k channels are modular proteins , composed of a central pore module ( pm ) surrounded by sensor domains that perceive external stimuli and convert them into changes of pore activity ( gating ) .
this feature and simple assessment of their performance by electrophysiological measurements make ion channels amenable for testing the hypothesis that the pore of voltage - gated k ( kv ) channels acquired the voltage - sensing domain ( vsd ) throughout evolution .
first , it suggests that single domains , once detached from the original channel , can function independently ; for instance , the pm of the bacterial kvlm channel forms a functional k channel when separated from its vsd domain ( santos et al . , 2008 ) .
second , regulatory modules , which control k channels , are also found in proteins with completely different enzymatic functions .
a vsd displaying the hallmarks of those of kv channels is connected to a soluble phosphatase in the sea squirt ciona intestinalis phosphatase ( ci - vsp ) protein ( murata et al . , 2005 ) .
third , modularity also implies that domains of different classes of channels can be swapped .
so far , attempts to exchange entire membrane modules between k channels have been unsuccessful , leaving this third implication unfulfilled ( caprini et al . ,
 motif of the vsd and the p loop with most of the transmembrane domains of the pore , could be exchanged between k and na channels ( lu et al .
it was concluded that membrane modules have coevolved so intimately in kv proteins that it is impossible to exchange them entirely without losing functionality of the chimeric channels .
here , we show that it is possible to obtain a voltage - gated channel in one step by fusing two full - length modules : the vsd of ci - vsp and the pm of the viral k channel kcv ( plugge et al . , 2000 ) .
these two membrane modules are evolutionarily unrelated , and there is not any  a priori  indication that they should be able to make a voltage - sensitive channel .
the ciona vsd regulates a soluble enzyme , yet it remains speculative if this sensor would gate a channel pore .
the second component , the viral channel kcv , is clearly not designed to be controlled by external stimuli , such as voltage .
apart from a 12amino acid - long n terminus , the channel comprises only the bare pm and has neither additional membrane nor cytosolic domains ( plugge et al . , 2000 ) .
all constructs were inserted into bamhi and xhoi restriction sites of psgem vector ( a modified version of pgem - he ) . in vitro transcription
was performed on linearized plasmids using t7 rna polymerase ( promega ) , and crnas were injected ( 50 ng per oocyte ) into xenopus laevis oocytes , as reported previously ( plugge et al . , 2000 ) .
mutations were inserted by site - directed mutagenesis ( quikchange site - directed mutagenesis kit ; agilent technologies ) and confirmed by sequencing .
chimeric constructs were generated by overlapping pcr from ci - vsp ( provided by d. minor , university of california , san francisco , san francisco , ca ) and pbcv-1 kcv .
kvsynth1 includes amino acids 1239 of ci - vsp and amino acids 194 of kcv .
two - electrode voltage - clamp ( tevc ) experiments were performed as indicated previously ( plugge et al . , 2000 ) using an amplifier ( geneclamp 500 ; axon instruments ) and filtered at 5 khz .
data acquisition and analysis were done using the pclamp8 software package ( axon instruments ) .
electrodes were filled with 3 m kcl and had a resistance of 0.20.8 m in 50 mm kcl .
the oocytes were perfused at room temperature ( 2527c ) at a rate of 2 ml / min with a bath solution containing 50 mm kcl ( or nacl ) , 1.8 mm cacl2 , 1 mm mgcl2 , and 5 mm hepes , adjusted to ph 7.4 with koh ( or naoh ) .
mannitol was used to adjust the osmolarity of the solution to 215 mosmol / l .
bacl2 diluted from 1-m stocks was added to the solution as indicated . the standard clamp protocol consisted of steps from the holding voltage of 20 mv to voltages in the range of 100 to + 100 mv ; tail currents
instantaneous and steady - state currents were sampled after 10 ms and at the end of the voltage step , respectively .
for single - channel recordings , the vitelline membrane of the oocytes was removed mechanically before the experiment .
bath and pipette solution for measurements with kcvpbcv1 contained 100 mm kcl , 1.8 mm cacl2 , 1 mm mgcl2 , and 10 mh hepes , adjusted to ph 7.4 with koh .
the slightly different k concentrations ( 150 mm for kvsynth and 100 mm for wt ) do not impede comparison . according to pagliuca et al .
