voltage - gated k channels ( vkcs ) constitute a structurally related family of intrinsic membrane proteins that respond to changes in transmembrane potential by opening and closing an ion - selective permeation pathway for k ions ( 1 ) .
the sensitivity to the membrane potential and the kinetics of the response to changes in potential vary substantially between the different vkc proteins , which means that cells expressing different vkcs repolarize at different rates and at different parts of an action potential . as such , vkcs are the primary agents in shaping action potentials in excitable cells of the eumetazoa .
vkcs are tetramers , each subunit of which comprises a core structural domain consisting of six transmembrane helices and a re - entrant loop that forms the ion - selective channel ( figure 1 ) and highly variable c- and n - terminal domains that play a role in appropriate assembly of newly synthesized tetrameric channels ( 2 ) , transport to the appropriate cell compartment ( usually the plasma membrane ) ( 3 ) and in modulating the core functionality of the channel ( 4 ) . the core structural domain is sufficiently conserved that robust multiple alignments between vkcs of single subfamilies can be obtained based on sequence , and alignments between families can be obtained based on a combination of sequence and structural similarity .
the n- and c - terminal domains are usually specific to a given subfamily , and thus give robust multiple alignments within subfamilies but not between subfamilies . 
 figure 1.schematic drawing of the conserved functional core of vkcs .
the dotted lines represent the lipid bilayer of the membrane , with the extracellular compartment at the top of the drawing , intracellular at the bottom .
the functional pore consists of the s5 and s6 transmembrane helices supporting the re - entrant loop that joins them and that forms the ion selective pore in the assembled tetramer .
the array of positive charges every third residue in the s4 helix is the primary sensor of the voltage , with the other three helices forming a supporting scaffold around the s4 helix .
the cytoplasmic n- and c - terminal domains ( not shown in this schematic ) are much more variable than the core structure .
the dotted lines represent the lipid bilayer of the membrane , with the extracellular compartment at the top of the drawing , intracellular at the bottom .
the functional pore consists of the s5 and s6 transmembrane helices supporting the re - entrant loop that joins them and that forms the ion selective pore in the assembled tetramer .
the array of positive charges every third residue in the s4 helix is the primary sensor of the voltage , with the other three helices forming a supporting scaffold around the s4 helix .
the cytoplasmic n- and c - terminal domains ( not shown in this schematic ) are much more variable than the core structure .
the existence of a diverse cohort of vkc paralogs within individual species of metazoa and of divergent orthologs between species indicates that the functional evolution of this family of proteins has been a significant factor in the evolution of electrophysiological excitability in the animal kingdom .
the original vkcdb ( 5 ) has been updated and expanded to provide support for evolutionary and comparative studies of the relationships between vkc sequence , electrophysiological characteristics of the individual channel proteins and ultimately the complex electrophysiological behavior of neurons and muscles in animals .
content update of vkcdb consists of two phases that are automated to run at quarterly intervals .
the first is update of the information for existing records . for each record , the last update date of the record in vkcdb
is compared to the last update date of the corresponding genbank record . if the genbank record has been replaced , the replacement record is parsed to update the vkcdb entry
. if the genbank record has only had new information added to it ( for example , additional bibliographic references or new sequence annotations ) , the full record is parsed and the new information is written into the vkcdb record along with a new date - of - last - update . if the genbank record has been removed , the vkcdb record is flagged as no longer being a recognized channel sequence , but it is left in the database , and the replacement entry ( if any ) is added to the database .
the second phase is addition of new records for vkcs that were added to genbank after the last vkcdb update .
protein sequences for the bacterial vkcs , archaeal vkcs and each of the eukaryotic subfamilies are aligned ( 6 ) using muscle v3.8 ( http://www.drive5.com/muscle/ ) used to create separate profiles ( 7 ) using hmmbuild from hmmer v3 ( http://hmmer.janelia.org/ ) .
these profiles are then used to search the most recent genbank non - redundant protein database ( downloaded in fasta format and searched locally ) using hmmsearch ( from hmmer ) to identify potential new entries .
each unique new record is tagged with name of the subfamily whose profile gives the highest full - sequence hmmer score and the results are then sorted based on this provisional subfamily designation followed by sorting on the basis of the hmmer score .
