Accumulation of inactivation in a cloned transient K+ channel (AKv1.1a) of Aplysia

1995 ◽  
Vol 74 (3) ◽  
pp. 1248-1257 ◽  
Author(s):  
Y. Furukawa
Keyword(s):  
Short Interval ◽  
Low Frequency ◽  
K Channel ◽  
Amino Terminal ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
A Cell ◽  
K Concentration ◽  
External K

1. Inactivation of a cloned Aplysia K+ channel, AKv1.1a, expressed in Xenopus oocytes was examined by a cell-attached macropatch recording. A fast macroscopic inactivation (the time constant for decay was in the range of 20-40 ms) in response to a depolarizing command pulse was insensitive to the concentration of external K+ (2-100 mM KCl). 2. By contrast, recovery from inactivation was extremely slow and dependent on external K+. When the concentration of external KCl was 2-3 mM, a patched membrane had to be held at hyperpolarized potential for > 40 s for a full recovery. The recovery was greatly accelerated if external K+ concentration was increased. A tail current following a command pulse long enough to inactivate most of the channels showed a marked rising phase. 3. A consequence of the slow recovery from inactivation was that AKv1.1a showed a marked accumulation of the inactivation following repetitive pulses, even at low frequency (< 0.1 Hz). When two depolarizing pulses were applied at a short interval, the current during a second pulse was smaller than the current at the end of the preceding pulse. This is a phenomenon called "cumulative inactivation." The onset and the extent of cumulative inactivation of AKv1.1a were voltage dependent but relatively insensitive to external K+ concentration. An amino terminal deletion mutant of AKv1.1a that lacks the fast N-type inactivation did not show cumulative inactivation. 4. These results suggest that the inactivation gating by the amino terminal region of AKv1.1a has a similarity to open-channel blockade, and that the cumulative inactivation can also be dependent on the amino terminal cytoplasmic domain of K+ channels.

scholarly journals Voltage-dependent inactivation gating at the selectivity filter of the MthK K+ channel

2010 ◽  
Vol 136 (5) ◽  
pp. 569-579 ◽  
Author(s):  
Andrew S. Thomson ◽  
Brad S. Rothberg
Keyword(s):  
Voltage Sensor ◽  
Selectivity Filter ◽  
K Channel ◽  
K Channels ◽  
Dependent Inactivation ◽  
Wide Range ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
External K ◽  
Conductive State

Voltage-dependent K+ channels can undergo a gating process known as C-type inactivation, which involves entry into a nonconducting state through conformational changes near the channel’s selectivity filter. C-type inactivation may involve movements of transmembrane voltage sensor domains, although the mechanisms underlying this form of inactivation may be heterogeneous and are often unclear. Here, we report on a form of voltage-dependent inactivation gating observed in MthK, a prokaryotic K+ channel that lacks a canonical voltage sensor and may thus provide a reduced system to inform on mechanism. In single-channel recordings, we observe that Po decreases with depolarization, with a half-maximal voltage of 96 ± 3 mV. This gating is kinetically distinct from blockade by internal Ca2+ or Ba2+, suggesting that it may arise from an intrinsic inactivation mechanism. Inactivation gating was shifted toward more positive voltages by increasing external [K+] (47 mV per 10-fold increase in [K+]), suggesting that K+ binding at the extracellular side of the channel stabilizes the open-conductive state. The open-conductive state was stabilized by other external cations, and selectivity of the stabilizing site followed the sequence: K+ ≈ Rb+ &gt; Cs+ &gt; Na+ &gt; Li+ ≈ NMG+. Selectivity of the stabilizing site is weaker than that of sites that determine permeability of these ions, suggesting that the site may lie toward the external end of the MthK selectivity filter. We could describe MthK gating over a wide range of positive voltages and external [K+] using kinetic schemes in which the open-conductive state is stabilized by K+ binding to a site that is not deep within the electric field, with the voltage dependence of inactivation arising from both voltage-dependent K+ dissociation and transitions between nonconducting (inactivated) states. These results provide a quantitative working hypothesis for voltage-dependent, K+-sensitive inactivation gating, a property that may be common to other K+ channels.

scholarly journals Interactions between Multiple Phosphorylation Sites in the Inactivation Particle of a K+ Channel