( 2007 ) , the difference in conductance for the wt would be 20% at most , which is well within the error bars .
currents were recorded with an amplifier ( dagan3900 ; dagan corporation ) and a digitizer unit ( lih8 + 8 ; heka ) .
all constructs were inserted into bamhi and xhoi restriction sites of psgem vector ( a modified version of pgem - he ) . in vitro transcription
was performed on linearized plasmids using t7 rna polymerase ( promega ) , and crnas were injected ( 50 ng per oocyte ) into xenopus laevis oocytes , as reported previously ( plugge et al . , 2000 ) .
mutations were inserted by site - directed mutagenesis ( quikchange site - directed mutagenesis kit ; agilent technologies ) and confirmed by sequencing .
chimeric constructs were generated by overlapping pcr from ci - vsp ( provided by d. minor , university of california , san francisco , san francisco , ca ) and pbcv-1 kcv .
kvsynth1 includes amino acids 1239 of ci - vsp and amino acids 194 of kcv .
two - electrode voltage - clamp ( tevc ) experiments were performed as indicated previously ( plugge et al . , 2000 ) using an amplifier ( geneclamp 500 ; axon instruments ) and filtered at 5 khz .
data acquisition and analysis were done using the pclamp8 software package ( axon instruments ) .
electrodes were filled with 3 m kcl and had a resistance of 0.20.8 m in 50 mm kcl .
the oocytes were perfused at room temperature ( 2527c ) at a rate of 2 ml / min with a bath solution containing 50 mm kcl ( or nacl ) , 1.8 mm cacl2 , 1 mm mgcl2 , and 5 mm hepes , adjusted to ph 7.4 with koh ( or naoh ) .
mannitol was used to adjust the osmolarity of the solution to 215 mosmol / l .
bacl2 diluted from 1-m stocks was added to the solution as indicated . the standard clamp protocol consisted of steps from the holding voltage of 20 mv to voltages in the range of 100 to + 100 mv ;
instantaneous and steady - state currents were sampled after 10 ms and at the end of the voltage step , respectively .
for single - channel recordings , the vitelline membrane of the oocytes was removed mechanically before the experiment .
bath and pipette solution for measurements with kcvpbcv1 contained 100 mm kcl , 1.8 mm cacl2 , 1 mm mgcl2 , and 10 mh hepes , adjusted to ph 7.4 with koh .
the slightly different k concentrations ( 150 mm for kvsynth and 100 mm for wt ) do not impede comparison . according to pagliuca et al .
( 2007 ) , the difference in conductance for the wt would be 20% at most , which is well within the error bars .
currents were recorded with an amplifier ( dagan3900 ; dagan corporation ) and a digitizer unit ( lih8 + 8 ; heka ) .
to test whether the voltage - dependent movement of the ciona vsd is able to gate a channel , we created the fusion protein kvsynth1 by adding amino acids 1239 of ci - vsp to the full - length kcv sequence ( amino acids 194 ) . the fusion protein and its individual components have been expressed in xenopus oocytes and tested by tevc .
1 a ) generate small currents , not different from typical water - injected oocytes .
oocytes expressing kcv alone ( fig . 1 b ) show the typical k currents , which have been characterized previously ( moroni et al .
, 2002 ; gazzarrini et al . , 2004 , 2006 ; kang et al . , 2004 ; abenavoli et al . ,
the i - v relationship of kcv is linear at moderate voltages . at extreme voltages ,
the ohmic behavior is lost because of a fast gating mechanism that occurs at the selectivity filter and results in a negative slope conductance ( abenavoli et al . , 2009 ) .
kvsynth1 generates currents that are very different from those of its single components and shows the typical properties of a delayed - rectifier k channel , slowly activating at positive potentials ( hille , 1992 ) ( fig .
depolarizing voltages generate large time - dependent currents on top of a small instantaneous component .
the corresponding steady - state i - v relationship shows a strong outward rectification . to quantify the degree of rectification
, we use the ratio of steady - state currents at + 60/100 mv ( i+60mv / i100mv ) . for kvsynth1 ,
it is 13.3  2 ( average of six oocytes ) ; this ratio is 1  0.1 in kcv .
