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1.
The interpretation of slow inactivation in potassium channels has been strongly influenced by work on C-type inactivation in Shaker channels. Slow inactivation in Shaker and some other potassium channels can be dramatically modulated by the state of the pore, including mutations at outer pore residue T449, which altered inactivation kinetics up to 100-fold. KV2.1, another voltage-dependent potassium channel, exhibits a biophysically distinct inactivation mechanism with a U-shaped voltage-dependence and preferential closed-state inactivation, termed U-type inactivation. However, it remains to be demonstrated whether U-type and C-type inactivation have different molecular mechanisms. This study examines mutations at Y380 (homologous to Shaker T449) to investigate whether C-type and U-type inactivation have distinct molecular mechanisms, and whether C-type inactivation can occur at all in KV2.1. Y380 mutants do not introduce C-type inactivation into KV2.1 and have little effect on U-type inactivation of KV2.1. Interestingly, two of the mutants tested exhibit twofold faster recovery from inactivation compared to wild-type channels. The observation that mutations have little effect suggests KV2.1 lacks C-type inactivation as it exists in Shaker and that C-type and U-type inactivation have different molecular mechanisms. Kinetic modeling predicts that all mutants inactivate preferentially, but not exclusively, from partially activated closed states. Therefore, KV2.1 exhibits a single U-type inactivation process including some inactivation from open as well as closed states. 相似文献
2.
Michel J. Roux Riccardo Olcese Ligia Toro Francisco Bezanilla Enrico Stefani 《The Journal of general physiology》1998,111(5):625-638
Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region. 相似文献
3.
Ben Short 《The Journal of general physiology》2021,153(9)
JGP modeling study suggests that selectivity filter constriction is a plausible mechanism for C-type inactivation of the Shaker voltage-gated potassium channel.In response to prolonged activation, many K+ channels spontaneously reduce the membrane conductance by undergoing C-type inactivation, a kinetic process crucial for the pacing of cardiac action potentials and the modulation of neuronal firing patterns. In the pH-activated bacterial channel KcsA, C-type inactivation appears to involve constriction of the channel’s selectivity filer that prohibits ion conduction, but whether voltage-gated channels like Drosophila Shaker use a similar mechanism is controversial (1). In this issue of JGP, a computational study by Li et al. suggests that filter constriction is indeed a plausible mechanism for the C-type inactivation of Shaker (2).(Left to right) Jing Li, Benoît Roux, and colleagues use computational modeling to show that selectivity filter constriction, allosterically promoted by opening of the intracellular activation gate, is a plausible mechanism for the C-type inactivation of voltage-gated K+ channels such as Drosophila Shaker. The selectivity filter is conductive (left) when the intracellular gate is partially open, but adopts a constricted conformation (right) when the gate is open wide.Various structural approaches have shown that C-type inactivation of KcsA channels is associated with the symmetrical constriction of all four channel subunits at the level of the central glycine residue in the selectivity filter. Benoît Roux and colleagues at The University of Chicago used MD simulations to show that the KcsA pore can transition from the conductive to the constricted conformation on an appropriate timescale, and that this transition is allosterically promoted by the wide opening of the pore’s intracellular gate (3). Modeling by Roux and colleagues suggests that C-type inactivation of cardiac hERG channels could also involve selectivity filter constriction, though in this case it appears to be an asymmetric process in which only two of the channel’s subunits move closer together (4).“In view of the high similarity between the pore domains of Shaker and KcsA (almost 40% sequence identity), we wanted to examine if it’s possible for the Shaker selectivity filter to constrict and, if so, how similar it is to KcsA,” Roux explains. Led by first author Jing Li—now an assistant professor at the University of Mississippi—Roux and colleagues developed several homology models of the Shaker pore domain with the intracellular gate open to various degrees (2).MD simulations and free energy calculations revealed that the Shaker selectivity filter can dynamically transition from a conductive to a constricted conformation, and that this transition is allosterically coupled to the intracellular gate; the constricted conformation is stable when the gate is wide open. “Our computations strongly suggest that constriction is a plausible mechanism for the C-type inactivation of Shaker,” Roux says. “There’s no reason based on the currently available information to reject the existence of a constricted state in Shaker channels.”As with KcsA, Shaker channels appear to constrict symmetrically at the level of the selectivity filter’s central glycine. But Li et al.’s simulations revealed some small variations between the two channels, including differences in the number of water molecules bound to each channel subunit and the arrangement of the hydrogen-bond network they form to stabilize the constricted state.Li et al. also modeled the pore domain of the Shaker W434F mutant, which is widely assumed to be trapped in a C-type inactivated state. The simulation suggests that the mutant channel’s filter adopts a stable constricted conformation even when the intracellular gate is only partially open, although the constriction is asymmetric and occurs at the level of a different filter residue (2).Constriction may therefore be a universal mechanism of C-type inactivation, even if the exact conformation varies from channel to channel. But, says Roux, confirming this will require more experimental work using the right conditions and mutations to capture the structure of inactivated channels. 相似文献
4.
