The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus (original) (raw)
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Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel
The Journal of General Physiology, 2010
MthK is a Ca 2+ -gated K + channel whose activity is inhibited by cytoplasmic H + . To determine possible mechanisms underlying the channel's proton sensitivity and the relation between H + inhibition and Ca 2+ -dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca 2+ ] (ranging from nominally 0 to 30 mM) at a given [H + ], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca 2+ ] and open probability (Po), with a mean Hill coefficient (n H ) of 9.9 ± 0.9. Neither the maximal Po (0.93 ± 0.005) nor n H changed significantly as a function of [H + ] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca 2+ was not H + sensitive over pH ranging from 7.3 to 9.0. However, increasing [H + ] raised the EC 50 for Ca 2+ activation by 4.7-fold per tenfold increase in [H + ], displaying a linear relation between log(EC 50 ) and log([H + ]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H + binding does not directly modulate either the channel's closedopen equilibrium or the allosteric coupling between Ca 2+ binding and channel opening. We can account for the Ca 2+ activation and proton sensitivity of MthK gating quantitatively by assuming that Ca 2+ allosterically activates MthK, whereas H + opposes activation by destabilizing the binding of Ca 2+ .
eLife
ASICs are proton-gated sodium channels expressed in neurons. Structures of chicken ASIC1 in three conformations have advanced understanding of proton-mediated gating; however, a molecular mechanism describing desensitization from open and pre-open states (steady-state desensitization or SSD) remains elusive. A distinct feature of the desensitized state is an 180o rotation of residues L415 and N416 in the β11- β12 linker that was proposed to mediate desensitization; whether and how it translates into desensitization has not been explored yet. Using electrophysiological measurements of injected Xenopus oocytes, we show that Q276 in β9 strand works with L415 and N416 to mediate both types of desensitization in ASIC1a, ASIC2a and ASIC3. Q276 functions as a valve that enables or restricts rotation of L415 and N416 to keep the linker compressed, its relaxation lengthens openings and leads to sustained currents. At low proton concentrations, the proposed mechanism working in only one of th...
Gating of the TrkH ion channel by its associated RCK protein TrkA
Nature, 2013
TrkH belongs to a superfamily of K + transport proteins required for growth of bacteria in low external K + concentrations. The crystal structure of TrkH from Vibrio parahaemolyticus showed that TrkH resembles a K + channel, and may have a gating mechanism substantially different from K + channels. TrkH assembles with TrkA, a cytosolic protein comprising two Regulate-the-Conductance-of-K + , or RCK domains, which are found in certain K + channels and control their gating. However, fundamental questions on whether TrkH is an ion channel and how it is regulated by TrkA remain unresolved. Here we show single-channel activity of TrkH that is upregulated by ATP via TrkA. We report two structures of the tetrameric TrkA ring, one in complex with TrkH and one in isolation, in which the ring assumes two dramatically different conformations. These results suggest a mechanism for how ATP increases TrkH activity by inducing conformational changes in TrkA. K + is concentrated in all living cells and is essential for many physiological processes. In bacteria, homeostasis of K + is largely mediated by specialized K + transport proteins known as the Superfamily of K + Transporters, or SKT proteins 1 . The bacterial TrkH/TrkG/KtrB proteins form the largest sub-family of SKT proteins, and the crystal structure of TrkH from Vibrio parahaemolyticus, VpTrkH, was reported recently 2 . Each of the four connected homologous domains of TrkH resembles a subunit of the KcsA K + channel, comprising two transmembrane helices connected by a re-entrant P-loop (M1-P-M2) 3 , and the four domains encircle a central ion permeation pathway. In addition, TrkH has two structural features atypical of K + channels: a dimeric quaternary structure, with each protomer containing its own pore; and a long membrane-embedded loop, inserted into the middle of a pore-lining
Amino-terminal Determinants of U-type Inactivation of Voltage-gated K+ Channels
Journal of Biological Chemistry, 2002
The T1 domain is a cytosolic NH 2-terminal domain present in all Kv (voltage-dependent potassium) channels, and is highly conserved between Kv channel subfamilies. Our characterization of a truncated form of Kv1.5 (Kv1.5⌬N209) expressed in myocardium demonstrated that deletion of the NH 2 terminus of Kv1.5 imparts a U-shaped inactivation-voltage relationship to the channel, and prompted us to investigate the NH 2 terminus as a regulatory site for slow inactivation of Kv channels. We examined the macroscopic inactivation properties of several NH 2-terminal deletion mutants of Kv1.5 expressed in HEK 293 cells, demonstrating that deletion of residues up to the T1 boundary (Kv1.5⌬N19, Kv1.5⌬N91, and Kv1.5⌬N119) did not alter Kv1.5 inactivation, however, deletion mutants that disrupted the T1 structure consistently exhibited inactivation phenotypes resembling Kv1.5⌬N209. Chimeric constructs between Kv1.5 and the NH 2 termini of Kv1.1 and Kv1.3 preserved the inactivation kinetics observed in fulllength Kv1.5, again suggesting that the Kv1 T1 domain influences slow inactivation. Furthermore, disruption of intersubunit T1 contacts by mutation of residues Glu 131 and Thr 132 to alanines resulted in channels exhibiting a U-shaped inactivation-voltage relationship. Fusion of the NH 2 terminus of Kv2.1 to the transmembrane segments of Kv1.5 imparted a U-shaped inactivation-voltage relationship to Kv1.5, whereas fusion of the NH 2 terminus of Kv1.5 to the transmembrane core of Kv2.1 decelerated Kv2.1 inactivation and abolished the U-shaped voltage dependence of inactivation normally observed in Kv2.1. These data suggest that intersubunit T1 domain interactions influence U-type inactivation in Kv1 channels, and suggest a generalized influence of the T1 domain on U-type inactivation between Kv channel subfamilies.
