Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels (original) (raw)
Related papers
Location of the 4 Transmembrane Helices in the BK Potassium Channel
Journal of Neuroscience, 2009
Large-conductance, voltage-and Ca 2ϩ-gated potassium (BK) channels control excitability in a number of cell types. BK channels are composed of ␣ subunits, which contain the voltage-sensor domains and the Ca 2ϩ-sensor domains and form the pore, and often one of four types of  subunits, which modulate the channel in a cell-specific manner. 4 is expressed in neurons throughout the brain. Deletion of 4 in mice causes temporal lobe epilepsy. Compared with channels composed of ␣ alone, channels composed of ␣ and 4 activate and deactivate more slowly. We inferred the locations of the two 4 transmembrane (TM) helices TM1 and TM2 relative to the seven ␣ TM helices, S0-S6, from the extent of disulfide bond formation between cysteines substituted in the extracellular flanks of these TM helices. We found that 4 TM2 is close to ␣ S0 and that 4 TM1 is close to both ␣ S1 and S2. At least at their extracellular ends, TM1 and TM2 are not close to S3-S6. In six of eight of the most highly crosslinked cysteine pairs, four crosslinks from TM2 to S0 and one each from TM1 to S1 and S2 had small effects on the V 50 and on the rates of activation and deactivation. That disulfide crosslinking caused only small functional perturbations is consistent with the proximity of the extracellular ends of TM2 to S0 and of TM1 to S1 and to S2, in both the open and closed states. Materials and Methods Constructs. Mutants of the BK ␣ subunit (mSlo1, KCNMA1; GenBank accession number NM_010610; 1169 residues; molecular weight 131,700) and human BK 4 subunit (KCNMB4, Open Biosystems clone/
Location of modulatory β subunits in BK potassium channels
Journal of General Physiology, 2010
Large-conductance voltage- and calcium-activated potassium (BK) channels contain four pore-forming α subunits and four modulatory β subunits. From the extents of disulfide cross-linking in channels on the cell surface between cysteine (Cys) substituted for residues in the first turns in the membrane of the S0 transmembrane (TM) helix, unique to BK α, and of the voltage-sensing domain TM helices S1–S4, we infer that S0 is next to S3 and S4, but not to S1 and S2. Furthermore, of the two β1 TM helices, TM2 is next to S0, and TM1 is next to TM2. Coexpression of α with two substituted Cys’s, one in S0 and one in S2, and β1 also with two substituted Cys’s, one in TM1 and one in TM2, resulted in two αs cross-linked by one β. Thus, each β lies between and can interact with the voltage-sensing domains of two adjacent α subunits.
Locations of the β1 transmembrane helices in the BK potassium channel
Proceedings of the National Academy of Sciences of the United States of America, 2008
Fig. 1. Mouse BK ␣and 1-subunits. (A) Scheme of the threading of BK ␣ through the membrane. The extracellular regions flanking S0-S6, in which Cys were substituted, are indicated by thick lines. (B) Scheme of the threading of BK 1 through the membrane. The extracellular regions flanking TM1 and TM2, in which Cys were substituted, are indicated by thick lines. Two disulfide bonds within the extracellular loop are shown.
Pharmacological Reviews, 2003
This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. 1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/. Almost a decade ago, a standardized nomenclature for the six-transmembrane domain (TM), voltage-gated K ϩ channel genes-the K V naming system-was widely adopted . This nomenclature was based on deduced phylogenetic relationships; channels that shared 65% sequence identity being assigned to one subfamily. A parallel nomenclature-KCN-was developed by the Human Genome Organisation (HUGO) . Since then, the K ϩ channel superfamily of genes has greatly expanded, requiring an update of the naming system.
FEBS Letters, 2012
The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming a-subunit is associated to an auxiliary b-subunit that modulates the voltage-and Ca 2+-dependent activation of the channel. Structural components present in b-subunits that are important for the physical association with the a-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the b2-subunit are dispensable for association with the a-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the b2-subunit physically binds to the transmembrane S1 domain of the a-subunit. Structured summary of protein interactions: BK channel subunit alpha physically interacts with BK channel subunit beta-2 by anti tag coimmunoprecipitation(View interaction)
Journal of General Physiology, 2012
Large-conductance voltage- and Ca2+-gated K+ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four addit...
Position and Role of the BK Channel α Subunit S0 Helix Inferred from Disulfide Crosslinking
The Journal of General Physiology, 2008
The position and role of the unique N-terminal transmembrane (TM) helix, S0, in large-conductance, voltage- and calcium-activated potassium (BK) channels are undetermined. From the extents of intra-subunit, endogenous disulfide bond formation between cysteines substituted for the residues just outside the membrane domain, we infer that the extracellular flank of S0 is surrounded on three sides by the extracellular flanks of TM helices S1 and S2 and the four-residue extracellular loop between S3 and S4. Eight different double cysteine–substituted alphas, each with one cysteine in the S0 flank and one in the S3–S4 loop, were at least 90% disulfide cross-linked. Two of these alphas formed channels in which 90% cross-linking had no effect on the V50 or on the activation and deactivation rate constants. This implies that the extracellular ends of S0, S3, and S4 are close in the resting state and move in concert during voltage sensor activation. The association of S0 with the gating charg...
Proceedings of the National Academy of Sciences, 1996
The pore-forming α subunit of large conductance voltage- and Ca 2+ -sensitive K (MaxiK) channels is regulated by a β subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming α subunit necessary for β-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to β-subunit modulation, and analyzed the topology of the α subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel α subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1–S6) usually found in voltage-dependent ion channels. The transmembrane nature of this unique S0 region was demonstrated by in vitro translation experiments. Moreover, normal functional expression of signal sequence fusions and in vitro N-linked glycosylation ...
Potassium channel opening: a subtle two-step
The Journal of Physiology, 2009
Voltage-gated K + channels undergo a voltage-dependent conductance change that plays a key role in modulating cellular excitability. While the Open state is captured in crystal structures of Kv1.2 and a chimeric Kv1.2/Kv2.1 channel, the Close state and the mechanism of this transition are still a subject of debate. Here, we propose a model based on mutagenesis combined with measurements of both ionic and gating currents which is consistent with the idea that the Open state is the default state, the energy of the electric field being used to keep the channel closed. Our model incorporates an 'Activated state' where the bulk of sensor movement is completed without channel opening. The model accounts for the well characterized electrophysiology of the 'V2' and 'ILT' mutations in Shaker, where sensor movement and channel opening occur over distinct voltage ranges. Moreover, the model proposes relatively small protein rearrangements in going from the Activated to the Open state, consistent with the rapid transitions observed in single channel records of Shaker type channels at zero millivolts.
Biochemical Journal, 2003
N-glycosylation is a post-translational modification that plays a role in the trafficking and/or function of some membrane proteins. We have shown previously that N-glycosylation affected the function of some Kv1 voltage-gated potassium (K+) channels [Watanabe, Wang, Sutachan, Zhu, Recio-Pinto and Thornhill (2003) J. Physiol. (Cambridge, U.K.) 550, 51–66]. Kv1 channel S1–S2 linkers vary in length but their N-glycosylation sites are at similar relative positions from the S1 or S2 membrane domains. In the present study, by a scanning mutagenesis approach, we determined the allowed N-glycosylation sites on the Kv1.2 S1–S2 linker, which has 39 amino acids, by engineering N-glycosylation sites and assaying for glycosylation, using their sensitivity to glycosidases. The middle section of the linker (54% of linker) was glycosylated at every position, whereas both end sections (46% of linker) near the S1 or S2 membrane domains were not. These findings suggested that the middle section of th...