1998 ◽  
Vol 112 (1) ◽  
pp. 71-84 ◽  
Author(s):  
Edward J. Beck ◽  
Roger G. Sorensen ◽  
Simon J. Slater ◽  
Manuel Covarrubias
Keyword(s):  
Tertiary Structure ◽  
Site Directed Mutagenesis ◽  
K Channel ◽  
Phosphorylation Sites ◽  
Free Energies ◽  
Channel Inactivation ◽  
Recovery From Inactivation ◽  
Pkc Isoforms ◽  
Inactivation Gating ◽  
Rate Of Recovery

Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, and at least two NH2-terminal serines (S15 and S21) appeared involved in this interaction (Covarrubias et al. 1994. Neuron. 13:1403–1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine → alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by ∼85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S → A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S → D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S → D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH2-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating.

scholarly journals Inactivation Gating of Kv4 Potassium Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 641-660 ◽  
Author(s):  
Henry H. Jerng ◽  
Mohammad Shahidullah ◽  
Manuel Covarrubias
Keyword(s):  
Time Course ◽  
Membrane Potentials ◽  
Structural Basis ◽  
K Channel ◽  
Inactivation Curve ◽  
Double Mutation ◽  
K Channels ◽  
Closed State ◽  
Recovery From Inactivation ◽  
Inactivation Gating

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (&gt;10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

scholarly journals Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel.

1988 ◽  
Vol 91 (3) ◽  
pp. 335-349 ◽  
Author(s):  
R MacKinnon ◽  
C Miller
Keyword(s):  
Lipid Bilayers ◽  
External Solution ◽  
Dissociation Rate ◽  
K Channel ◽  
Internal Solution ◽  
Conduction Pathway ◽  
Voltage Dependent ◽  
K Concentration ◽  
A Site ◽  
Low K

The mechanism of charybdotoxin (CTX) block of single Ca2+-activated K+ channels from rat muscle was studied in planar lipid bilayers. CTX blocks the channel from the external solution, and K+ in the internal solution specifically relieves toxin block. The effect of K+ is due solely to an enhancement of the CTX dissociation rate. As internal K+ is raised, the CTX dissociation rate increases in a rectangular hyperbolic fashion from a minimum value at low K+ of 0.01 s-1 to a maximum value of approximately 0.2 s-1. As the membrane is depolarized, internal K+ more effectively accelerates CTX dissociation. As the membrane is hyperpolarized, the toxin dissociation rate approaches 0.01 s-1, regardless of the K+ concentration. When internal K+ is replaced by Na+, CTX dissociation is no longer voltage dependent. The permeant ion Rb also accelerates toxin dissociation from the internal solution, while the impermeant ions Li, Na, Cs, and arginine do not. These results argue that K ions can enter the CTX-blocked channel from the internal solution to reach a site located nearly all the way through the conduction pathway; when K+ occupies this site, CTX is destabilized on its blocking site by approximately 1.8 kcal/mol. The most natural way to accommodate these conclusions is to assume that CTX physically plugs the channel's externally facing mouth.

Changes in the intracellular free calcium concentration of Aplysia and leech neurones measured with calcium-sensitive microelectrodes

1987 ◽  
Vol 65 (5) ◽  
pp. 934-939 ◽  
Author(s):  
Joachim W. Deitmer ◽  
Roger Eckert ◽  
Wolf-R. Schlue
Keyword(s):  
Calcium Concentration ◽  
Neuronal Membrane ◽  
Free Calcium ◽  
Voltage Dependent ◽  
Intracellular Ca ◽  
K Current ◽  
Intracellular Free Calcium Concentration ◽  
Free Calcium Concentration ◽  
K Concentration ◽  
External K

The intracellular free Ca concentration was measured in invertebrate neurones using single-barrelled and double-barrelled neutral-carrier microelectrodes. The electrodes were calibrated in solutions containing different Ca concentrations between 1 mM and 0.01 μM. The electrode responses were also tested at different ionic strengths and at varying Na concentrations. The electrodes responded with 25–30 mV per 10-fold change in Ca concentration between 1 mM and 1 μM and with 10–25 mV between 1 and 0.1 μM Ca. The intracellular free Ca concentration was measured to be between 0.1 and 0.7 μM in the neurones. The changes of intracellular Ca in identified voltage-clamped neurones of Aplysia californica were recorded during iontophoretic injections of Ca2+ or EGTA. The decrease of intracellular Ca following EGTA injection was correlated with the suppression of the Ca-dependent K current and with the reduction of Ca-induced inactivation of voltage-dependent Ca current. In identified neurones of the leech Hirudo medicinalis a reversible increase of intracellular Ca2+ was recorded after inhibition of the Na–K pump, either by addition of ouabain (0.5 mM) or by lowering the external K concentration (0.2 mM). This rise in intracellular Ca2+ did not occur, and was even reversed, in the absence of external Na, suggesting the existence of Na–Ca exchange across the leech neuronal membrane.

