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.

Voltage dependent activation of potassium channels is coupled to T1 domain structure.

The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.

Case of the month: June 1997--a 42 year old man with left facial weakness.

A 42 yr old male presented with left facial weakness. MRI showed lesions affecting the distal seventh nerve and third division of the trigeminal nerve. The seventh nerve was biopsied and showed a malignant epithelioid schwannoma. The patient underwent extensive resection followed by irradiation. This is one of very few examples of intracranial malignant peripheral nerve sheath tumors and the first reported example of an intracranial malignant epithelioid schwannoma. The literature is reviewed and completeness of resection appears to be the most pertinent prognostic factor.

Shaker K+ channel T1 domain self-tetramerizes to a stable structure.

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.

Post-Salmonella reactive arthritis: late clinical sequelae in a point source cohort.

PURPOSE: To define the natural history of post-Salmonella-infection reactive arthritis (ReA) in a point source cohort concurrently exposed to the same microorganism, and to determine any relationship between anti-Salmonella humoral immune response to the organism and clinical outcome at 5 years. PATIENTS AND METHODS: A cohort of 423 Ontario Provincial Police officers with a clinical diagnosis of Salmonella food poisoning were defined in 1984. Five years following the food poisoning, a mail and telephone survey was carried out to determine all those who developed ReA within 3 months of the onset of dysentery. Medical and physiotherapy charts from an earlier study on the same cohort were incorporated. All patients with a history compatible with reactive arthritis were interviewed and examined. Serum was taken to determine the presence of isotypic antibodies to the lipopolysaccharide of the causative Salmonella typhimurium. RESULTS: Twenty-seven of the 423 individuals with dysentery were identified as developing acute ReA. In one third of them, the arthritis resolved within 4 months of onset. Two thirds continued to have subjective complaints, mostly of minor significance. However, symptoms were severe enough to force a change in work for 4 patients. Another 4 patients had objective damage to joints radiographically. Objective changes to joints were documented on physical examination in 37% of ReA patients 5 years following onset of disease. IgA antilipopolysaccharide antibodies correlated with the severity and duration of disease. Tests of cellular immune function did not correlate with clinical variables. CONCLUSIONS: Chronic symptoms persist 5 years after the onset of ReA in the majority of patients. Joint damage by physical examination and radiographic assessment correlate with functional disability. Some early clinical features of disease, including prolonged diarrhea during the acute illness, may predict a worse outcome. IgA antilipopolysaccharides may serve as a disease marker for late post-Salmonella-infection ReA.

Spontaneous amplification of interstitial telomeric bands in Chinese hamster ovary cells.

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.