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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.

The potassium channel T1 domain plays an important role in the regulated assembly of subunit proteins. We have examined the assembly properties of the Shaker channel T1 domain to determine if the domain can self-assemble, the number of subunits in a multimer, Ns and the mechanism of assembly. High pressure liquid chromatography (HPLC) size exclusion chromotography (SEC) separates T1 domain proteins into two peaks. By co-assembly assays, these peaks are identified to be a high molecular weight assembled form and a low molecular weight monomeric form. To determine the Ns of the assembled protein peak on HPLC SEC, we first cross-linked the T1 domain proteins and then separated them on HPLC. Four evenly spaced bands co-migrate with the assembled protein peak; thus, the T1 domain assembles to form a tetramer. The absence of separate dimeric and trimeric peaks of assembled T1 domain protein suggests that the tetramer is the stable assembled state, most probably a closed ring structure.

Chinese hamster cell lines have been widely used to explore the mechanism of gene amplification under stress conditions. Investigations of gene amplification in Chinese hamster cells under nonstress conditions, however, have been limited due to technical reasons. We initiated studies on the amplification mechanism by examining the frequency of interstitial telomeric bands (ITBs) in continuously growing cultures by fluorescence in situ hybridization (FISH), using a (TTAGGG)n probe. We found that one of the chromosomes uniquely exhibit amplification of ITBs in Chinese hamster ovary (CHO) cells but not in drug-resistant mutant CHO cells or in Chinese hamster V79 cells. Amplification of ITBs could not be induced by DNA damaging agents or DNA synthesis inhibitors. To investigate the early events that mediate ITB amplification, we developed clones and determined that the frequency of acquisition of additional ITBs on the marker chromosome was in the range of 10–5 to 10–3 per cell doubling. Using DNA strand-specific FISH, we found that one of the initial events of amplification is isochromosome formation. Our observations support the involvement of sister chromatid fusion in the initiation of spontaneous ITB amplification.