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Cooperative effect of S4–S5 loops in domains D3 and D4 on fast inactivation of the Na + channel
Author(s) -
Oana Popa M.,
Alekov Alexi K.,
Bail Sigrid,
LehmannHorn Frank,
Lerche Holger
Publication year - 2004
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2004.065912
Subject(s) - chemistry , reagent , cysteine , biophysics , kinetics , protein subunit , cytoplasm , oxidizing agent , mutation , patch clamp , biochemistry , receptor , biology , enzyme , physics , organic chemistry , quantum mechanics , gene
Cytoplasmic S4–S5 loops have been shown to be involved in fast inactivation of voltage‐gated ion channels. We studied mutations in these loops and their potential cooperative effects in domains D3 (N1151C, A1152C, I1160C/A) and D4 (F1473C, L1482C/A) of the human skeletal muscle Na + channel α‐subunit (hNa v 1.4) using expression in tsA201 cells and the whole cell patch‐clamp technique. All cysteine mutations were accessible to intracellularly applied sulfhydryl reagents which considerably destabilized fast inactivation. For different combinations of corresponding D3/D4 double mutations, fast inactivation could be almost completely removed. Thermodynamic cycle analysis indicated an additive effect for N1151C/F1473C and a significant cooperative effect for I1160/L1482 double mutations. Application of oxidizing reagents such as Cu‐phenanthroline to link two cysteines via a disulfide bridge did not reveal evidence for a direct physical interaction of cysteines in D3 and D4. In addition to the pronounced alterations of fast inactivation, mutations of I1160 shifted steady‐state activation in the hyperpolarizing direction and slowed the kinetics of both activation and deactivation. Sulfhydryl reagents had charge‐dependent effects on I1160C suggesting interaction with negative charges in another protein region. We conclude that fast inactivation of the Na + channel involves both S4–S5 loops in D3 and D4 in a cooperative manner. D3/S4–S5 also plays an important role in activation and deactivation.