Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 M) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis-i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.Transthyretin (TTR) is present in human plasma (0.2 mg͞ml; 3.63 M, tetramer) and is composed of four identical ␤-sheetrich subunits that bind and transport thyroxine (T4) and the retinol binding protein (1). In unfortunate individuals, TTR is converted into an insoluble fibrillar structure called amyloid. These fibrils putatively cause senile systemic amyloidosis (wild-type TTR composes the fibrils-late onset) and familial amyloid polyneuropathy (FAP; predominantly variant TTR composing the fibrils-earlier onset) by virtue of the amyloid's neurotoxicity and͞or by physically interfering with normal organ function (2-8). A TTR amyloid fibril is Ϸ130Å in diameter and made up of four protofilaments, each having a twisted cross-␤-helix structure (9, 10). TTR amyloid fibril formation is observed during partial acid denaturation from a conformational intermediate formed under conditions simulating a lysosome (pH 5.5 Ϯ 0.5), which has been implicated in fibril formation in vivo (11,12). TTR amyloid fibril formation can be avoided under acidic conditions by working at low TTR concentrations and low temperature (25ЊC) allowing identification of the quaternary, tertiary, and secondary structure of the intermediate(s) that can form amyloid (12). These studies reveal that tetrameric TTR is nonamyloidogenic; however, the dissociation of the tetramer into a monomeric intermediate having an altered, but defined, tertiary structure is capable of amyloid fibril formation and is therefore called the amyloidogenic intermediate (Fig. 1). Several of the 50 FAP-associated TTR single-site mutations still adopt a normal tetrameric s...

KChIP proteins regulate Shal, Kv4.x, channel expression by binding to a conserved sequence at the N terminus of the subunit. The binding of KChIP facilitates a redistribution of Kv4 protein to the cell surface, producing a large increase in current along with significant changes in channel gating kinetics. Recently we have shown that mutants of Kv4.2 lacking the ability to bind an intersubunit Zn 2؉ between their T1 domains fail to form functional channels because they are unable to assemble to tetramers and remain trapped in the endoplasmic reticulum. Here we find that KChIPs are capable of rescuing the function of Zn 2؉ site mutants by driving the mutant subunits to assemble to tetramers. Thus, in addition to known trafficking effects, KChIPs play a direct role in subunit assembly by binding to monomeric subunits within the endoplasmic reticulum and promoting tetrameric channel assembly. Zn 2؉ -less Kv4.2 channels expressed with KChIP3 demonstrate several distinct kinetic changes in channel gating, including a reduced time to peak and faster entry into the inactivated state as well as extending the time to recover from inactivation by 3-4 fold.The formation of voltage-gated potassium (Kv) 1 channels is a multistep process with many different interactions and folding events required to form the completed channel (1). The common functional core of all Kv channels assembles as a tetramer of pore-forming ␣-subunits. This tetramer is the core of the future ion channel signal transduction complex, but additional folding steps as well as interactions with auxiliary proteins occur before the final functional channel complex at the cell surface is formed. Many auxiliary subunit proteins that bind to Kv ␣-subunits have been identified, but precisely when these interactions occur during channel complex formation and what role these interactions play in helping the channels to assemble, traffic, and function are topics of great interest (1-4). Through the use of heterologous expression systems and mutagenesis studies, we can expose many of these important interactions and folding events, and reveal the processes by which Kv channel complexes form. A comparison of channel expression and functional properties with and without specific auxiliary proteins reveals how these different processes contribute to the formation and function of ion channel complexes.An early step in Kv channel formation involves the tetramerization of the ␣-subunit T1 domains at the cytoplasmic N terminus of the protein (5-7). For Kv4.2 channels, a critical component of the T1 domain interaction involves the coordination of an intersubunit Zn 2ϩ ion found on non-Shaker type Kv channel T1 domains (8 -10). Although Zn 2ϩ binding sites are common in proteins, intersubunit Zn 2ϩ binding sites, as found in the T1 domain, are relatively rare. To determine what functions might be regulated by the T1 intersubunit Zn 2ϩ site, we generated a series of mutations to the Zn 2ϩ coordination residues and tested them for cell surface expression (8). We found that mut...