scholarly journals Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

1999 ◽  
Vol 113 (2) ◽  
pp. 333-346 ◽  
Author(s):  
G. Richard Benzinger ◽  
Gayle S. Tonkovich ◽  
Dorothy A. Hanck
Keyword(s):  
Time Course ◽  
Genetic Diseases ◽  
Low Frequency ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Na Channels ◽  
Cell Recovery ◽  
Reverse Transcription Pcr ◽  
Recovery From Inactivation ◽  
Voltage Dependent

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

scholarly journals Fast inactivation causes rectification of the IKr channel.

1996 ◽  
Vol 107 (5) ◽  
pp. 611-619 ◽  
Author(s):  
P S Spector ◽  
M E Curran ◽  
A Zou ◽  
M T Keating ◽  
M C Sanguinetti
Keyword(s):  
Outward Current ◽  
K Channel ◽  
Delayed Rectifier ◽  
Fast Inactivation ◽  
Time Constants ◽  
Recovery From Inactivation ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Cardiac Delayed Rectifier ◽  
Cytoplasmic Side

The mechanism of rectification of HERG, the human cardiac delayed rectifier K+ channel, was studied after heterologous expression in Xenopus oocytes. Currents were measured using two-microelectrode and macropatch voltage clamp techniques. The fully activated current-voltage (I-V) relationship for HERG inwardly rectified. Rectification was not altered by exposing the cytoplasmic side of a macropatch to a divalent-free solution, indicating this property was not caused by voltage-dependent block of outward current by Mg2+ or other soluble cytosolic molecules. The instantaneous I-V relationship for HERG was linear after removal of fast inactivation by a brief hyperpolarization. The time constants for the onset of and recovery from inactivation were a bell-shaped function of membrane potential. The time constants of inactivation varied from 1.8 ms at +50 mV to 16 ms at -20 mV; recovery from inactivation varied from 4.7 ms at -120 mV to 15 ms at -50 mV. Truncation of the NH2-terminal region of HERG shifted the voltage dependence of activation and inactivation by +20 to +30 mV. In addition, the rate of deactivation of the truncated channel was much faster than wild-type HERG. The mechanism of HERG rectification is voltage-gated fast inactivation. Inactivation of channels proceeds at a much faster rate than activation, such that no outward current is observed upon depolarization to very high membrane potentials. Fast inactivation of HERG and the resulting rectification are partly responsible for the prolonged plateau phase typical of ventricular action potentials.

Functional properties of a brain-specific NH2-terminally spliced modulator of Kv4 channels

2003 ◽  
Vol 285 (1) ◽  
pp. C161-C170 ◽  
Author(s):  
Linda M. Boland ◽  
Min Jiang ◽  
So Yeong Lee ◽  
Scott C. Fahrenkrug ◽  
Mark T. Harnett ◽  
...  
Keyword(s):  
Terminal Region ◽  
K Channel ◽  
The Novel ◽  
Splice Form ◽  
Interacting Protein ◽  
Current Decay ◽  
Amino Terminal ◽  
Human Genes ◽  
Kv4 Channels ◽  
Inactivation Gating

Kv4/K channel-interacting protein (KChIP) potassium channels are a major class of rapidly inactivating K channels in brain and heart. Considering the importance of alternative splicing to the quantitative features of KChIP gating modulation, a previously uncharacterized splice form of KChIP1 was functionally characterized. The KChIP1b splice variant differs from the previously characterized KChIP1a splice form by the inclusion of a novel amino-terminal region that is encoded by an alternative exon that is conserved in mouse, rat, and human genes. The expression of KChIP1b mRNA was high in brain but undetectable in heart or liver by RT-PCR. In cerebellar tissue, KChIP1b and KChIP1a transcripts were expressed at nearly equal levels. Coexpression of KChIP1b or KChIP1a with Kv4.2 channels in oocytes slowed K current decay and destabilized open-inactivated channel gating. Like other KChIP subunits, KChIP1b increased Kv4.2 current amplitude and KChIP1b also shifted Kv4.2 conductance-voltage curves by —10 mV. The development of Kv4.2 channel inactivation accessed from closed gating states was faster with KChIP1b coexpression. Deletion of the novel amino-terminal region in KChIP1b selectively altered the subunit's modulation of Kv4.2 closed inactivation gating. The role of the KChIP1b NH2-terminal region was further confirmed by direct comparison of the properties of the NH2-terminal deletion mutant and the KChIP1a subunit, which is encoded by a transcript that lacks the novel exon. The features of KChIP1b modulation of Kv4 channels are likely to be conserved in mammals and demonstrate a role for the KChIP1 NH2-terminal region in the regulation of closed inactivation gating.