this allows for fairly easy manual evaluation of cutoffs that distinguish between real vkc sequences and other , more distantly related sequences .
the gi numbers of the manually selected set of new entries from the hmmer search are then used to retrieve the relevant genbank records and create new entries in vkcdb .
for the bacterial domain , the archaeal domain and each of the eukaryotic subfamilies , all previous entries of substantial length and all new entries are then subjected to a large multiple alignment to identify which of the new entries span a substantial portion of the core vkc structure ( coded as 
small fragmentary records [ those that are gapped in substantial areas of compact alignment of the core region , s1s6 ( figure 1 ) ] are flagged as not of sufficient length , thus providing the user with the option of selecting either nearly full length , fragmentary , or both in the standard web access .
the remaining protein sequences are then realigned , the multiple alignment is pruned to remove any sites with > 5% gaps , and the resulting data matrix is used for a bayesian search ( 8) to generate a phylogenetic tree for each subfamily . this tree is used to check subfamily assignment made on the basis of the hmmer searches .
table 1 gives the number of channel entries that have a substantial ( fullish_length  =  y ) amount of protein sequence for channels in each distinct phylogenetic subfamily . 

table 1.table of different vkc families in current release of vkcdbchannel familynumber of entries ( fullish length)kv1 	 270kv2 	 71kv3 	 180kv4 	 132kv5 	 21kv6 	 78kv7 	 191kv8 	 20kv8.2 	 18kv9 	 40kv10 	 99kv11 	
147kv12 	 107kv13 	 19bk 	 151archaea 	 18bacteria 	 477kcnh ( unicellular ) 	 24total 	 2063these family designations are based on hmmer searches using profiles generated from well characterized , channel sequences .
the kcnh family includes the kv10 , kv11 and kv12 families from metazoa , but these families arose after the origin of metazoa , so the unicellular kcnh channels do not fall into any of these families .
table of different vkc families in current release of vkcdb these family designations are based on hmmer searches using profiles generated from well characterized , channel sequences .
the kcnh family includes the kv10 , kv11 and kv12 families from metazoa , but these families arose after the origin of metazoa , so the unicellular kcnh channels do not fall into any of these families .
utilizing a customized lamp ( linux , apache , mysql and php ) setup to ensure stability , maintainability and future scaling needs , we have revised the web interface to vkcdb to provide a finer level of selection , detail and usability than the previous version . corresponding sets of protein and nucleotide sequence data are now available for download from any viewed record on any page .
there are three options for searching vkcdb , accessible from links at the top of every web page .
search database allows for searching on the contents of various fields ( figure 2 ) .
this is designed provide users the ability to find entries for which they already have some identifying information , including the vkc i d number , the genbank i d number , accession number or authors of the paper reporting the sequence . 

figure 2.screen capture of results of a search for a particular channel based on its vkc i d number . to select any entry that has been returned by a search
it is necessary to check the box at the left side of the entry and choose either protein sequence or nucleotide sequence before clicking on the download button .
screen capture of results of a search for a particular channel based on its vkc i d number . to select any entry that has been returned by a search it is necessary to check the box at the left side of the entry and choose either protein sequence or nucleotide sequence before clicking on the download button .
browse database is designed to allow comprehensive retrieval of sets of vkc sequences that are of general use in comparative and phylogenetic analyses .
the  browse database section is divided into three subsections :  by family ,  by organism and  by electrophysiology. the  by organism page allows all channels from a single organism to be retrieved with a single query ( figure 3 ) .
note that this can return multiple alternatively spliced transcripts from a single gene , or occasionally different variant sequences .
the only entries that are flagged as duplicates in vkcdb are those with identical amino acid sequences from the same organism . 

