Cooperative effect of S4-S5 loops in domains D3 and D4 on fast inactivation of the Na+ channel.
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 alpha-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.
Department of Applied Physiology, University of Ulm, D-89069 Ulm, Germany.
M Oana
MO
Popa
Alexi K
AK
Alekov
Sigrid
S
Bail
Frank
F
Lehmann-Horn
Holger
H
Lerche
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D003300:Copper; D003545:Cysteine / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D062553:NAV1.4 Voltage-Gated Sodium Channel; D018408:Patch-Clamp Techniques; D010618:Phenanthrolines; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents
D009124:Muscle Proteins; D062553:NAV1.4 Voltage-Gated Sodium Channel; D010618:Phenanthrolines; C106251:SCN4A protein, human; D015222:Sodium Channels; D013439:Sulfhydryl Reagents; D003300:Copper; D003545:Cysteine