Circular Dichroism Spectroscopy: Units (original) (raw)
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A novel calcium-sensing domain in the BK channel
Biophysical Journal, 1997
The high-conductance Ca2"-activated K+ channel (mSlo) plays a vital role in regulating calcium entry in many cell types. mSlo channels behave like voltage-dependent channels, but their voltage range of activity is set by intracellular free calcium. The mSlo subunit has two parts: a "core" resembling a subunit from a voltage-dependent K+ channel, and an appended "tail" that plays a role in calcium sensing. Here we present evidence for a site on the tail that interacts with calcium. This site, the "calcium bowl," is a novel calcium-binding motif that includes a string of conserved aspartate residues. Mutations of the calcium bowl fall into two categories: 1) those that shift the position of the G-V relation a similar amount at all [Ca2+], and 2) those that shift the position of the G-V relation only at low [Ca2+]. None of these mutants alters the slope of the G-V curve. These mutant phenotypes are apparent in calcium ion, but not in cadmium ion, where mutant and wild type are indistinguishable. This suggests that the calcium bowl is sensitive to calcium ion, but insensitive to cadmium ion. The presence and independence of a second calcium-binding site is inferred because channels still respond to increasing levels of [Ca2+] or [Cd2+], even when the calcium bowl is mutationally deleted. Thus a low level of activation in the absence of divalent cations is identical in mutant and wild-type channels, possibly because of activation of this second Ca2+-binding site.
Elimination of the BK Ca Channel's High-Affinity Ca 2 � Sensitivity
We report here a combination of site-directed mutations that eliminate the high-affinity Ca 2 ϩ response of the large-conductance Ca 2 ϩ -activated K ϩ channel (BK Ca ), leaving only a low-affinity response blocked by high concentrations of Mg 2 ϩ . Mutations at two sites are required, the "Ca 2 ϩ bowl," which has been implicated previously in Ca 2 ϩ binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK Ca channel contains three types of Ca 2 ϩ binding sites, one of low affinity that is Mg 2 ϩ sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca 2 ϩ to influence channel opening. Estimates of the binding characteristics of the BK Ca channel's high-affinity Ca 2 ϩ -binding sites are provided.
Proceedings of the National Academy of Sciences of the United States of America, 2016
Large-conductance voltage- and calcium-activated K(+) (BK) channels are key physiological players in muscle, nerve, and endocrine function by integrating intracellular Ca(2+) and membrane voltage signals. The open probability of BK channels is regulated by the intracellular concentration of divalent cations sensed by a large structure in the BK channel called the "gating ring," which is formed by four tandems of regulator of conductance for K(+) (RCK1 and RCK2) domains. In contrast to Ca(2+) that binds to both RCK domains, Mg(2+), Cd(2+), or Ba(2+) interact preferentially with either one or the other. Interaction of cations with their binding sites causes molecular rearrangements of the gating ring, but how these motions occur remains elusive. We have assessed the separate contributions of each RCK domain to the cation-induced gating-ring structural rearrangements, using patch-clamp fluorometry. Here we show that Mg(2+) and Ba(2+) selectively induce structural movement of ...
Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel
Nature, 2011
High conductance voltage-and Ca 2+-activated K + channels (Slo1 or BK channels) function in many physiological processes that link cell membrane voltage and intracellular Ca 2+ , including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning 1-8. Like other voltage-dependent K + (Kv) channels, BK channels open when the cell membrane depolarizes, but in contrast to other Kv channels they also open when intracellular Ca 2+ levels rise. Channel opening by Ca 2+ is conferred by a structure called the gating ring, located in the cytoplasm. Recent structural studies have defined the Ca 2+-free, closed conformation of the gating ring, but the open conformation is not yet known 9. Here we present the Ca 2+-bound, open conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca 2+ , opens like the petals of a flower. The magnitude of opening explains how Ca 2+ binding can open the pore. These findings present amolecular basis of Ca 2+ activation and suggest new possibilities for targeting the gating ring to treat diseases such as asthma and hypertension. Regulators of K + conductance (RCK) domains are ubiquitous among ion channels and transporters in prokaryotic cells 10-14. Eight RCK domains assemble in the cytoplasm to form a closed-ring structure that changes its diameter upon ligand binding, thus enabling the 'gating ring' to regulate allosterically the transmembrane component of the transport protein. In prokaryotic cells the gating ring most often consists of eight identical RCK domains, arranged as a tetrad of pairs, giving rise to a four-fold symmetric ring with identical top and bottom layers of RCK domains 12,15. Intracellular ligands such as Ca 2+ or small organic molecules bind in a cleft between RCK pairs forming the top and bottom layers to affect the shape of the ring 12,15-17. RCK domains are also found in higher eukaryotes in the Slo family of K + channels 9,10,18,19. There, two non-identical RCK domains are encoded in the C-terminus of the K + channel subunit. A tetrameric K + channel thus provides eight RCK domains to make a gating ring, but in contrast to most prokaryotic gating rings with identical RCK domains, the eukaryotic gating ring has one kind of RCK domain (RCK1) forming its top layer and another kind Correspondence and requests for materials should be addressed to R.M.
Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution
Science (New York, N.Y.), 2010
High-conductance voltage-and Ca 2+ -activated K + (BK) channels encode negative feedback regulation of membrane voltage and Ca 2+ signaling, playing a central role in numerous physiological processes. We determined the x-ray structure of the human BK Ca 2+ gating apparatus at a resolution of 3.0 angstroms and deduced its tetrameric assembly by solving a 6angstrom resolution structure of a Na + -activated homolog. Two tandem C-terminal regulator of K + conductance (RCK) domains from each of four channel subunits form a 350-kilodalton gating ring at the intracellular membrane surface. A sequence of aspartic amino acids that is known as the Ca 2+ bowl, and is located within the second of the tandem RCK domains, creates four Ca 2+ binding sites on the outer perimeter of the gating ring at the "assembly interface" between RCK domains. Functionally important mutations cluster near the Ca 2+ bowl, near the "flexible interface" between RCK domains, and on the surface of the gating ring that faces the voltage sensors. The structure suggests that the Ca 2+ gating ring, in addition to regulating the pore directly, may also modulate the voltage sensor.
Large conductance Ca 2-activated K (BK) channel: Activation by Ca 2 and voltage
Biol. Res, 2006
Large conductance Ca 2+ -activated K + (BK) channels belong to the S4 superfamily of K + channels that include voltage-dependent K + (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH 2 -terminus. The BK channel is activated by internal Ca 2+ , and using chimeric channels and mutagenesis, three distinct Ca 2+ -dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca 2+ -binding domains activate the BK channel when cytoplasmic Ca 2+ reaches micromolar concentrations, and a low Ca 2+ affinity mechanism may be involved in the physiological regulation by Mg 2+ . The presence in the BK channel of multiple Ca 2+ -binding sites explains the huge Ca 2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca 2+ . Therefore, Ca 2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.