The RCK2 domain of the human BKCa channel is a calcium sensor

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intracellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming α subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca2+ sensitivity, possibly participating in the formation of a Ca2+-binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca2+-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed

A Common Ca2+-Driven Interdomain Module Governs Eukaryotic NCX Regulation

Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants

Potentiation of large conductance KCa channels by niflumic, flufenamic, and mefenamic acids

Large conductance calcium-activated K+ (KCa) channels are rapidly activated by niflumic acid dose-dependently and reversibly. External niflumic acid was about 5 times more potent than internal niflumic acid, and its action was characterized by an increase in the channel affinity for [Ca2+], a parallel left shift of the voltage-activation curve, and a decrease of the channel long-closed states. Niflumic acid applied from the external side did not interfere with channel block by charybdotoxin, suggesting that its site of action is not at or near the charybdotoxin receptor. Accordingly, partial tetraethylammonium blockade did not interfere with channel activation by niflumic acid. Flufenamic acid and mefenamic acid also stimulated KCa channel activity and, as niflumic acid, they were more potent from the external than from the internal side. Fenamates applied from the external side displayed the following potency sequence: flufenamic acid approximately niflumic acid >> mefenamic acid. These results indicate that KCa channels possess at least one fenamatereceptor whose occupancy leads to channel opening

Voltage-controlled gating in a large conductance Ca(2+)-sensitive K(+)channel (hslo)

Large conductance calcium- and voltage-sensitive K(+) (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca(2+) favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca(2+) concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges