Regulation of mammalian Shaker-related K+ channels: evidence for non-conducting closed and non-conducting inactivated states.
1. Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K+ channels induced by the removal of extracellular potassium, K+o. 2. In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3. To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mM K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4. The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild-type Kv1.3 channel. 5. CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6. We conclude that removal of K+o induces the Kv1.3 channel to transition to a non-conducting 'closed' state which can switch into a non-conducting 'inactivated' state upon depolarization.
Jäger
H
H
Department of Applied Physiology, University Ulm, Germany.
Rauer
H
H
Nguyen
A N
AN
Aiyar
J
J
Chandy
K G
KG
Grissmer
S
S
D016475:3T3 Cells; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004594:Electrophysiology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D006639:Histidine / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D015640:Ion Channel Gating / Q000502:physiology; D051662:Kv1.1 Potassium Channel; D051663:Kv1.2 Potassium Channel; D051664:Kv1.3 Potassium Channel; D051665:Kv1.4 Potassium Channel; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D024642:Potassium Channels, Voltage-Gated; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured
C496947:Kcna1 protein, mouse; C496970:Kcna2 protein, mouse; C496974:Kcna3 protein, mouse; C496978:Kcna4 protein, mouse; D051663:Kv1.2 Potassium Channel; D051664:Kv1.3 Potassium Channel; D051665:Kv1.4 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051662:Kv1.1 Potassium Channel; D006639:Histidine; D011188:Potassium