( a ) vsd is the voltage - sensing domain ( amino acids 1239 ) of ci - vsp ; s1s4 indicate the four transmembrane domains of the vsd .
( b ) kcv is the full - length ( amino acids 194 ) viral k channel ; tm1 , tm2 , and p indicate , respectively , the two transmembrane domains and the pore loop of kcv .
voltage protocol : vh , 20 mv ; test voltages , + 100 to 100 mv ( 200 mv for kcv ) ; tail , 80 mv .
test pulse length : a and c , 8.5 s ; b , 0.6 s. ( bottom ) corresponding steady - state i - v relationships .
replacement of external k with na shifts the reversal voltage of the fully activated kvsynth1 current by 80 mv  6 ( sd , n = 3 oocytes ) ( fig .
2 a ) and reduces the outward current , another typical feature of kcv ( plugge et al . , 2000 ) .
the estimated relative permeability pna / pk of 0.05  0.01 closely resembles the value of 0.03  0.01 reported previously for kcv ( chatelain et al . , 2009 ) .
another feature of kcv that is preserved in kvsynth1 is the aforementioned negative slope conductance of the open channel , in this case already visible at approximately 100 mv ( fig .
2 b showing clear flickering behavior with a consequent reduction of the unitary channel conductance at voltages negative of approximately 100 mv .
current reversal potential recorded from the same oocyte was 20 mv in 50 mm [ k]out (  ) and 98 mv in 50 mm [ na]out (  ) .
voltage protocol : prepulse voltage step at + 60 mv , followed by testing voltages from + 60 to 180 mv .
( left ) representative opening burst at different membrane voltages , with arrows denoting the baseline .
the low - pass filter was set to 20 khz , the sampling rate was 100 khz , and traces were digitally filtered at 2 khz for clarity .
( right ) comparison of the i - v curves of kvsynth1 in 150 mm k (  ) and kcv in 100 mm k (  ) .
( c ) nernst plot : current reversal potential ( erev ) of macroscopic kvsynth1 current plotted as a function of external k concentration ( lg[k]out ) .
black line is the linear regression to erev mean values ( n = 3 ) .
steady - state currents recorded in (  ) control solution ( 50 mm k ) and (  ) plus 1 mm bacl2 .
currents recorded from a holding potential ( vh ) of 20 mv to the indicated test voltages .
test pulse length was 8.5 s. the addition of barium blocks the inward current and moderately affects the outward current , as reported previously for kcv channel ( plugge et al . , 2000 ) .
( e ) western blot analysis performed on total protein extracts with a custom - made antibody ( 8d6 ) recognizing the tetramer , but not the monomer , of kcv .
the expected molecular mass of kcv and kvsynth1 tetramers are 42.5 and 149 kd , respectively .
the single - channel i - v relationship ( i - v curve ) of kvsynth1 reported in fig .
2 b together with that of kcv for comparison shows that at membrane voltages more negative than 60 mv , the current of kvsynth1 decreases , resulting in a negative slope conductance .
the kcv channel shows this behavior only at more negative voltages , beyond 100 mv ( abenavoli et al . , 2009 ) .
this is consistent with the observation that the instantaneous currents of kvsynth1 in tevc experiments ( fig .
the conductance of the synthetic channel ( 112  25 ps between 50 and + 50 mv ) is only slightly smaller than that of kcv ( 133  8 ps , also reported previously by abenavoli et al .
this confirms that the general filter structure and function are not dramatically altered by the addition of the voltage sensor .
2 summarizes other kcv properties preserved in kvsynth1 , including k selectivity shown by the slope of 58.5 mv in the nernstian plot of fig .
2 c , voltage - dependent barium block of the instantaneous inward current shown in fig .
2 e shows a western blot decorated with an antibody raised against the kcv tetramer that does not recognize the monomer .
the same antibody recognized kvsynth1 wt at about the predicted molecular mass of the tetramer ( 149 kd ) .
interestingly , mutation f305a in the k channel signature sequence ( gfg ) of kvsynth1 prevented antibody recognition , indicating that either the protein is not synthesized or , more likely , does not form a stable tetramer .
