Ile-177 and Ser-180 in the S1 Segment Are Critically Important in Kv1.1 Channel Function

Mathur, Rajesh S.; Zhou, Jun; Babila, Tamar; Koren, Gideon

doi:10.1074/jbc.274.17.11487

Cited by 9 publications

(9 citation statements)

References 32 publications

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“…The immunoprecipitation experiments were carried out as described previously (29,31). Protein samples were heated in SDS sample buffer, and the proteins were resolved by electrophoresis on 8% or 10% SDS-PAGE gels.…”

Section: Methodsmentioning

confidence: 99%

“…PalmϪ stable CHO cells were grown to 50 -60% confluence on glass coverslips in six-well plates, fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100 in PBS for 5 min at room temperature, and blocked with 5% donkey serum in PBS for 1 h at room temperature (29,31). The cells were then incubated with rabbit polyclonal or mouse monoclonal antibodies at 1:100 -250 in 5% donkey serum for 1 to 2 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

Jindal

Folco²,

Liu³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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Jindal HK, Folco EJ, Liu GX, Koren G. Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOHterminal palmitoylation modulates its biological properties. Am J Physiol Heart Circ Physiol 294: H2012-H2021, 2008. First published March 14, 2008 doi:10.1152/ajpheart.01374.2007.-The physiological function of ion channels is affected by protein-protein and protein-membrane interactions that modulate their activity and/or localization. Palmitoylation modulates protein function by facilitating targeted membrane association, interaction with other proteins, and determining subcellular localization. In this study, we demonstrate that the voltage-dependent potassium (Kv) channel Kv1.5 is palmitoylated and that the mutation of COOH-terminal cysteines is sufficient to abolish the palmitoylation of the Kv1.5 polypeptide in Chinese hamster ovary (CHO) cells. The labeling represented the thioester linkage of the labeled palmitic acid to cysteine rather than amide and oxygen ester linkages as judged by the release of the palmitic acid upon the treatment of the gel with hydroxylamine at a neutral pH. Site-directed mutagenesis and radiolabeling studies revealed that C593 was the sole site of palmitoylation. The elucidation of the biological function of palmitoylation revealed that the expression of the FLAG-Kv1.5 palmitoylation-deficient mutant (FLKv1.5PalmϪ ) in stable CHO cells increased membrane expression as determined by the biotinylation of surface proteins and quantitative immunofluorescence analyses of these cells, in turn enhancing the outward potassium current. This enhanced surface expression and the currents were consequential to the slower rate of internalization, causing an increased localization of PalmϪ in the plasma membrane compared with the wild-type FL-Kv1.5 channels. We conclude that the Kv1.5 channel is palmitoylated and that its palmitoylation modulates its biological functions and, therefore, might provide a physiological link between the metabolic state and the expression of Kv1.5 on the plasma membrane.internalization; trafficking VOLTAGE-DEPENDENT potassium (Kv) channels are involved in establishing the resting potential, shaping action potentials, and cellular repolarization in the heart and peripheral vascular tissues (21, 33) by regulating potassium ion fluxes in response to alterations of membrane potentials.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

Jindal

Folco²,

Liu³

et al. 2008

American Journal of Physiology-Heart and Circulatory Physiology

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“…The importance of Ile177 for Kv1.1 channel function has been inferred from the expression characteristics of several mutations of this residue [21]. However, this study did not report biophysical evidence supporting this conclusion, as the mutations analysed abolished channel function.…”

Section: Resultsmentioning

confidence: 68%

Functional characterization of an episodic ataxia type-1 mutation occurring in the S1 segment of hKv1.1 channels

Imbrici

Cusimano

D’Adamo

et al. 2003

Pflugers Arch - Eur J Physiol

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Voltage-gated potassium channels (Kv) play important roles in neurotransmission, nerve cell excitability and disease. Several missense mutations in the Kv1.1 gene have been associated with episodic ataxia type-1 syndrome (EA-1), which is characterized by continuous myokymia, episodic attacks of ataxic gait and spastic contractions of skeletal muscles. In this study we show that I177N, an EA-1 mutation located in S1 segment, alters the expression and gating properties of the channel expressed in Xenopus oocytes. In particular, it reduces ~17-fold the current amplitude, accelerates ~4-fold the deactivation kinetics of the channel and shifts the voltage dependence of activation ~60 mV to more depolarized potentials. Single-channel recordings also showed that the mean open duration of I177N channels was ~2.6-fold smaller than the wild-type. These results demonstrate that both reduced current levels and specific gating defects are the likely causes of EA-1 symptoms in patients bearing the I177N mutation. Furthermore, the data suggest that the I177N substitution may alter the gating properties of the channel that are specifically defined by the S1 segment.

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“…No gating currents could be detected in 15 out of 114 mutants (minus signs "-" in Table 1). Among them, the homologous mutations I237R, S240L, S240P, and S240R in the related potassium channel Kv1.1 were previously shown to abolish K + conduction (49).…”

Section: Resultsmentioning

confidence: 99%

Moving gating charges through the gating pore in a Kv channel voltage sensor

Lacroix

Hyde

Campos

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.energy landscape | kinetic model | global fit

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Ile-177 and Ser-180 in the S1 Segment Are Critically Important in Kv1.1 Channel Function

Cited by 9 publications

References 32 publications

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

Posttranslational modification of voltage-dependent potassium channel Kv1.5: COOH-terminal palmitoylation modulates its biological properties

Functional characterization of an episodic ataxia type-1 mutation occurring in the S1 segment of hKv1.1 channels

Moving gating charges through the gating pore in a Kv channel voltage sensor

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