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 ...
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