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...

Voltage-gated potassium channels are formed by the tetramerization of their ␣ subunits, in a process that is controlled by their conserved N-terminal T1 domains. The crystal structures of Shaker and Shaw T1 domains reveal interesting differences in structures that are contained within a highly conserved BTB/POZ domain fold. The most surprising difference is that the Shaw T1 domain contains an intersubunit Zn 2؉ ion that is lacking in the Shaker T1 domain. The Zn 2؉ coordination motif is conserved in other non-Shaker channels making this the most distinctive difference between these channels and Shaker. In this study we show that Zn 2؉ is an important co-factor for the tetramerization of isolated Shaw and Shal T1 domains. Addition of Zn 2؉ increases the amount of tetramer formed, whereas chelation of Zn 2؉ with phenanthroline blocks tetramerization and causes assembled tetramers to disassemble. Within an intact cell, full-length Shal subunits containing Zn 2؉ site mutations also fail to form functional channels, with the majority of the protein found to remain monomeric by size exclusion chromatography. Therefore, zinc-mediated tetramerization also is a physiologically important event for full-length functional channel formation.Voltage-dependent potassium channels (Kv channels) play important roles in regulating the excitability of electrically active cells such as neurons and muscle cells (1-3). We are interested in the unique properties and biological roles for the different classes of voltage-dependent potassium channels that have existed as distinct groups for hundreds of millions of years. Kv channel ␣ subunits assemble into tetramers, and they are classified into different subfamilies based on sequence homology and assembly specificity (3, 4 -6). In general, the subunits in specific subfamilies form functional channels by the selective tetramerization of these subunits only with other members of the same subfamily.Previous work on Shaker channel assembly has identified the T1 domain, a highly conserved N-terminal cytoplasmic domain, as a critical determinant for subunit self-association into tetrameric channels (7-9). Recently, we reported a notable structural difference between the Shaker (Kv1) and Shaw (Kv3) type ␣ subunits based on crystallographic analyses of the T1 domains of these subunits (10,11). Comparisons between these structures revealed that an intersubunit Zn 2ϩ atom is coordinated between the subunits of the tetrameric T1 domain of Kv3 but not Kv1. By sequence analysis, it was noted that the Zn 2ϩ coordination residues are highly conserved in all Shaw (Kv3), Shab (Kv2), and Shal (Kv4) subunits but are never seen in Shaker (Kv1) subunits. The lack of an intersubunit Zn 2ϩ site in the Shaker T1 domain is the primary structural feature that we have identified to distinguish this channel gene family from the other voltage-gated potassium channels (12).Given the central role for the T1 domain in the assembly of Kv channels, we hypothesize that the interfacial Zn 2ϩ coordination site is likely to ...

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 fulllength functional channel.The structural elements of potassium channels have begun to be characterized in atomic detail, allowing much increased sophistication in our understanding of their mechanism of action and biological function. For voltage-dependent potassium (Kv) 1 channels, the structure of the highly conserved cytoplasmic N-terminal T1 domain has been determined as a rotationally symmetric tetramer from three different Kv channels and in complex with an auxiliary ␤-subunit protein (1-4). Because the T1 domain structures are determined from isolated soluble protein domains, questions arise as to the relevance of the determined structures to the ultrastructure of the full-length Kv channel and in how the tetrameric domain is integrated into the remainder of the channel. Recent published studies have suggested that the T1 structure within the channel is likely to be very similar to the tetrameric structure of the isolated domain (3, 5-7). However, crystallography also has shown that the T1 domain can adopt several different conformations within the cha...

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 selfassemble, the number of subunits in a multimer, N s 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 N s of the assembled protein peak on HPLC SEC, we first crosslinked 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.Voltage-gated K ϩ channel proteins are multisubunit ion channel proteins. The core channel consists of an apparent tetramer of ␣-subunit proteins that assembles to form the K ϩ ion-selective aqueous pore across the cell's plasma membrane (1). In addition, ␤-subunit proteins, which are apparently not transmembrane proteins, can be attached to each ␣-subunit protein (2). Molecular cloning has revealed a large diversity of K ϩ channel subunit proteins. Sequence comparisons among ␣-subunits has revealed that the similarities among encoded proteins cluster into a variety of K ϩ channel subfamilies (3). Biophysical studies have indicated that these subfamilies are in fact functional subsets of channel proteins in that functional heteromultimeric channels have only been formed by co-expression of two ␣-subunit proteins from the same subfamily (4).The mechanisms that govern the assembly and function of voltage-gated ion channels are poorly understood. Recently we and others have identified a conserved molecular domain, the T1 domain, encoded within the cytoplasmic N terminus of the ␣-subunit protein that plays an important role in the assembly of K ϩ channel subunit proteins (5-10). Our studies have suggested that the T1 domain, translated by itself, can self-assemble (6). Sucrose density gradients reveal the formation of a high molecular weight complex; co-immunoprecipitation studies show that a tagged T1 domain protein can co-precipitate another un-tagged T1 domain protein. In addition, the T1 domain contains the molecular recognition sequences required for the subfamily-specific assembly of voltage-gated K ϩ channel proteins (11). Chimeras made with swapped N-terminal sequences show the assembly specificity of the N-terminal donor; the soluble T1 domain translated by itself only co-assembles with T1 domain proteins made from the same subfamily. These results have prompted our hypothesis that the T1 domain is the primary site for organized tetramerization of K ϩ channel subunit proteins along subfamily-specific lines.Other recent studies have ...

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.