The Role of Zn2+ in Shal Voltage-gated Potassium Channel Formation

Abstract

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 ...

Ca2+-selective Transient Receptor Potential V Channel Architecture and Function Require a Specific Ankyrin Repeat

“…8), a theory that is supported by the partial biochemical but not functional rescue of the interaction in the ϩ5p2 construct. In contrast to the tetramerization via T1 seen in voltage-gated potassium channels where Zn 2ϩ ions are an important cofactor (23), tetramerization of TRPV6 via ANK 3 appears not to require a divalent ion. Not much is known about a role for ankyrin repeats in multimerization of proteins; so far only for the chloroplast protein SRP43 has it been shown that its third and fourth ankyrin repeats are involved in dimerization of the protein (30).…”

“…We also positively retested the interaction of 116 -191 with itself in a novel Bacteriomatch reporter strain that grows on minimal medium (His drop-out) with too little divalent ions for detecting ion-dependent interactions, suggesting that, in contrast to the Zn 2ϩ dependence of the potassium channel Shal and Shaw T1 tetramerization domain (23), multimerization via ANK 3 is independent of divalent cations.…”

Zinc Activates TREK-2 Potassium Channel Activity

“…Single-channel recordings in the outside-out configuration might distinguish between these possibilities. In the case of Shaker-and Shaw-type K ϩ channels, Zn 2ϩ activates channel activity by increasing subunit tetramerization (Jahng et al, 2002;Strang et al, 2003). Therefore, the question of whether the Zn 2ϩ activation effect on TREK-2 is correlated with dimerization of its subunits should be tested by biochemical approaches.…”

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

“…Thus, we hypothesized that the negative charge at position 95 that is provided by phosphorylation would be needed at some step during the trafficking of the channel from the endoplasmic reticulum and later must be removed to allow for the correct expression of the KChIP-channel complex in the plasma membrane. To explore this possibility, we used a Kv4.2 tetramerization mutant (ZnB3) unable to incorporate into the plasma membrane because of its incapacity to multimerize within the endoplasmic reticulum, the first event before the channel can be carried toward the plasma membrane (40). Coexpression of DREAM with the Kv4.2-ZnB3 mutant, however, rescues the tetramerization of the channel (25).…”

“…Channels to the Plasma Membrane-Increased current expression of Kv4 channels at the plasma membrane after cotransfection with KChIP auxiliary proteins is a complex process involving enhanced channel assembly, enhanced folding of the assembled channels, and increased surface expression of the channel complex (37,38,24,39,40). To investigate whether the reduced net conductance observed after cotransfection with S95D DREAM mutant could be related to a deficit of channel expression at the plasma membrane, we analyzed the subcellular localization of DREAM-EGFP fusion proteins in the absence or in the presence of Kv4.2.…”