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An intermolecular Zn2؉ -binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1). T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules. In this structure, a Zn 2؉ ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX 5 CX 20 CC) of the T1 domain. This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels. The presence of this conserved Zn 2؉ -binding site is a primary molecular determinant that distinguishes the tetrameric assembly of nonShaker Kv channel subunits from that of Shaker channels. We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn 2؉ , and the addition/removal of Zn 2؉ reversibly switches the protein between a stable tetrameric or monomeric state. We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly. The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent. Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn 2؉ . The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn 2؉ to be removed more effectively by EDTA. The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels.Voltage-gated, potassium ion selective (Kv) channels are found throughout most of the eukaryotic kingdom and display the widest range of channel properties among all voltage-gated ion channels (1-3). They are involved in various physiological functions such as the regulation of action potential firing and duration, endocrine and exocrine secretion, cardiac excitability, learning and memory, as well as the control of synaptic efficacy. Functional Kv channels are formed by the tetramerization of their pore-forming ␣-subunits, a process regulated by the T1 domain of the channel (4, 5). Four major gene subfamilies (Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4)) are designated by sequence similarity and their ability to homo-and heterotetramerize exclusively within subfamily members. In the crystal structures of Shaker (6) and Shaw T1 tetramers (7), four T1 subunits form a rotationally symmetric tetramer. The interfacial interaction is highly polar, and the interface residues show a high degree of sequence conservation within a subfamily, thus providing a structural explanation for the subfamily-specific assembly of Kv channels. Most recently, studi...

The hemolytic plaque assay technique can be used to detect specific hormone release from single pituitary cells. Using antisera raised against murine GH or rat PRL, we have enumerated the active lactotropes and somatotropes from male and female rat pituitary glands. These studies reveal sex-related differences in the number of cells exporting GH and PRL among anterior pituitary cells in culture. In the presence of human GH-releasing factor (hGRF), the mean percentage of GH cells was 53% in males and 30% in females (P less than 0.005). The mean percentage of PRL cells was 15% in males and 39% in females (P less than 0.008). These values were not significantly altered when hGRF was omitted. The sum of GH and PRL cells identified in separate plaque assays significantly exceeds the number obtained when GH and PRL cells were determined concurrently with a simultaneous plaque assay for both hormones. This difference is dependent on the presence of hGRF, since there was no difference when hGRF was omitted. These data identify the mammosomatotrope in numbers lower than previous reports. By this approach, the mammosomatotrope subpopulation numbers about 5% of all cells in culture. In summary, we demonstrate a sex-related difference in the number of cells exporting GH or PRL among pituitary cells in culture. This difference corresponds with and may underly sex-related differences in the responsiveness of GH and PRL secretion from the pituitary gland. Furthermore, a minor subpopulation of normal pituitary cells appears capable of simultaneous secretion of both GH and PRL.

Although survival from primary breast cancer has improved with earlier diagnosis and treatment, the management of the opposite breast is still in question. The risk factors for bilaterality are known, and preoperative mammography is occasionally helpful, but identification of early second breast cancer is very limited. Contralateral biopsy may provide a reasonable answer to the problem. During a 5-year period, 62 elective contralateral biopsies were performed in patients having mastectomies for primary breast cancer. This consisted of either a mirror image biopsy or, more commonly, a biopsy of the upper outer quadrant. Thirteen patients had simultaneous contralateral cancers, of whom two had clinically overt bilateral cancers and 11 (18%) had clinically occult malignancy. Seven of these 11 had both radiologically and clinically normal breasts. Thus, 11.3% had radiologically and clinically occult cancer demonstrated by biopsy. Surgical management consisted of total mastectomy with low axillary dissection for noninvasive cancers and modified radical mastectomy for invasive cancers. Pathologic findings of the dominant breast cancer and the contralateral lesion were: bilateral, noninvasive: three patients; invasive, noninvasive: (seven patients), and invasive, invasive: three patients. Although follow-up is short (median of 40 months), 82% of the patients who had clinically occult second-breast cancer remain free of disease. During a previous 8-year period, 37 of 500 primary breast cancer patients (7.4%) developed metachronous (33) or synchronous (4) second-breast primary cancers primarily diagnosed clinically or radiologically. Of these, 35 were invasive and two noninvasive cancers; 41% had nodal metastases. A selected "favorable group," 28 of these patients who were free of disease 3 years after their first cancer, was analyzed. The analysis showed that only 10 (36%) were surviving free of disease at 7 years; 25% were free of disease at 10 years. Although the incidence of clinically-recognized, second-primary breast cancer is relatively low, development of a second invasive cancer severely impairs patient survival. Contralateral biopsy would appear useful to identify patients with early invasive or preinvasive cancer in the second breast, which appears normal after clinical observation or mammography. It provides opportunity to reduce the risk of invasive cancer in that breast, as well as to provide important diagnostic and prognostic information.