Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.
1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III-IV linker). Specifically, we measured whole-cell and single-channel currents through wild-type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild-type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild-type. The fraction of non-inactivating current is substantial (37 +/- 2% of peak current at -20 mV) in F1304Q. 3. Cell-attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1-subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild-type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in 'inactivation-deficient' F1304Q channels. 5. The steady-state voltage dependence of F1304Q inactivation is right-shifted by 26 +/- 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild-type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q-induced rightward shift in steady-state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.
Nuss
H B
HB
Department of Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.
Balser
J R
JR
Orias
D W
DW
Lawrence
J H
JH
Tomaselli
G F
GF
Marban
E
E
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005260:Female; D015640:Ion Channel Gating; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials; D018482:Muscle, Skeletal / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015222:Sodium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors; D014982:Xenopus laevis
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D015222:Sodium Channels