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

Voltage-gated potassium channels are formed by the tetramerization of their α 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 Zn2+ ion that is lacking in the Shaker T1 domain. The Zn2+ 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 Zn2+ is an important co-factor for the tetramerization of isolated Shaw and Shal T1 domains. Addition of Zn2+ increases the amount of tetramer formed, whereas chelation of Zn2+ with phenanthroline blocks tetramerization and causes assembled tetramers to disassemble. Within an intact cell, full-length Shal subunits containing Zn2+ 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-gated potassium channels are formed by the tetramerization of their α 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 Zn2+ ion that is lacking in the Shaker T1 domain. The Zn2+ 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 Zn2+ is an important co-factor for the tetramerization of isolated Shaw and Shal T1 domains. Addition of Zn2+ increases the amount of tetramer formed, whereas chelation of Zn2+ with phenanthroline blocks tetramerization and causes assembled tetramers to disassemble. Within an intact cell, full-length Shal subunits containing Zn2+ 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 (1Yi B.A. Minor Jr., D.L. Lin Y.F. Jan Y.N. Jan L.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11016-11023Crossref PubMed Scopus (70) Google Scholar, 2Levitan E.S. Takimoto K. J. Neurobiol. 1998; 37: 60-68Crossref PubMed Scopus (55) Google Scholar, 3Barry D.M. Nerbonne J.M. Annu. Rev. Physiol. 1996; 58: 363-394Crossref PubMed Scopus (202) Google Scholar). 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 α subunits assemble into tetramers, and they are classified into different subfamilies based on sequence homology and assembly specificity (3Barry D.M. Nerbonne J.M. Annu. Rev. Physiol. 1996; 58: 363-394Crossref PubMed Scopus (202) Google Scholar, 4Salkoff L. Baker K. Butler A. Covarrubias M. Pak M. Wei A. Trends Neurosci. 1992; 15: 161-166Abstract Full Text PDF PubMed Scopus (253) Google Scholar, 5Jan L.Y. Jan Y.N. Trends Neurosci. 1990; 13: 415-419Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 6Jan L.Y. Jan Y.N. Annu. Rev. Neurosci. 1997; 20: 91-123Crossref PubMed Scopus (457) Google Scholar). 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 (7Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (158) Google Scholar, 8Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Crossref PubMed Scopus (392) Google Scholar, 9Xu J. Yu W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Recently, we reported a notable structural difference between the Shaker (Kv1) and Shaw (Kv3) type α subunits based on crystallographic analyses of the T1 domains of these subunits (10Kreusch A. Pfaffinger P.J. Stevens C.F. Choe S. Nature. 1998; 392: 945-948Crossref PubMed Scopus (264) Google Scholar, 11Bixby K.A. Nanao M.H. Shen N.V. Kreusch A. Bellamy H. Pfaffinger P.J. Choe S. Nat. Struct. Biol. 1999; 6: 38-43Crossref PubMed Scopus (144) Google Scholar). Comparisons between these structures revealed that an intersubunit Zn2+ atom is coordinated between the subunits of the tetrameric T1 domain of Kv3 but not Kv1. By sequence analysis, it was noted that the Zn2+ 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 Zn2+ 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 (12Choe S. Kreutsch A. Pfaffinger P.J. Trends Biochem. Sci. 1999; 24: 345-349Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Given the central role for the T1 domain in the assembly of Kv channels, we hypothesize that the interfacial Zn2+ coordination site is likely to be important for channel subunit assembly. However, given that Shaker type subunits assemble and function without a T1 Zn2+ site, the question remains as to the significance for the Zn2+ contribution toward subunit association into tetrameric channel. In these studies we have demonstrated the functional importance of Zn2+ for Shaw T1 tetramerization and extended these results to the Shal type Kv channel Kv4.2. DNA Subcloning—Rat Kv4.2 cDNA was obtained as a gift from the L. Jan laboratory (pBS-rKv4.2). A bacterial T1 expression construct containing amino acids 41–162 of the Kv4.2 N terminus was made by PCR adaptor subcloning into the pET20 vector and was confirmed by full-length sequencing. The T1 constructs from the Aplysia and rat Kv3.1 sequences were cloned into pCITE2 vectors by PCR and confirmed by full-length sequencing. The