figure 3.screen capture of top of results page for a search for all channels from callithrix jacchus , the common marmoset .
this search requires ( a ) prefiltering the search for eukaryotes , ( b ) choosing long , fragmentary or both records for retrieval and then ( c ) selection of species from a pull - down menu .
screen capture of top of results page for a search for all channels from callithrix jacchus , the common marmoset .
this search requires ( a ) prefiltering the search for eukaryotes , ( b ) choosing long , fragmentary or both records for retrieval and then ( c ) selection of species from a pull - down menu .
alternatively , all members of a particular family of channels can be selected and downloaded ( figure 4 ) from the  by subfamily page , or all sequences for which quantitative functional parameters ( half - activation voltage , half - inactivation voltage and activation threshold ) are identified in the database . 
 figure 4.(a )
screen capture of top of search page for bk family with download options set to retrieve protein sequence with the genbank gi number , genbank accession number , definition line from genbank record and the channel subfamily name as part of the fasta header and with all the entries checked for retrieval .
( b ) top of the text file that was retrieved with the above settings .
note that the sequence is on one unbroken line , so this figure has truncated the right - hand side of the image .
( a ) screen capture of top of search page for bk family with download options set to retrieve protein sequence with the genbank gi number , genbank accession number , definition line from genbank record and the channel subfamily name as part of the fasta header and with all the entries checked for retrieval .
( b ) top of the text file that was retrieved with the above settings .
note that the sequence is on one unbroken line , so this figure has truncated the right - hand side of the image . in all cases
the amino acid sequence or the nucleic acid sequence can be retrieved in the same order with the same identifiers , to facilitate a variety of evolutionary analyses by making it straightforward to align nucleic acid sequences against a pre - existing protein sequence alignment .
all downloaded sequences are in plain text fasta format , so combining multiple sets into a single file can be accomplished by simple concatenation of the individual output files .
the identifier line for the fasta - formatted data always begins with the vkc i d number , which is the primary key for protein sequences , and can be configured to also include the gi number , the accession number , the definition line , and the channel subfamily ( kv1 through kv13 and bk ) ( figure 4 ) .
the third search method is vkcdb - blast , which is simply the blast+ search tools blastp and blastn ( 9 ) implemented over all vkcdb entries ( either protein or nucleic acid ) that have been confirmed as potassium channels .
this allows for rapid searches using query sequences that are potential potassium channels and a rapid first - order determination of possible affinities to other vkcs .
the full vkcdb database , including entries that have been annotated as invalid or obsolete , is available in xml or sql format , downloaded from the 
the main enhancements planned over the next year are : 
 addition of pre - aligned sequences for each of s1 , s2 , s3 , s4 and the s5-pore - s6 module , to allow easy access to a standardized data set for phylogenetic and comparative analysis , andaddition of more functional electrophysiological parameters to provide a standardized set of data for structure / function analyses andrefinement of subfamily designations .
addition of pre - aligned sequences for each of s1 , s2 , s3 , s4 and the s5-pore - s6 module , to allow easy access to a standardized data set for phylogenetic and comparative analysis , and addition of more functional electrophysiological parameters to provide a standardized set of data for structure / function analyses and refinement of subfamily designations .
natural sciences and engineering research council of canada ( grant number 36402 - 2010 to w.j.g . ) ; canadian institutes for health research ( grant number mop-184491 to w.j.g . ) . funding for open access charge : canadian institutes for health research operating grant , natural sciences and engineering research council of canada operating grant .
 the voltage - gated k+ channel database ( vkcdb ) ( http://vkcdb.biology.ualberta.ca ) makes a comprehensive set of sequence data readily available for phylogenetic and comparative analysis . 
 the current update contains 2063 entries for full - length or nearly full - length unique channel sequences from bacteria ( 477 ) , archaea ( 18 ) and eukaryotes ( 1568 ) , an increase from 346 solely eukaryotic entries in the original release . 
 in addition to protein sequences for channels , corresponding nucleotide sequences of the open reading frames corresponding to the amino acid sequences are now available and can be extracted in parallel with sets of protein sequences . 
 channels are categorized into subfamilies by phylogenetic analysis and by using hidden markov model analyses . 
 although the raw database contains a number of fragmentary , duplicated , obsolete and non - channel sequences that were collected in early steps of data collection , the web interface will only return entries that have been validated as likely k+ channels . 
 the retrieval function of the web interface allows retrieval of entries that contain a substantial fraction of the core structural elements of vkcs , fragmentary entries , or both . 
 the full database can be downloaded as either a mysql dump or as an xml dump from the web site . 
 we have now implemented automated updates at quarterly intervals . 