Jing Li Rong Shen Ahmed Rohaim Ramon Mendoza Uriarte Mikolai Fajer Eduardo Perozo Benoît Roux 《The Journal of general physiology》2021,153(9)
C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D. 相似文献
5.
Christopher A. Ahern Amy L. Eastwood Dennis A. Dougherty Richard Horn 《The Journal of general physiology》2009,134(6):461-469
Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations. 相似文献
6.
E. Dietlind Koch Baldomero M. Olivera Heinrich Terlau Franco Conti 《Biophysical journal》2004,86(1):191-209
κ-Conotoxin PVIIA (κ-PVIIA), a 27-amino acid peptide identified from the venom of Conus purpurascens, inhibits the Shaker K+ channel by blocking its outer pore. The toxin appears as a gating modifier because its binding affinity decreases with relatively fast kinetics upon channel opening, but there is no indication that it interferes with the gating transitions of the wild-type channels (WT), including the structural changes of the outer pore that underlie its slow C-type inactivation. In this report we demonstrate that in two outer pore mutants of Shaker-IR (M448K and T449S), that have high toxin sensitivity and fast C-type inactivation, the latter process is instead antagonized by and incompatible with κ-PVIIA binding. Inactivation is slowed by the necessary preliminary unbinding of κ-PVIIA, whereas toxin rebinding must await recovery from inactivation causing a double-exponential relaxation of the second response to double-pulse stimulations. Compared with the lack of similar effects in WT, these results demonstrate the ability of peptide toxins like κ-PVIIA to reveal possibly subtle differences in structural changes of the outer pore of K+ channels; however, they also warn against a naive use of fast inactivating mutants as models for C-type inactivation. Unfolded from the antagonistic effect of inactivation, toxin binding to mutant noninactivated channels shows state- and voltage-dependencies similar to WT: slow and high affinity for closed channels; relatively fast dissociation from open channels at rate increasing with voltage. This supports the idea that these properties depend mainly on interactions with pore-permeation processes that are not affected by the mutations. In mutant channels the state-dependence also greatly enhances the protection of toxin binding against steady-state inactivation at low depolarizations while still allowing large responses to depolarizing pulses that relieve toxin block. Although not obviously applicable to any known combination of natural channel and outer-pore blocker, our biophysical characterization of such highly efficient mechanism of protection from steady-state outer-pore inactivation may be of general interest. 相似文献
7.
Voltage-dependent ion channels transduce changes in the membrane electric field into protein rearrangements that gate their transmembrane ion permeation pathways. While certain molecular elements of the
voltage sensor and gates have been identified, little is known about either the nature of their conformational rearrangements or about how the voltage sensor is coupled to the gates. We used voltage clamp fluorometry to examine the voltage sensor (S4) and pore region (P-region) protein motions that underlie the slow inactivation of the
Shaker K+ channel. Fluorescent probes in both the P-region and S4 changed emission intensity in parallel with the
onset and recovery of slow inactivation, indicative of local protein rearrangements in this gating process. Two sequential rearrangements were observed, with channels first entering the P-type, and then the C-type inactivated
state. These forms of inactivation appear to be mediated by a single gate, with P-type inactivation closing the gate
and C-type inactivation stabilizing the gate''s closed conformation. Such a stabilization was due, at least in part, to
a slow rearrangement around S4 that stabilizes S4 in its activated transmembrane position. The fluorescence reports of S4 and P-region fluorophore are consistent with an increased interaction of the voltage sensor and inactivation gate upon gate closure, offering insight into how the voltage-sensing apparatus is coupled to a channel
gate. 相似文献
8.
Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels
Riccardo Olcese Ramón Latorre Ligia Toro Francisco Bezanilla Enrico Stefani 《The Journal of general physiology》1997,110(5):579-589
Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation. 相似文献
9.
Claydon TW Vaid M Rezazadeh S Kwan DC Kehl SJ Fedida D 《The Journal of general physiology》2007,129(5):437-455
Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state. 相似文献
10.
John G. Starkus Lioba Kuschel Martin D. Rayner Stefan H. Heinemann 《The Journal of general physiology》1998,112(1):85-93
C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions. 相似文献
11.
Alain J. Labro Jerome J. Lacroix Carlos A. Villalba-Galea Dirk J. Snyders Francisco Bezanilla 《The Journal of general physiology》2012,140(5):481-493
Voltage-dependent potassium (Kv) channels provide the repolarizing power that shapes the action potential duration and helps control the firing frequency of neurons. The K+ permeation through the channel pore is controlled by an intracellularly located bundle-crossing (BC) gate that communicates with the voltage-sensing domains (VSDs). During prolonged membrane depolarizations, most Kv channels display C-type inactivation that halts K+ conduction through constriction of the K+ selectivity filter. Besides triggering C-type inactivation, we show that in Shaker and Kv1.2 channels (expressed in Xenopus laevis oocytes), prolonged membrane depolarizations also slow down the kinetics of VSD deactivation and BC gate closure during the subsequent membrane repolarization. Measurements of deactivating gating currents (reporting VSD movement) and ionic currents (BC gate status) showed that the kinetics of both slowed down in two distinct phases with increasing duration of the depolarizing prepulse. The biphasic slowing in VSD deactivation and BC gate closure was strongly correlated in time and magnitude. Simultaneous recordings of ionic currents and fluorescence from a probe tracking VSD movement in Shaker directly demonstrated that both processes were synchronized. Whereas the first slowing originates from a stabilization imposed by BC gate opening, the subsequent slowing reflects the rearrangement of the VSD toward its relaxed state (relaxation). The VSD relaxation was observed in the Ciona intestinalis voltage-sensitive phosphatase and in its isolated VSD. Collectively, our results show that the VSD relaxation is not kinetically related to C-type inactivation and is an intrinsic property of the VSD. We propose VSD relaxation as a general mechanism for depolarization-induced slowing of BC gate closure that may enable Kv1.2 channels to modulate the firing frequency of neurons based on the depolarization history. 相似文献
12.
《Biophysical journal》2020,118(10):2612-2620
Voltage-gated potassium (Kv) channels display several types of inactivation processes, including N-, C-, and U-types. C-type inactivation is attributed to a nonconductive conformation of the selectivity filter (SF). It has been proposed that the activation gate and the channel’s SF are allosterically coupled because the conformational changes of the former affect the structure of the latter and vice versa. The second threonine of the SF signature sequence (e.g., TTVGYG) has been proven to be essential for this allosteric coupling. To further study the role of the SF in U-type inactivation, we substituted the second threonine of the TTVGYG sequence by an alanine in the hKv2.1 and hKv3.1 channels, which are known to display U-type inactivation. Both hKv2.1-T377A and hKv3.1-T400A yielded channels that were resistant to inactivation, and as a result, they displayed noninactivating currents upon channel opening; i.e., hKv2.1-T377A and hKv3.1-T400A remained fully conductive upon prolonged moderate depolarizations, whereas in wild-type hKv2.1 and hKv3.1, the current amplitude typically reduces because of U-type inactivation. Interestingly, increasing the extracellular K+ concentration increased the macroscopic current amplitude of both hKv2.1-T377A and hKv3.1-T400A, which is similar to the response of the homologous T to A mutation in Shaker and hKv1.5 channels that display C-type inactivation. Our data support an important role for the second threonine of the SF signature sequence in the U-type inactivation gating of hKv2.1 and hKv3.1. 相似文献
13.