N terminus of rKv4.2 was cloned into the vector pEGFP-N1 (Clontech) by PCR subcloning, both replacing EGFP 1The abbreviations used are: EGFP, enhanced green fluorescent protein; CHO, Chinese hamster ovary; SEC, size exclusion chromatography; FPLC, fast protein liquid chromatography; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline. within the vector to make wild type Kv4.2 and in-frame with the C terminus of EGFP to make EGFP-rKv4.2. The remainder of rKv4.2 was subcloned as an EcoN1, MfeI cassette. The resultant cloning was confirmed by full-length sequencing. We noted a discrepancy in these clones from the originally published sequence for rKv4.2, where the codon for residue 537 is actually ATC and not TCT. This changes the residue at this position from Ser to Thr. This sequence difference is present in the original DNA for rKv4.2,and in other rKv4.2 sequences; therefore, we feel the most likely explanation is a sequencing error in the original publication. Kv3.1 T1 constructs were cloned into pCITE2a vectors (Novagen). cRNA was synthesized and The was made by cloning in-frame with a on the sequence the and of and by size differences on site were made the and with that were in mutations were confirmed by DNA sequence the and were on cells with were an and and was with and cells were identified by to EGFP an for constructs not with EGFP, pEGFP-N1 cDNA was in the to for were as a with EGFP and and were by to from a of and were from cells to the channel In not functional channels, we also studies with a of and to that the cells were not We not for active channels with Zn2+ site was in the of on a for by to the of containing Kv4.2 T1 protein as was in phosphate-buffered potassium in the same as the same in therefore, for were by and T1 the protein was from cRNA with as (7Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (158) Google Scholar). studies were on a liquid as J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). studies were on a with of from the were by and on as (7Shen N.V. Pfaffinger P.J. Neuron. 1995; 14: 625-633Abstract Full Text PDF PubMed Scopus (158) Google Scholar). Kv4.2 T1 domain in vector was into cells the from the and were by on with and were in the to an A and with with at at for were by at and with and as were in the same with for and with on for to The was to to was by the of to and the was to with the of and with at for at The was at for and the was and a and were to and and the was a at at The was with with and and the protein was in of the to was and the protein was by within into for in this was and it was tetramer and was on to the from the cells were into and with as by the the wild type the of rKv4.2 the site channel the cells were by the cells with PBS, and a of were with a of the same a of and and for on The cells were by with a and for to a by to the cells were at for to A at for to the and the resultant was in at a of analysis, the were with an of analysis, the was as and was to a of The were on for at as and a a used were to the used for T1 domain for the of to of the The of was by of Zn2+ site an enhanced was to the protein was used to the and the was a and were in and of cells on were with of with A was as 2001; 14: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). were at as the were with PBS, for on in PBS, The cells were with and in for the were with of and The cells were with a a for were obtained as and on a were of the Aplysia Shaw Kv channel T1 domain revealed a surprising intersubunit Zn2+ present at the tetramerization K.A. Nanao M.H. Shen N.V. Kreusch A. Bellamy H. Pfaffinger P.J. Choe S. Nat. Struct. Biol. 1999; 6: 38-43Crossref PubMed Scopus (144) Google Scholar). an intersubunit Zn2+ was not present in the Shaker Kv channel T1 domain and was not to this of other T1 domains that the Zn2+ coordination residues found in the Shaw T1 domain are conserved in all non-Shaker type Kv channel T1 domains (12Choe S. Kreutsch A. Pfaffinger P.J. Trends Biochem. Sci. 1999; 24: 345-349Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The of the Zn2+ at the assembly in the crystal that Zn2+ play a role in subunit assembly. of Shaw T1 in to for the of Zn2+ on Shaw Shaker T1 we T1 domain in in the of Zn2+ Zn2+ chelation and we the of the to assemble to tetramer by size exclusion on as J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). In SEC, protein are a with a of that the the protein in to from the are from most the the protein the the and the the protein from the In all T1 