependent Ca 2+ channel (VDCC) activity, and elevated global cytosolic calcium (Knot and Nelson, 1998 , the NIH (R01 HL078983, R01 HL078983-05S1, R01 ...

Large conductance, voltage- and Ca2+-activated K+ (BKCa) channels regulate blood vessel tone, synaptic transmission, and hearing owing to dual activation by membrane depolarization and intracellular Ca2+. Similar to an archeon Ca2+-activated K+ channel, MthK, each of four α subunits of BKCa may contain two cytosolic RCK domains and eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient with one another upon Ca2+ binding to change the gating ring conformation and open the activation gate. Here we report that the conformational changes of the NH2 terminus of RCK1 (AC region) modulate BKCa gating. Such modulation depends on Ca2+ occupancy and activation states, but is not directly related to the Ca2+ binding sites. These results demonstrate that AC region is important in the allosteric coupling between Ca2+ binding and channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an important step in addition to the reorientation of RCK domains leading to the opening of the BKCa activation gate. Our observations are consistent with a mechanism for Ca2+-dependent activation of BKCa channels such that the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca2+; Ca2+ binding and depolarization relieve this inhibition.

Krishnamoorthy G, Sonkusare SK, Heppner TJ, Nelson MT. Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries. Am J Physiol Heart Circ Physiol 306: H981-H988, 2014. First published February 7, 2014 doi:10.1152 doi:10. /ajpheart.00866.2013polarized smooth muscle cells of pressurized cerebral arteries, ryanodine receptors (RyRs) generate "Ca 2ϩ sparks" that activate largeconductance, Ca 2ϩ -, and voltage-sensitive potassium (BK) channels to oppose pressure-induced (myogenic) constriction. Here, we show that BK channels and RyRs have opposing roles in the regulation of arterial tone in response to sympathetic nerve activation by electrical field stimulation. Inhibition of BK channels with paxilline increased both myogenic and nerve-induced constrictions of pressurized, resistance-sized mesenteric arteries from mice. Inhibition of RyRs with ryanodine increased myogenic constriction, but it decreased nerveevoked constriction along with a reduction in the amplitude of nerve-evoked increases in global intracellular Ca 2ϩ . In the presence of L-type voltage-dependent Ca 2ϩ channel (VDCC) antagonists, nerve stimulation failed to evoke a change in arterial diameter, and BK channel and RyR inhibitors were without effect, suggesting that nerve-induced constriction is dependent on activation of VDCCs. Collectively, these results indicate that BK channels and RyRs have different roles in the regulation of myogenic versus neurogenic tone: whereas BK channels and RyRs act in concert to oppose myogenic vasoconstriction, BK channels oppose neurogenic vasoconstriction and RyRs augment it. A scheme for neurogenic vasoregulation is proposed in which RyRs act in conjunction with VDCCs to regulate nerve-evoked constriction in mesenteric resistance arteries.

Regulation of cochlear blood flow is critical for hearing due to its exquisite sensitivity to ischemia and oxidative stress. Many forms of hearing loss such as sensorineural hearing loss and presbyacusis may involve or be aggravated by blood flow disorders. Animal experiments and clinical outcomes further suggest that there is a gender preference in hearing loss, with males being more susceptible. Autoregulation of cochlear blood flow has been demonstrated in some animal models in vivo, suggesting that similar to the brain, blood vessels supplying the cochlea have the ability to control flow within normal limits, despite variations in systemic blood pressure. Here, we investigated myogenic regulation in the cochlear blood supply of the Mongolian gerbil, a widely used animal model in hearing research. The cochlear blood supply originates at the basilar artery, followed by the anterior inferior cerebellar artery, and inside the inner ear, by the spiral modiolar artery and the radiating arterioles that supply the capillary beds of the spiral ligament and stria vascularis. Arteries from male and female gerbils were isolated and pressurized using a concentric pipette system. Diameter changes in response to increasing luminal pressures were recorded by laser scanning microscopy. Our results show that cochlear vessels from male and female gerbils exhibit myogenic regulation but with important differences. Whereas in male gerbils, both spiral modiolar arteries and radiating arterioles exhibited pressure-dependent tone, in females, only radiating arterioles had this property. Male spiral modiolar arteries responded more to L-NNA than female spiral modiolar arteries, suggesting that NO-dependent mechanisms play a bigger role in the myogenic regulation of male than female gerbil cochlear vessels.

Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-NG-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.