Dimerization of TWIK-1 K+ channel subunits via a disulfide bridge (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...
Proceedings of the National Academy of Sciences, 2009
The cardiac-delayed rectifier K + current (I KS ) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic I KS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1–S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1–S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cys-flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation...
TWIK-1, a ubiquitous human weakly inward rectifying K+ channel with a novel structure
The EMBO Journal, 1996
A new human weakly inward rectifying K+ channel, TWIK-1, has been isolated. This channel is 336 amino acids long and has four transmembrane domains. Unlike other mammalian K+ channels, it contains two pore-forming regions called P domains. Genes encoding structural homologues are present in the genome of Caenorhabditis elegans. TWIK-1 currents expressed in Xenopus oocytes are time-independent and present a nearly linear I-V relationship that saturated for depolarizations positive to 0 mV in the presence of internal Mg2". This inward rectification is abolished in the absence of internal Mg2'. TWIK-1 has a unitary conductance of 34 pS and a kinetic behaviour that is dependent on the membrane potential. In the presence of internal Mg2+, the mean open times are 0.3 and 1.9 ms at-80 and +80 mV, respectively. The channel activity is up-regulated by activation of protein kinase C and down-regulated by internal acidification. Both types of regulation are indirect. TWIK-1 channel activity is blocked by Ba2+ (IC50 = 100 FM), quinine (IC50 = 50 FM) and quinidine (IC50 = 95 piM). This channel is of particular interest because its mRNA is widely distributed in human tissues, and is particularly abundant in brain and heart. TWIK-1 channels are probably involved in the control of background K+ membrane conductances. Keywords: background conductance/cloning/pH/PKC P domains. From the functional point of view, this channel exhibits weak inward rectification properties and was denoted TWIK-1 (for Tandem of P domains in a Weak Inward rectifying K+ channel). Its abundance and wide tissue distribution suggest a role for this channel in setting the background membrane K+ conductance in many cell types.
The structure, function and distribution of the mouse TWIK-1 K + channel
Febs Letters, 1997
The two P domain K + channel mTWIK-1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK-1 currents are K + -selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba 2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK-1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK-1 subunits dimerize via a disulfide bridge. TWIK-1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK-1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.
Mapping of interactions between the N- and C-termini and the channel core in HERG K+ channels
Biochemical Journal, 2013
The characteristic gating properties of the HERG [human eag (ether-a-go-go)-related gene] potassium channel determine its contribution to cardiac repolarization and in setting the electrical behaviour of a variety of cells. In the present study we analysed, using a site-directed cysteine and disulfide chemistry approach, whether the eag/PAS (Per/Arnt/Sim) and proximal domains at the HERG N-terminus exert a role in controlling the access of the N-terminal flexible tail to its binding site in the channel core for interaction with the gating machinery. Whereas the eag/PAS domain is necessary for disulfide bridging, plus the cysteine residues introduced at positions 3 and 542 of the HERG sequence, the presence of the proximal domain seems to be dispensable. The state-dependent formation of a disulfide bridge between Cys3 and an endogenous cysteine residue at position 723 in the C-terminal C-linker suggests that the N-terminal tail of HERG can also get into close proximity with the C-lin...
TWIK-2, an inactivating 2P domain K+ channel
The Journal of biological chemistry, 2000
We cloned human and rat TWIK-2 and expressed this novel 2P domain K(+) channel in transiently transfected COS cells. TWIK-2 is highly expressed in the gastrointestinal tract, the vasculature, and the immune system. Rat TWIK-2 currents are about 15 times larger than human TWIK-2 currents, but both exhibit outward rectification in a physiological K(+) gradient and mild inward rectification in symmetrical K(+) conditions. TWIK-2 currents are inactivating at depolarized potentials, and the kinetic of inactivation is highly temperature-sensitive. TWIK-2 shows an extremely low conductance, which prevents the visualization of discrete single channel events. The inactivation and rectification are intrinsic properties of TWIK-2 channels. In a physiological K(+) gradient, TWIK-2 is half inhibited by 0.1 mm Ba(2+), quinine, and quinidine. Finally, cysteine 53 in the M1P1 external loop is required for functional expression of TWIK-2 but is not critical for subunit self-assembly. TWIK-2 is the f...
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/
A Central Role for the T1 Domain in Voltage-gated Potassium Channel Formation and Function
Journal of Biological Chemistry, 2001
To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the fulllength functional channel.
Journal of Biological Chemistry, 2012
Background: Voltage-gated Na ϩ channels are composed of ␣ and  subunits. Results: We identified the cysteine residue in 2 responsible for disulfide linkage to ␣. Conclusion: ␣ and 2 associate through a single disulfide bridge to achieve proper subcellular targeting in neurons. Significance: Understanding how Na ϩ channel complexes are formed in neurons is crucial for understanding the development of excitability. Voltage-gated Na ؉ channels in the brain are composed of a single pore-forming ␣ subunit, one non-covalently linked  subunit (1 or 3), and one disulfide-linked  subunit (2 or 4). The final step in Na ؉ channel biosynthesis in central neurons is concomitant ␣-2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding 2) null mice have reduced Na ؉ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant 2 cDNA constructs to investigate the cysteine residue(s) responsible for ␣-2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between ␣ and 2 subunits. Loss of ␣-2 covalent complex formation disrupts the targeting of 2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with ␣ is required for normal 2 subcellular localization in vivo. WT 2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant 2 subunits, which cannot form disulfide bonds with ␣, are removed by detergent. Taken together, our results demonstrate that ␣-2 covalent association via a single, extracellular disulfide bond is required for 2 targeting to specialized neuronal subcellular domains and for 2 association with the neuronal cytoskeleton within those domains.