Positions of the cytoplasmic end of BK α S0 helix relative to S1-S6 and of β1 TM1 and TM2 relative to S0-S6 (original) (raw)
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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...
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...
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.
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 ...
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/
Proceedings of the National Academy of Sciences
The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels is regulated by a  subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming ␣ subunit necessary for -subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to -subunit modulation, and analyzed the topology of the ␣ subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel ␣ 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 experiments indicate that S0 leads to an exoplasmic N terminus. Therefore, we propose a new model where MaxiK channels have a seventh transmembrane segment at the N terminus (S0). Chimeric exchange of 41 N-terminal amino acids, including S0, from the human MaxiK channel to the Drosophila homologue transfers -subunit regulation to the otherwise unresponsive Drosophila channel. Both the unique S0 region and the exoplasmic N terminus are necessary for this gain of function.
A BK (Slo1) channel journey from molecule to physiology
Channels, 2013
Abbreviations: BK, big conductance voltage and Ca 2+ -dependent potassium channel; Charybdotoxin, ChTx; Iberotoxin, IbTx; regulator of the conductance of K + channels, RCK; voltage sensing domain, VSD; leucine-rich repeat proteins, LRRC; nitric oxide, NO; cyclic guanosin mono-phosphate, cGMP www.landesbioscience.com Channels 443
β1-subunit-induced structural rearrangements of the Ca2+- and voltage-activated K+ (BK) channel
Proceedings of the National Academy of Sciences of the United States of America, 2016
Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α- or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.
PloS one, 2013
The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not...
Coupling of Ca2+ and voltage activation in BK channels through the αB helix/voltage sensor interface
Large conductance Ca2+ and voltage activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is comprised of a voltage sensor domain (VSD), a central pore gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We now explore mechanism by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the HCA model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high affinity Ca2+ sites transduce their effect, at least ...