, kvsynth1 displays the pore properties of kcv , such as selectivity , fast gating , and overall pore architecture ; these are not modified by the addition of the vsd .
the new property introduced by the vsd is a strong voltage dependency of the currents .
the voltage sensor closes the channel at negative potentials and opens it with a slow kinetics at voltages more positive than 0 mv ( t1/2 = 450  74 ms at + 60 mv ; n = 4 oocytes ) ( fig .
3 a was determined from the tail currents after subtraction of the instantaneous component ( inset of fig .
even though full saturation of the open probability could not be reached for the presence of endogenous conductances in oocytes , the activation curve could be fitted with a boltzmann equation .
kvsynth1 has an apparent half - activation voltage ( v1/2 ) of + 56 mv , and channel opening is caused by the movement of the equivalent of about one electronic charge across the membrane ( z = 0.92 ) .
these parameters strongly resemble those reported for the sensing currents of ci - vsp , either with or without the phosphatase ( murata et al . , 2005 ; villalba - galea et al . ,
2008 ) . to confirm that the vsd is responsible for the voltage - dependent properties of kvsynth1 , we replaced two of the four arginines in the s4 segment , r229 and r232 , with glutamines . in ci - vsp
, this double mutation eliminates the  sensing  currents and generates a voltage - insensitive phosphatase ( murata et al . , 2005 ; murata and okamura , 2007 ) .
in kvsynth1 , the double mutation r229q / r232q results in a very small and quasi - ohmic current ( 3b ) ; the mutated vsd has apparently lost its tight control on channel opening and voltage dependency . another interesting mutation that shifts the q - v curve of ci - vsp to the left by 46 mv is the single neutralization of the gating charge r217 to q ( villalba - galea et al . , 2008 ) .
the same mutation in kvsynth1 resulted in a channel that is already more open at the holding voltage because the activation curve is shifted by 38 mv to the left ( fig .
3 , c and d ) . judging from the behavior of these mutants , we conclude that the properties of the vsd are transmitted to the gating of the pore . to examine further details of the causal interplay between vsd and channel gating , we tested whether kvsynth1 also reveals the mode shift , which has been described for the sensor domain ( villalba - galea et al . , 2008 ) . during a long - lasting depolarization
conformation in which the voltage dependency is markedly shifted toward negative potentials . to test this , we measured the tail currents of kvsynth1 r217q after holding the same oocyte at two different preconditioning voltages , 100 and + 40 mv for 5 s. tail currents were collected at 80 mv after prepulse at different voltages that were kept short ( 1.2 s ) to limit the effect on the relaxation .
3 e shows that the resulting itail / v curve is shifted to more negative potentials when the oocyte is preconditioned at + 40 mv .
the observed negative shift of 30 mv ( n = 3 oocytes ) matches quite well the direction and the amplitude of the shift reported for the vsd ( villalba - galea et al . , 2008 ) .
these data further confirm that the properties of the sensor are transmitted to the gating of the pore .
( a ) activation curve of kvsynth1 constructed from tail currents ( inset ) after subtracting the instantaneous current offset .
1 c. datasets ( n = 6 ) were jointly fitted to a two - state boltzmann equation ( dotted line ) of the form y = ( 1 + e 

 1/2 ) , where z is the effective charge ; v1/2 is the half - activation voltage ; and f , r , and t have their usual thermodynamic meaning .
( b ) steady - state i - v relationships of the single mutant f305a (  ) , the double mutant r229q / r232q (  ) , and the wt kvsynth1 (  ) .
currents were recorded from a holding potential ( vh ) of 20 mv to the indicated test voltages ( pulse length , 600 ms ) .
