Structure of the Human BK Channel Ca ²⁺ -Activation Apparatus at 3.0 Å Resolution

Abstract

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 6-angstrom 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.High-conductance voltage-and calcium-activated K + channels (BK or Slo1 channels) participate in numerous physiological processes, including neuronal excitability, smooth muscle contractility, and hair cell tuning (1-6). BK channels have an unusually high singlechannel conductance, but their most important physiological property is dual regulation through membrane voltage and intracellular Ca 2+ (7)(8)(9). Depolarization of the membrane voltage and increased intracellular Ca 2+ levels both cause BK channels to open, which hyperpolarizes the membrane and closes voltage-dependent channels, including Ca 2+ channels, reducing Ca 2+ influx into the cell. Thus, BK channels are negative-feedback regulators of electrical excitation (membrane depolarization) as well as the numerous biochemical pathways that are stimulated through Ca 2+ acting as a second messenger.The complexity of BK channel function mirrors the complexity of its protein structure. The amino acid sequence includes the integral membrane pore shared by all K + channels, the integral membrane voltage sensor domains present in voltage-dependent channels, and also a cytoplasmic domain (CTD) consisting of approximately 800 amino acids per subunit, which accounts for the C-terminal two thirds of the entire channel. The CTD structure † To whom correspondence should be addressed. confers upon the BK channel its ability to respond to changes in intracellular Ca 2+ . It is also the source of functional heterogeneity through alternate splicing, polymorphisms, phosphorylation, and protein interactions, which modulate BK channel activity (10-12).The only information currently available on BK channel structure is either low resolution from cryogenic electron microscopy (cryo-EM) (13) or indirect through homology models. The pore and voltage sensors of the BK channel will undoubtedly resemble the corresponding regions of oth...

A heme-binding domain controls regulation of ATP-dependent potassium channels

“…2 A and B). In this regard, there are similarities to the structure of human Slo1, because the proposed CXXCH heme-binding motif in Slo1 is either invisible or only partly visible in crystal structures (37)(38)(39) and is thus assumed to be located in an unstructured (conformationally mobile) region of the molecule.…”

The structure of the KtrAB potassium transporter

“…We have uncovered several differences between KtrAB and the BK [13][14][15] and MthK 11,12,24 channels which have an impact on their mechanisms of transport activation. In channels, the RCK rings are covalently tethered to the C terminus of a single channel pore domain and the rings expand symmetrically upon binding of an activating ligand.…”

“…The membrane proteins in both superfamilies share the same architecture 2,5-9 : four subunits or repeats, each with the TM-P loop-TM (where TM indicates transmembrane helix) structural motif first seen in the KcsA potassium channel structure 10 , assemble to form an ion pore with a 'selectivity filter' along its central axis. In addition, the cytosolic regulatory proteins of the Trk and Ktr transporters and of the MthK 11,12 and large-conductance Ca 2+ -gated (BK) K + channels [13][14][15] are RCK (regulate conductance of K+) domains.Ktr ion transporters are crucial K + transport systems in some bacteria 16,17 , with a role in resistance to osmotic stress and high salinity by mediating the early uptake of K + (refs 16, 18). The Ktr ion transporters are composed of two essential components 3,16 : a membrane protein (KtrB or KtrD) and a cytosolic regulatory protein (KtrA or KtrC).…”

“…The membrane proteins in both superfamilies share the same architecture 2,5-9 : four subunits or repeats, each with the TM-P loop-TM (where TM indicates transmembrane helix) structural motif first seen in the KcsA potassium channel structure 10 , assemble to form an ion pore with a 'selectivity filter' along its central axis. In addition, the cytosolic regulatory proteins of the Trk and Ktr transporters and of the MthK 11,12 and large-conductance Ca 2+ -gated (BK) K + channels [13][14][15] are RCK (regulate conductance of K+) domains.…”

“…The interactions between KtrB and the KtrA ring are an important difference relative to K + channels [11][12][13][14][15] . In channels the pore subunit and one or two RCK domains are encoded in the same polypeptide and the RCK octameric ring hangs from the cytoplasmic face of the membrane protein through a polypeptide tether.…”