Calcium ions open a selectivity filter gate during activation of the MthK potassium channel
Nature Communications, 2015
Ion channel opening and closing are fundamental to cellular signalling and homeostasis. Gates that control K þ channel activity were found both at an intracellular pore constriction and within the selectivity filter near the extracellular side but the specific location of the gate that opens Ca 2 þ-activated K þ channels has remained elusive. Using the Methanobacterium thermoautotrophicum homologue (MthK) and a stopped-flow fluorometric assay for fast channel activation, we show that intracellular quaternary ammonium blockers bind to closed MthK channels. Since the blockers are known to bind inside a central channel cavity, past the intracellular entryway, the gate must be within the selectivity filter. Furthermore, the blockers access the closed channel slower than the open channel, suggesting that the intracellular entryway narrows upon pore closure, without preventing access of either the blockers or the smaller K þ. Thus, Ca 2 þ-dependent gating in MthK occurs at the selectivity filter with coupled movement of the intracellular helices.
Journal of Neuroscience, 2014
It has recently been proposed that post-translational modification of not only the M3-M4 linker but also the M1-M2 linker of pentameric ligand-gated ion channels modulates function in vivo. To estimate the involvement of the M1-M2 linker in gating and desensitization, we engineered a series of mutations to this linker of the human adult-muscle acetylcholine receptor (AChR), the ␣34 AChR and the homomeric ␣1 glycine receptor (GlyR). All tested M1-M2 linker mutations had little effect on the kinetics of deactivation or desensitization compared with the effects of mutations to the M2 ␣-helix or the extracellular M2-M3 linker. However, when the effects of mutations were assessed with 50 Hz trains of ϳ1 ms pulses of saturating neurotransmitter, some mutations led to much more, and others to much less, peak-current depression than observed for the wild-type channels, suggesting that these mutations could affect the fidelity of fast synaptic transmission. Nevertheless, no mutation to this linker could mimic the irreversible loss of responsiveness reported to result from the oxidation of the M1-M2 linker cysteines of the ␣3 AChR subunit. We also replaced the M3-M4 linker of the ␣1 GlyR with much shorter peptides and found that none of these extensive changes affects channel deactivation strongly or reduces the marked variability in desensitization kinetics that characterizes the wild-type channel. However, we found that these large mutations to the M3-M4 linker can have pronounced effects on desensitization kinetics, supporting the notion that its post-translational modification could indeed modulate ␣1 GlyR behavior.
N-type Inactivation of the Potassium Channel KcsA by the Shaker B “Ball” Peptide
Journal of Biological Chemistry, 2008
The effects of the inactivating peptide from the eukaryotic Shaker B K ؉ channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr 8 , which exhibit the strongest STD effects; the C4H in the imidazole ring of His 16 ; the methyl protons of Val 4 , Leu 7 , and Leu 10 and the side chain amine protons of one, if not both, the Lys 18 and Lys 19 residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val 4 and Leu 10 , whereas His 16 seems similarly affected as before. Conversely, STD effects on Tyr 8 are strongly diminished, and those on Lys 18 and/or Lys 19 are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of 13 C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a -hairpin structure when bound to the KcsA channel. Indeed, docking such a -hairpin structure into an open pore model for K ؉ channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.