A peptide segment critical for sodium channel inactivation functions as an inactivation gate in a potassium channel

doi:10.1016/0896-6273(93)90125-B

Neuron

Volume 11, Issue 5, November 1993, Pages 967-974

https://doi.org/10.1016/0896-6273(93)90125-B Get rights and content

Abstract

The short cytoplasmic peptide segment connecting domains III and IV of voltage-gated sodium channels (III–IV linker) is essential for fast inactivation. To test the functional similarity between the III–IV linker and the potassium channel inactivation particle, we attached the III–IV linker to the amino terminus of a noninactivating potassium channel. This chimeric channel inactivated rapidly and displayed biophysical properties similar to Shaker A-type potassium channels. Recovery from inactivation in the chimeric channels was accelerated by high external potassium, consistent with the idea that potassium ions passing through the channel displaced the III–IV linker inactivation particle. A mutation that completely abolishes fast inactivation in rat brain sodium channels also completely abolished inactivation in the chimera. These results demonstrate that the sodium channel III–IV linker can function as a fast inactivation gate and suggest a functional relationship between the fast inactivation processes of sodium and potassium channels.

References (40)

V.J. Auld et al.
A rat brain Na⁺ channel α subunit with novel gating properties
Neuron
(1988)
R.L. Barchi
Sodium channel gene defects in the periodic paralyses
Curr. Opin. Neurobiol.
(1992)
S.C. Cannon et al.
Functional expression of sodium channel mutations identified in families with periodic paralysis
Neuron
(1993)
S.C. Cannon et al.
A sodium channel defect in hyperkalemic periodic paralysis: potassium-induced failure of inactivation
Neuron
(1991)
K.L. Choi et al.
The internal quaternary ammonium receptor site of Shaker potassium channels
Neuron
(1993)
S.D. Demo et al.
The inactivation gate of the Shaker K⁺ channel behaves like an open-channel blocker
Neuron
(1991)
A.L. Goldin
Expression of ion channels by injection of mRNA into Xenopus oocytes
Meth. Cell Biol.
(1991)
A. Kamb et al.
Multiple products of the Drosophila Shaker gene may contribute to potassium channel diversity
Neuron
(1988)
R. Morello et al.
Interaction of nonylguanidine with the sodium channel
Biophys. J.
(1980)
D.E. Patton et al.
A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes
Neuron
(1991)

W. Stühmer et al.

Potassium channels expressed from rat brain cDNA have delayed rectifier properties

FEBS Lett.

(1988)

R.W. Aldrich et al.

A reinterpretation of mammalian sodium channel gating based on single channel recording

Nature

(1983)

C.M. Armstrong

Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons

J. Gen. Physiol.

(1969)

C.M. Armstrong

Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons

J. Gen. Physiol.

(1971)

C.M. Armstrong et al.

Inactivation of the sodium channel. II. Gating current experiments

J. Gen. Physiol.

(1977)

V.J. Auld et al.

A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel

F. Bezanilla et al.

Inactivation of the sodium channel-sodium current experiments

J. Gen. Physiol.

(1977)

K.G. Chandy et al.

A family of three mouse potassium channel genes with intronless coding regions

Science

(1990)

D.C. Eaton et al.

Arginine-specific reagents remove sodium channel inactivation

Nature

(1978)

A.L. Hodgkin et al.

A quantitative description of membrane current and its application to conduction and excitation in nerve

J. Physiol.

(1952)

