A central role for the T1 domain in voltage-gated potassium channel formation and function.
To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the full-length functional channel.
Strang
C
C
Division of Neuroscience, Baylor College of Medicine, Houston, Texas 77030, USA.
Cushman
S J
SJ
DeRubeis
D
D
Peterson
D
D
Pfaffinger
P J
PJ
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D051662:Kv1.1 Potassium Channel; D018613:Microscopy, Confocal; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D017354:Point Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D014162:Transfection; D014357:Trypsin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D051662:Kv1.1 Potassium Channel; D014357:Trypsin