Gating of voltage-dependent K ϩ channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn 2 ϩ coordination. Also, added Zn 2 ϩ or a potent Zn 2 ϩ chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is ‫ف‬ 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact ␣ -subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1-T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn 2 ϩ binding. I N T R O D U C T I O NActivation of voltage-gated potassium channels (Kv channels) is directly controlled by the movements of their S4 voltage sensors, and a subsequent concerted conformational change that opens an internal gate (Yellen, 1998;Horn, 2000;Bezanilla and Perozo, 2003). The bundle-crossing of four transmembrane S6 segments constitutes the main activation gate that controls K ϩ passage at the internal opening of the tetrameric pore structure (Jiang et al., 2002;Webster et al., 2004). Just beneath the main activation gate, the NH 2 -terminal tetramerization domain (T1) of Kv channels is a fourfold symmetric structure that is responsible for the subfamily-specific coassembly of Kv subunits (Li et al., 1992;Shen et al., 1993). The "side windows" between the T1 domain and the transmembrane core domain provide direct access to the internal mouth of the pore (Kreusch et al., 1998;Gulbis et al., 2000;Kobertz et al., 2000;Sokolova et al., 2001;Kim et al., 2004a). Recent studies have suggested that the T1 domain and other intracellular regions also contribute to the function of Kv channels (Cushman et al., 2000;Gulbis et al., 2000;Minor et al., 2000;Kurata et al., 2002;Hatano et al., 2003;Wray, 2004). However, the underlying molecula...

Electron micrographs are shown ofthe first component of human complement (CI) which has been crosslinked with a water-soluble carbodiimide to prevent dissociation into its Clq and Clr2Cls2 subunits. Two projections of the crosslinked molecule are seen in the electron micrographs, which are called "top" and "profile. " In both views, the Clq heads are visible. From the top, the Clr2Cls2 tetrameric subunit appears to be located centrally on the Clq and folded to form a compact mass obscuring most of the arms and central bundle. In profile, the tetramer appears to be located in the region of the arms between the Clq heads and the central bundle. Both the heads and the rod-like central bundle appear to be free of Clr2Cls2 in these profile projections. Sometimes it is possible to count more than six domains in the region ofthe Clq heads, as though a portion ofthe tetramer had unfolded to protrude among the heads.Excellent electron micrographs ofClq (1-4) and ofthe Clr2Cls2 tetramer have been published (5), as well as hydrodynamic studies on Clq, Clr2Cls2, and C1 (5-12). Clq has the appearance of a bouquet of tulips (13); Clr2Cls2 resembles a rod with bent ends (5). Reassembled C1, which spontaneously re-forms when one subunit of Clq is mixed with one tetrameric subunit of Clr2Cls2 in the presence of Ca2+, has hydrodynamic properties consistent with a 1-to-i complex (ref. 12; unpublished data) but the ultrastructure of the complex has remained an intriguing mystery, perhaps due to a tendency of C1 to dissociate when it adheres to a carbon-coated grid. We have successfully stabilized the C1 complex by treatment with a water-soluble carbodiimide and have succeeded in obtaining electron micrographs of this fascinating and unusual macromolecule. METHODSPreparation of Clr2C1s2. A modification of the procedure described by Medicus and Chapius (14) was used to purify Clr2Cls2. Diluted serum was pumped at a flow rate of200 mV hr, through a 2 x 30 cm rabbit IgG-Sepharose affinity column, strictly at 40C, in the presence of 30 ,uM p-nitrophenylguanidinobenzoate (NPGB). Next, the column was washed with 200 ml of a 1:1 mixture of 0.1% gelatin/0.15 mM CaCl2/L mM MgCl2 in Veronal-buffered saline (GVB) and 9.7% sucrose/ 0.15 mM CaCl2/l mM MgCl2 in Veronal-buffered saline (SVB) and then 500 ml of Veronal-buffered saline (VBS), containing NPGB. The subcomponents Clr and Cls were eluted with VBS/ 25 mM EDTA/4.85% sucrose/30 ,uM NPGB (250 ml), concentrated by ultrafiltration through a YM 10 membrane (Amicon, Lexington, MA), and reconstituted to form the Clr2Cls2 complex by dialysis against 0.01 M Tris/0. 15 M NaCl (TBS) at pH 7.3 containing 5 mM Ca2+. The reconstituted Clr2Cls2 was loaded onto a 2 X 100 cm Sepharose 4B column and eluted with TBS at pH 7.3 containing 5 mM Ca2+. The symmetrical portion of the first protein peak was pooled and concentrated by ultrafiltration. The preparation was shown to contain Clr and Cls by immunodiffusion with specific anti-Cir and anti-Cis antisera (Atlantic Antibodies, Scarborough, ME)....

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...