domains are as isolated whereas full-length are into and be with to assembly Shaw T1 protein is in the protein is found in as seen with the Shaker T1 domain with of the protein in the of tetramer and the in the of The of these in the are to the of the and tetramer for the Shaker T1 domain that the is a T1 domain, and not to the Shaw subunit in a with other protein present in the in we a to that the to the assembled Shaw T1 the assembly of Shaw T1 we subunits with and without a an Shaw T1 domain is to a it not and protein is from the In the same protein is with a Shaw T1 the subunit is with the subunit that the are assembled into a By this same to in the of and Shaw T1 we the assembly for protein in these seen with Shaker T1 domain we that Shaw protein was only in the the of this is to the Shaker tetrameric we that both Shaker and Shaw T1 domains assemble into tetramers in to Shaker T1 the Shaw T1 domain protein is into tetramer and on with of Shaw T1 that the tetramer and of Shaw T1 be by FPLC, we the assembly of the protein a Zn2+ and of the Shaw T1 protein is found in the tetrameric Addition of Zn2+ to the changes the of the between the in of the Shaw protein at the tetrameric of the Zn2+ Zn2+ the of the Shaw T1 domain to with of the protein found in the monomeric This that Zn2+ present in the in is to assembly We in the of tetrameric protein with different of in that be to in Zn2+ this is with of of a Zn2+ for Shaw T1 assembly. was by of as in show a distinct Zn2+ for Shaw T1 domain protein assembly that is not seen with Shaker T1 domains is at for Shaw T1 domain and for Shaker T1 domain. is at for Shaw T1 domain and for Shaker T1 We the of the assembly. Shaw protein that was in the of Zn2+ was for of on we that the assembly by Zn2+ is by chelation of Zn2+ with phenanthroline the is In to the Aplysia Shaw we also the rat Kv3.1 Shaw T1 domain seen in results are obtained with the rat Shaw Kv3.1 T1 domain. the assembly of rat Shaw T1 and phenanthroline causes the protein to to In a Shaker T1 domain, the Zn2+ site, assembly in and to assemble in the of Zn2+ and of T1 distinguish the assembly of Shaw T1 domains in the of Zn2+ is to Zn2+ the assembly of T1 domains at a we on the Zn2+ coordination residues and for their to and their to Zn2+ The original is of a and of is the amino that to the of the subunit to make the intersubunit the for the Kv3.1 Zn2+ site and the In both the majority of the protein is found in the monomeric However, of the protein in the tetrameric The of these is not to increases in Zn2+ chelation of Zn2+ with that the protein for tetramerization in the of of Shal T1 to the Zn2+ for assembly is for channels, Kv channel that the conserved Zn2+ coordination site within the T1 domain. Shal subunits are to form potassium channels in neurons and and has found in on the of of cells M. Jan Y.N. Jan L.Y. Neuron. 1992; Full Text PDF PubMed Scopus Google Scholar, Nature. 1997; PubMed Scopus Google Scholar). the potassium to the and of the this that are differences in the excitability and that in a specific an work S. M. J. Physiol. 1999; Scopus Google has revealed the importance of channels in of the into and it is that the of this In to crystallographic studies on T1 and to the assembly of the T1 domain, we have on the of the rat Kv4.2 T1 domain made by in from the T1 domain is found to as a at a for a T1 tetramer We confirmed that this protein also contained at the for the intersubunit that was in the crystal of the Kv T1 domain. The T1 domain of Kv4.2 was found to of Zn2+ of tetrameric protein and for the and an of for a protein at the Kv3 type T1 the Kv4.2 T1 domain protein be to a by with phenanthroline in a of of of Kv4.2 In to the Zn2+ for Kv4.2 T1 domains is with of subunit tetramerization Zn2+ chelation with Given the of Kv4.2 protein in these at these results that Zn2+ be present to Kv4.2 T1 domain tetramerization at physiologically protein We have confirmed the of phenanthroline on Kv4.2 T1 association by both and J. and A. Pfaffinger Choe S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google by all of and we and only a of tetrameric and monomeric subunit studies we have show a association a for the assembly of this protein A. Pfaffinger Choe S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). We the monomeric Kv4.2 is for Zn2+ and We that the Kv4.2 Zn2+ and into a of the amino acids to an the and to of the Kv4.2 T1 domains as by on in the of the at and the subunits with Addition of to the subunits protein and in