scholarly journals Energetics of Shaker K channels block by inactivation peptides.

1993 ◽  
Vol 102 (6) ◽  
pp. 977-1003 ◽  
Author(s):  
R D Murrell-Lagnado ◽  
R W Aldrich
Keyword(s):  
Ionic Strength ◽  
Electrostatic Interactions ◽  
Time Course ◽  
Free Peptide ◽  
Terminal Domain ◽  
Voltage Dependent ◽  
Inactivation Process ◽  
K Concentration ◽  
Association Rate Constants ◽  
External K

A synthetic peptide of the NH2-terminal inactivation domain of the ShB channel blocks Shaker channels which have an NH2-terminal deletion and mimics many of the characteristics of the intramolecular inactivation reaction. To investigate the role of electrostatic interactions in both peptide block and the inactivation process we measured the kinetics of block of macroscopic currents recorded from the intact ShB channel, and from ShB delta 6-46 channels in the presence of peptides, at different ionic strengths. The rate of inactivation and the association rate constants (k(on)) for the ShB peptides decreased with increasing ionic strength. k(on) for a more positively charged peptide was more steeply dependent on ionic strength consistent with a simple electrostatic mechanism of enhanced diffusion. This suggests that a rate limiting step in the inactivation process is the diffusion of the NH2-terminal domain towards the pore. The dissociation rates (k(off)) were insensitive to ionic strength. The temperature dependence of k(on) for the ShB peptide was very high, (Q10 = 5.0 +/- 0.58), whereas k(off) was relatively temperature insensitive (Q10 approximately 1.1). The results suggest that at higher temperatures the proportion of time either the peptide or channel spends in the correct conformation for binding is increased. There were two components to the time course of recovery from block by the ShB peptide, indicating two distinct blocked states, one of which has similar kinetics and dependence on external K+ concentration as the inactivated state of ShB. The other is voltage-dependent and at -120 mV is very unstable. Increasing the net charge on the peptide did not increase sensitivity to knock-off by external K+. We propose that the free peptide, having fewer constraints than the tethered NH2-terminal domain binds to a similar site on the channel in at least two different conformations.

scholarly journals Voltage-activated K+ conductances in freshly isolated embryonic chicken osteoclasts

1989 ◽  
Vol 86 (17) ◽  
pp. 6821-6825 ◽  
Author(s):  
J H Ravesloot ◽  
D L Ypey ◽  
T Vrijheid-Lammers ◽  
P J Nijweide
Keyword(s):  
Time Constant ◽  
Membrane Potentials ◽  
Maximal Conductance ◽  
Exponential Process ◽  
Embryonic Chicken ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
K Concentration ◽  
Cs Ions ◽  
Full Activation

Patch-clamp measurements on freshly isolated embryonic chicken osteoclasts revealed three distinct types of voltage-dependent K+ conductance. The first type of conductance, present in 72% of the cells, activated at membrane potentials less negative than -30 to -20 mV and reached full activation at +40 mV. It activated with a delay, reached a peak value, and then inactivated with a time constant of approximately 1.5 s. Inactivation was complete or almost so. Recovery from inactivation, at -70 mV, had a time constant of roughly 1 s. The conductance could be blocked, at least partly, by 4 mM 4-aminopyridine. The second type of conductance (present in all cells) activated at membrane potentials more negative than -40 to -80 mV and reached full activation at -130 mV. Activation potential and maximal conductance were dependent on the extracellular K+ concentration. Inactivation of the conductance first became apparent at membrane potentials more negative than -100 mV and was a two-exponential process. The conductance could be blocked by external 5 mM Cs+ ions. The third type of conductance (present in all cells) activated at membrane potentials more positive than +30 mV. Generally, the conductance did not inactivate.

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