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Nav channels inactivate by both fast and slow mechanisms (Ahern et al., 2016; Ulbricht, 2005). The IFM motif on the III-IV linker was characterized to be critical for fast inactivation (Patton et al., 1993; Stühmer et al., 1989; Vassilev et al., 1989; West et al., 1992), and a number of residues on S4-S5 and S6 segments in repeats III and IV were shown to be involved in this process (McPhee et al., 1995, 1998; Smith and Goldin, 1997; Tang et al., 1996). In the structures of NavPaS and Cav1.1, a conserved helix on the III-IV linker interacts with the CTD, but neither channel contains the IFM motif (Shen et al., 2017; Wu et al., 2016).
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Competitive and non-competitive regulation of calcium-dependent inactivation in Ca<inf>V</inf>1.2 L-type Ca2+ channels by calmodulin and Ca2+-binding protein 1
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Multiple molecular determinants for inactivation have been identified. However, unlike for some voltage-dependent K+ and Na+ channels (4), the dynamics of conformational changes that culminate in VDI and CDI are controversial and remain the subject of intense research (5–10). CaM and CaBP1 are members of a large family of Ca2+ sensors and regulators that diversify Ca2+ signaling and neuronal activity through VGCCs and other pathways (11).
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Crystal structure of a fibroblast growth factor homologous factor (FHF) defines a conserved surface on FHFs for binding and modulation of voltage-gated sodium channels
2009, Journal of Biological Chemistry
Voltage-gated sodium channels (Na_v) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Na_v α subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Na_vs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Na_vs.
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Mathematical modeling suggests that alterations in the rate constants governing the transition from closed and open states into the inactivated states is sufficient to cause the electrophysiological changes observed in Fhf1−/−Fhf4−/− mice. Sodium channel inactivation is governed by interaction between an inactivation “particle” housed in the III-IV cytoplasmic loop and the inner pore of the channel (Patton et al., 1993; Vassilev et al., 1988; West et al., 1992). The channel C-terminal tail also contributes to inactivation (An et al., 1998; Glaaser et al., 2006; Mantegazza et al., 2001), potentially through contacts between the tail and the III-IV loop (Motoike et al., 2004).
Neurons integrate and encode complex synaptic inputs into action potential outputs through a process termed “intrinsic excitability.” Here, we report the essential contribution of fibroblast growth factor homologous factors (FHFs), a family of voltage-gated sodium channel binding proteins, to this process. Fhf1^−/−Fhf4^−/− mice suffer from severe ataxia and other neurological deficits. In mouse cerebellar slice recordings, WT granule neurons can be induced to fire action potentials repetitively (∼60 Hz), whereas Fhf1^−/−Fhf4^−/− neurons often fire only once and at an elevated voltage spike threshold. Sodium channels in Fhf1^−/−Fhf4^−/− granule neurons inactivate at more negative membrane potential, inactivate more rapidly, and are slower to recover from the inactivated state. Altered sodium channel physiology is sufficient to explain excitation deficits, as tested in a granule cell computer model. These findings offer a physiological mechanism underlying human spinocerebellar ataxia induced by Fhf4 mutation and suggest a broad role for FHFs in the control of excitability throughout the CNS.
Block of inactivation-deficient cardiac Na+ channels by acetyl-KIFMK-amide
2005, Biochemical and Biophysical Research Communications
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Despite these uncertainties, it is worth noting that the block of the Na+ channel by acetyl-KIFMK amide is quite similar to the action of the inactivation peptide in K+ channels responsible for the N-type fast inactivation process [22]. A transposed Na+ channel D3–D4 linker also acts as an inactivation gate in chimeric K+ channels, suggesting a functional relationship between fast inactivation process of Na+ and K+ channels [23]. The open Na+ channel may have an entryway of ∼15 Å in diameter toward the inner cavity [24].
The Na⁺ channel α-subunit contains an IFM motif that is critical for the fast inactivation process. In this study, we sought to determine whether an IFM-containing peptide, acetyl-KIFMK-amide, blocks open cardiac Na⁺ channels via the inner cavity. Intracellular acetyl-KIFMK-amide at 2 mM elicited a rapid time-dependent block (τ = 0.24 ms) of inactivation-deficient human heart Na⁺ channels (hNav1.5-L409C/A410W) at +50 mV. In addition, a peptide-induced tail current appeared conspicuously upon repolarization, suggesting that the activation gate cannot close until acetyl-KIFMK-amide is cleared from the open pore. Repetitive pulses (+50 mV for 20 ms at 1 Hz) produced a substantial use-dependent block of both peak and tail currents by ∼65%. A F1760K mutation (hNav1.5-L409C/A410W/F1760K) abolished the use-dependent block by acetyl-KIFMK-amide and hindered the time-dependent block. Competition experiments showed that acetyl-KIFMK-amide antagonized bupivacaine binding. These results are consistent with a model that two acetyl-KIFMK-amide receptors exist in proximity within the Na⁺ channel inner cavity.
A model of voltage gating developed using the KvAP channel crystal structure
2004, Biophysical Journal
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In Shaker channels, residues in the latter portion of S1 and S3, in the initial part of S2 and S4, and in the S1-S2 and S3-S4 loops are accessible from the extracellular solution in all conformations (Gandhi et al., 2003) (analogous KvAP residues in Structure 1 are located on or near the opposite side of the membrane). Residues immediately preceding S1 in Shaker channels are located in the cytoplasm (Patton et al., 1993) (in Structure 1 analogous KvAP residues are in the center of the transmembrane region). Except for the S3-S4 hairpin, the tertiary structure of the voltage-sensing domain in Structure 1 deviates substantially from the crystal structure (see Fig. 2 B) of the isolated KvAP voltage-sensing domain, Structure 2 (Jiang et al., 2003a).
Having inspected the crystal structure of the complete KvAP channel protein, we suspect that the voltage-sensing domain is too distorted to provide reliable information about its native tertiary structure or its interactions with the central pore-forming domain. On the other hand, a second crystal structure of the isolated voltage-sensing domain may well correspond to a native open conformation. We also observe that the paddle model of gating developed from these two structures is inconsistent with many experimental results, and suspect it to be energetically unrealistic. Here we show that the isolated voltage-sensing domain crystal structure can be docked onto the pore domain portion of the full-length KvAP crystal structure in an energetically favorable way to create a model of the open conformation. Using this as a starting point, we have developed rather conventional models of resting and transition conformations based on the helical screw mechanism for the transition from the open to the resting conformation. Our models are consistent with both theoretical considerations and experimental results.

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^†: Present address: Department of Physiology, UCLA School of Medicine, Los Angeles, California 90024.

^§: Present address: Cell Therapeutics Inc., Seattle, Washington 98102.

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Neuron

ArticleA peptide segment critical for sodium channel inactivation functions as an inactivation gate in a potassium channel

Abstract

Neuron

Curr. Opin. Neurobiol.

Neuron

Neuron

Neuron

Neuron

Meth. Cell Biol.

Neuron

Biophys. J.

Neuron

FEBS Lett.

A reinterpretation of mammalian sodium channel gating based on single channel recording

Nature

Inactivation of the potassium conductance and related phenomena caused by quaternary ammonium ion injection in squid axons

J. Gen. Physiol.

Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons

J. Gen. Physiol.

Inactivation of the sodium channel. II. Gating current experiments

J. Gen. Physiol.

A neutral amino acid change in segment IIS4 dramatically alters the gating properties of the voltage-dependent sodium channel

Inactivation of the sodium channel-sodium current experiments

J. Gen. Physiol.

A family of three mouse potassium channel genes with intronless coding regions

Science

Arginine-specific reagents remove sodium channel inactivation

Nature

A quantitative description of membrane current and its application to conduction and excitation in nerve

J. Physiol.

Article
A peptide segment critical for sodium channel inactivation functions as an inactivation gate in a potassium channel