of the to monomeric subunits of The of this Zn2+ within the is not is the for this is that the site for Zn2+ in these In making the same mutations in the Shaw T1 domain, the subunits in were found in chelation of Zn2+ from the wild type Shaw T1 domain the protein to of in a of be noted that the in Shaw T1 domain to of with Zn2+ that with the protein are between subunits. are with the studies on the domains that were in as the Shaw T1 that was whereas the Shal T1 only of Kv4.2 on results of the functional importance for Zn2+ in the tetramerization of the T1 domains from non-Shaker type Kv We to the importance of Zn2+ in the functional expression of full-length Shal channels in intact for the importance of the T1 Zn2+ site, we the of Zn2+ site mutations on the functional expression of Kv4.2 We a of and mutations of the Zn2+ coordination residues within the full-length Kv4.2 to the are with a of of assembly. We for the of the subunits with site mutations to form functional channels as by the expression of the A cells were with of Kv4.2 subunit expression construct by to and to of cells were identified by by with a of EGFP expression of an N-terminal EGFP expression of Kv4.2 in cells is on the of at In cells with Kv4.2 subunits containing Zn2+ site mutations results that Kv4.2 subunits not form channels without a functional Zn2+ and of Kv4.2 to the Zn2+ site channels were in assembly we Zn2+ site mutations into Kv4.2 subunits, and we the protein expression by and with revealed an of the of with expression in the of the and J. J. Biol. 1992; PubMed Scopus Google Scholar, S. S. K. J. J. Biol. 2001; PubMed Scopus Google was confirmed by of cells with is only fluorescent in and of the and the and subunits of the was In we have that Shaker channel subunits with assembly mutations are in The for a be and in general, the Kv subunit be in cells assembled the subunits were as we from cells wild type and Kv4.2 subunits, and we to size by in the of wild type a at a of was with the protein the Zn2+ site a that at a for was of were seen with of as for the In this the protein a with a but at the position where tetramers the that as as have formed in this Given that are to at expression it likely that this is to a these in size to the of functional A channels, we on cells green expression of wild type channels with EGFP is in these as for cells of channel subunit In of fluorescent green cells Kv4.2 Zn2+ site a A of for and site Kv4.2 subunits. were the and into and of of the are type Kv4.2 protein of that from the Zn2+ site show a of with most cells of wild of Kv4.2 subunits containing Zn2+ site mutations in wild type and Zn2+ site subunits were in in and on with in the is found in a at a as for a assembled channel Zn2+ site and to as a at a as for The of these an enhanced that they are in with where the protein be by are as for the to these a tetrameric is as as a of the into to subunits. of are the to Kv4.2 we to changes in Zn2+ in cells a to the of channels to the functional properties of channels at the in a of was to cells with Kv4.2 and were phenanthroline in cells that the and obtained were by making of and cells to and their for A in cells of these cells not be to and of the cells that be the of these cells not to and to making it to these We channels from these cells for analysis, and we by phenanthroline an on Kv4.2 channel assembly. from the that channels from cells not at the tetramer position but at for and This was found to be of the phenanthroline was to the it was only at the of and of the The channel was for both Kv4.2 and as phenanthroline was to the it to a in the protein the and with the and of but not the of the channel. these a channel from all and only a of the cells be from it is to the and the We distinguish between the that the functional we have from a of cells making tetramer that is not on that phenanthroline all channels to their in the of the to the channels for We of phenanthroline on Kv4.2 channels on the from to was to cells Kv4.2 the in the to changes in properties were that the channel tetrameric and in a the also not the that was found cells that to of phenanthroline this the of phenanthroline to channels the tetrameric of these on is to phenanthroline in cells has are to the these studies we have the that of Zn2+ to the T1 domain of non-Shaker voltage-gated potassium channels is an important in functional Kv channel assembly. The importance of a highly conserved Zn2+ site present in non-Shaker Kv channels was in Shaw and Shal T1 domains by chelation of and Zn2+ site by the the tetramerization of these T1 domains was to the intersubunit In the of most of the T1 domain was whereas Zn2+ chelation the T1 domain to The Zn2+ for T1 tetramerization was not by the Shaker T1 domain that the conserved Zn2+ In assembly for the Shaker T1 domain was in the of an that be to the of the assembly by at the of of full-length channel such as tetramerization of the T1 domain, of the and of the cytoplasmic domain. most studies on the importance of T1 domain for the assembly of Kv channels, studies have that functional expression of Kv channels with the T1 domain W. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar). we also were interested in the role of Zn2+ in full-length channel expression and function in Zn2+ and other are for a of in we found it to cells for extended with without their properties is an on channel on to and the of results in channels that are in of The lack of of phenanthroline on channel function that the intact channel has a site that is to in the without the channel has it to function the without Zn2+ to this This be with studies with channels, where we found that of the T1 domain from assembled channels not the assembled channels to to but make to by Pfaffinger J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). obtained by of the Zn2+ site were of the Shal Kv4.2 channel T1 Zn2+ site functional channel The mutations to of and conserved that is is found in both of these structural and is the only for of the amino Therefore, we not from this type of of from the of this functional is for a of mutations the amino the residues from different structural Given that the only is that these residues are in the T1 Zn2+ coordination site, we feel that the of this site is the most likely for these functional We site full-length subunit assembly the We Kv4.2 subunits in cells and for assembly by type subunits are found in the with tetramerization of these subunits. Zn2+ site subunits, on the other are found in the monomeric difference between these is that the protein by the are for Zn2+ site wild also were by of cells in phenanthroline they were and Kv subunits, and assembly of tetrameric channel was This is to has seen with Shaker channel assembly where is a lack of for in and other with as Pfaffinger J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, D.L. Lin Y.F. A. Jan Y.N. Jan L.Y. J.M. 6: Scholar). the of protein is for Zn2+ site the amount of subunit protein found in the monomeric was for the we found with the wild results that Zn2+ site a of size as a of between subunits to the of the contribution of toward that is for In the where we of as by the of the we a of the subunits into where be to be found as as a amount of channel protein that to We hypothesize that such subunits the of subunits their T1 domains. the found in the tetrameric assembled channels, we on cells EGFP we that wild type on the of the This that association is not to functional A we the important question of non-Shaker channels an intersubunit site to their assembly. is that the Zn2+ site be an important for these In the of functional channel tetramer at the be a function of the However, we not changes in channel function by of and it is not that cells show changes in Zn2+ in the Therefore, Zn2+ is an of channel function this site studies that tetramerization of subunits is important to subunit of Kv subunits that fail to assemble show protein expression Therefore, Zn2+ be a used to and of synthesized channel subunits. are studies that that important work from the of and Science. 1996; PubMed Scopus Google Scholar, J.M. J. PubMed Scopus Google Neurobiol. 2001; 24: PubMed Google and 2001; 14: PubMed Scopus Google and PubMed Scopus Google have that are and to cells of In the where is in the of the of the likely is in a with other and this type of a biological for between the primary the are by and other biological of J. Physiol. Scholar, Biol. PubMed Scopus Google Scholar, M. J. Physiol. PubMed Scopus Google Scholar). is such changes in Zn2+ changes in voltage-gated potassium channel functional it is that Zn2+ be by at of the coordination and this be by the Zn2+ site be used to functional channel an of such a other studies with the Shal T1 domain have that these subunits show a for to of the residue that is of the Zn2+ coordination site A. Pfaffinger Choe S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies are at such to the Zn2+ coordination residues are an important for Kv channel