Cooperative effect of S4-S5 loops in domains D3 and D4 on fast inactivation of the Na+ channel

Author(s): Popa MO, Alekov AK, Bail S, Lehmann-Horn F, Lerche H

Abstract

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.

Similar Articles

Sodium channelopathies of skeletal muscle result from gain or loss of function

Author(s): Jurkat-Rott K, Holzherr B, Fauler M, Lehmann-Horn F

The skeletal muscle sodium and chloride channel diseases

Author(s): Hudson AJ, Ebers GC, Bulman DE

Primary structure of the adult human skeletal muscle voltage-dependent sodium channel

Author(s): George AL Jr, Komisarof J, Kallen RG, Barchi RL

A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation

Author(s): West JW, Patton DE, Scheuer T, Wang Y, Goldin AL, et al.

Sodium channel mutations in paramyotoniacongenita uncouple inactivation from activation

Author(s): Chahine M, George AL Jr, Zhou M, Ji S, Sun W, et al.

Sodium channel mutations in paramyotoniacongenita exhibit similar biophysical phenotypes in vitro

Author(s): Yang N, Ji S, Zhou M, Ptácek LJ, Barchi RL, et al.

Thr1313Met mutation in skeletal muscle sodium channels in a Japanese family with paramyotoniacongenita

Author(s): Kinoshita M, Sasaki R, Nagano T, Matsuda A, Nakamura S, et al.

Voltage-gated ion channels and hereditary disease

Author(s): Lehmann-Horn F, Jurkat-Rott K