Hongxia Bao Atiya Hakeem Mark Henteleff John G. Starkus Martin D. Rayner 《The Journal of general physiology》1999,113(1):139-151
Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (P
o). Although P
o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P
o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain. 相似文献
14.
The mutation W434F produces an apparently complete block of potassium current in Shaker channels expressed in Xenopus oocytes. Tandem tetrameric constructs containing one or two subunits with this mutation showed rapid inactivation, although the NH2-terminal inactivation domain was absent from these constructs. The inactivation showed a selective dependence on external cations and was slowed by external TEA; these properties are characteristic of C-type inactivation. Inactivation was, however, incompletely relieved by hyperpolarization, suggesting the presence of a voltage-independent component. The hybrid channels had near-normal conductance and ion selectivity. Single-channel recordings from patches containing many W434F channels showed occasional channel openings, consistent with open probabilities of 10−5 or less. We conclude that the W434F mutation produces a channel that is predominantly found in an inactivated state. 相似文献
15.
Vincenzo Macri Hamed Nazzari Evan McDonald Eric A. Accili 《The Journal of biological chemistry》2009,284(23):15659-15667
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels resemble Shaker K+ channels in structure and function. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. However, HCNs open when hyperpolarized, whereas Shaker opens when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In Shaker, an alanine/valine scan of residues across the pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. Because most mutations likely destabilize protein packing, the Shaker pore is most stable when closed, and the voltage sensor works to open it. In HCN channels, the pore energetics and vector of work by the voltage sensor are unknown. Accordingly, we performed a 22-residue alanine/valine scan of the distal pore of the HCN2 isoform and show that the effects of mutations on channel opening and on the steepness of the response of the channel to voltage are mixed and smaller than those in Shaker. These data imply that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and the interactions between the pore and voltage sensor are weak. Moreover, cAMP binding to the channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.Hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channels are similar in structure and function to Shaker K+ channels (1–3). As in Shaker, HCN channels are comprised of four subunits, which each consist of six predicted membrane-spanning segments (S1–S6). The S1–S4 segments form the voltage-sensing domain, and the S5 and S6 segments, the pore-forming domain. The S4 segment in both channels contains positive charges that move similarly in response to changes in membrane voltage (4–6), to then alter the pore structure at the intracellular side of the S6 segment; this region functions as a voltage-controlled gate to cation flow (7–10). Despite these similarities, HCN channels are opened by hyperpolarization of the membrane potential, whereas Shaker channels open in response to depolarization. Thus, the electromechanical coupling between the voltage sensor and the gate is reversed in these two channels.A key determinant of this coupling is the intrinsic stability of the closed and open conformations of the pore. In Shaker channels, it has been proposed that the pore is intrinsically most stable when closed and that the voltage sensor works to open the pore during depolarization (11, 12). Results from an alanine/valine scan of residues across the entire Shaker pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. It was argued that, because most mutations likely destabilize protein packing, the closed conformation must be the stable state; this is consistent with the observed crystal structures of Shaker-related channels KcsA and MthK, in the closed and open states, respectively, wherein more optimally and tightly packed helices were seen in the closed conformation (13–15).Because of presumed shared architecture of the gate between HCN and Shaker channels, HCN channels might also be most stable when closed, and thus the voltage sensor would work to open the pore upon hyperpolarization. To test this hypothesis, we performed an alanine/valine scan of the C-terminal 22 amino acids of the S6 segment in HCN2, used as a prototype, and examined pore energetics as described previously in Shaker (11). Choice of this region for mutation was based on: 1) in Shaker, the corresponding region harbors one of two clusters of gating-sensitive residues and 2) it contains the voltage-controlled gate. Surprisingly, the effects of the mutations on channel opening and on the steepness of the channel''s response to voltage are mixed and smaller than those in Shaker. These findings imply that, in HCN2, the stabilities of the open and closed pore are similar, the interactions between the pore and voltage sensor, both structural and functional, are weaker than in Shaker, and that the voltage sensor must apply force to the pore to close it. Thus, Shaker is closed and HCN2 is open in the absence of input from the voltage sensor. Moreover, cAMP binding to the HCN2 channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state. 相似文献
16.
N-type Inactivation Features of Kv4.2 Channel Gating 总被引:12,自引:0,他引:12
Manuel Gebauer Dirk Isbrandt Kathrin Sauter Britta Callsen Andreas Nolting Olaf Pongs Robert Bhring 《Biophysical journal》2004,86(1):210-223
We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation. 相似文献
17.
Jinling Wang Matthew C. Trudeau Angelina M. Zappia Gail A. Robertson 《The Journal of general physiology》1998,112(5):637-647
Abnormalities in repolarization of the cardiac ventricular action potential can lead to life-threatening arrhythmias associated with long QT syndrome. The repolarization process depends upon the gating properties of potassium channels encoded by the human ether-à-go-go–related gene (HERG), especially those governing the rate of recovery from inactivation and the rate of deactivation. Previous studies have demonstrated that deletion of the NH2 terminus increases the deactivation rate, but the mechanism by which the NH2 terminus regulates deactivation in wild-type channels has not been elucidated. We tested the hypothesis that the HERG NH2 terminus slows deactivation by a mechanism similar to N-type inactivation in Shaker channels, where it binds to the internal mouth of the pore and prevents channel closure. We found that the regulation of deactivation by the HERG NH2 terminus bears similarity to Shaker N-type inactivation in three respects: (a) deletion of the NH2 terminus slows C-type inactivation; (b) the action of the NH2 terminus is sensitive to elevated concentrations of external K+, as if its binding along the permeation pathway is disrupted by K+ influx; and (c) N-ethylmaleimide, covalently linked to an aphenotypic cysteine introduced within the S4–S5 linker, mimics the N deletion phenotype, as if the binding of the NH2 terminus to its receptor site were hindered. In contrast to N-type inactivation in Shaker, however, there was no indication that the NH2 terminus blocks the HERG pore. In addition, we discovered that separate domains within the NH2 terminus mediate the slowing of deactivation and the promotion of C-type inactivation. These results suggest that the NH2 terminus stabilizes the open state and, by a separate mechanism, promotes C-type inactivation. 相似文献
18.
In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology
of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels
using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result
consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type
of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing
either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block.
Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this
block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology.
The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge,
with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged
vestibule with pharmacological properties approaching those of other types of K channels. 相似文献
19.
John G. Starkus Lioba Kuschel Martin D. Rayner Stefan H. Heinemann 《The Journal of general physiology》1997,110(5):539-550
C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel. 相似文献
20.
Block of K+ channels can be influenced by the ability of charged residues on the protein surface to accumulate cationic blocking ions
to concentrations greater than those in bulk solution. We examined the ionic strength dependence of extracellular block of
Shaker K+ channels by tetraethylammonium ions (TEA+) and by a trivalent quaternary ammonium ion, gallamine3+. Wild-type and mutant channels were expressed in Xenopus oocytes and currents recorded with the cut-open oocyte technique. Channel block by both compounds was substantially increased
when the bathing electrolyte ionic strength was lowered, but with a much larger effect for trivalent gallamine. These data
were quantitatively well described by a simple electrostatic model, accounting for accumulation of blocking ions near the
pore of the channel by surface charges. The surface charge density of the wild-type channel consistent with the results was
−0.1 e nm−2. Shaker channels with T449Y mutations have an increased affinity for both TEA and gallamine but the ionic strength dependence of
block was described with the same surface charge density as wild-type channels. Much of the increased sensitivity of Shaker K+ channels to gallamine may be due to a larger local accumulation of the trivalent ion. The negative charge at position 431
contributes to the sensitivity of channels to TEA (MacKinnon & Yellen, 1990). A charge reversal mutation at this location
had little effect on the ionic strength dependence of quaternary ammonium ion block, suggesting that the charge on this amino
acid may directly affect binding affinity but not local ion accumulation.
Received: 7 December 2000/Revised: 27 April 2001 相似文献