( c ) exemplary currents recorded from wt kvsynth1 ( control ) and its r217q mutant . to compare currents from different constructs ,
the traces have been normalized to the current value recorded at + 60 mv and expressed in arbitrary units ( a.u . ) .
voltage protocol as in fig . 1 c. ( d ) activation curve of the r217q mutant (  ) constructed as in a from four datasets : v1/2 = 18 mv and z = 1.1 .
the curve of the wt ( dotted line ) is replotted from a for comparison .
the same r217q - expressing oocyte was subjected to the following protocol : preconditioning ( 5 s ) at either 100 or + 40 mv and test pulses from 120 to + 30 mv .
tail currents ( at 80 mv ) are plotted for preconditioning at 100 mv (  ) or at + 40 mv (  ) .
the test pulses were kept short ( 1.2 s ) to maintain the effect of the preconditioning voltages ; hence , data in e do not reflect full activation of the channel .
considerable evidence supports the role of the s4s5 linker in kv channels as the transducing element between sensor movement and pore gating ( long et al . , 2005 ) .
in kvsynth1 , the vsd is directly connected to kcv after removal of the 16amino acid linker that in ci - vsp couples the vsd to the phosphatase ( fig .
the 12amino acid - long n terminus of kcv acts here as the s4s5 linker of kv channels and transmits the conformational changes of the sensor to the pore . to study if and how the linker influences gating , we varied its length and composition by combining the n terminus of kcv and the linker of the vsd , as shown in fig .
4 b. all constructs produced functional channels , apart from that with the 28amino acid linker that barely expressed and was not further analyzed .
4 c shows exemplary currents from constructs with 4 , 6 , and 20amino acid linkers and their corresponding i - v relationships .
the first and most cogent observation is that there is a minimum linker length required for efficient control of the voltage sensor on the pore , and that the transition is quite sharp .
the construct with 4 amino acids is quite similar to the original channel kcv , whereas that with 6 amino acids shows the typical features of kvsynth1 , rectification and slow kinetics .
extending the linker up to 12 amino acids does nt change significantly the properties of kvsynth1 ( not depicted ) , whereas trespassing this length results in a gradual disappearance of the properties , as shown by the construct with a 20amino acid linker , with a predominant instantaneous component and an increase in inward current ( see also the corresponding i - v curves in fig . 4 c ) .
4 d , where we have plotted the degree of rectification ( i+60mv / i100mv ) as a function of linker length , for all measurable constructs . interestingly ,
rectification was not appreciably influenced by the amino acid composition because two constructs with a linker of the same length , 20 amino acids , but of different sequence ( see fig .
the length of the linker connecting the vsd to the pore affects the degree of rectification in kvsynth1 constructs .
( a ) expanded view of the sequences of the two sequences that where variably combined to form the kvsynth1 linker .
( b ) list of linkers that have been tested in kvsynth1 , their amino acid ( aa ) length , and amino acid sequences ( color coded as in a ) .
( c ) exemplary current traces recorded from kvsynth1 constructs with the 4 , 6 , and 20amino acid linkers and their corresponding i - v relationships ( color coded ) . to compare currents from different constructs ,
the traces have been normalized to the current value recorded at + 60 mv and expressed in arbitrary units ( a.u . ) .
voltage protocol as in fig . 1 c. ( d ) the degree of current rectification ( i+60mv / i100mv ) is plotted as a function of linker length for all functional constructs .
experimental data ( with the exclusion of the value corresponding to the construct with a 4amino acid linker that loses the rectification ) have been interpolated with a logistic function ( black line ) in which the lower asymptote was set to 1 , corresponding to the value measured in kcv that lacks rectification ( dotted line ) .
the present data show that the coupling of ci - vsd to kcv is necessary and sufficient to transform a voltage - independent k channel into a kv - type outward rectifier .
first , it shows unambiguously that the shallow voltage dependency of the s4 of ciona vsd is sufficient to gate a channel pore .
the activation curves of kvsynth1 closely reflect the charge movement obtained with the isolated vsd in its wt and mutant form .
hence , the voltage - dependent features of the sensor are fully transmitted to the gating of the channel pore .
kvsynth1 exhibits the features of an outward rectifier , which conducts best at depolarizing voltages .
this gain of function over the ohmic kcv conductance can be explained with a model according to which the coupling with vs closes the pore of kcv ; the movement of the vs then causes a slow and voltage - dependent opening at positive voltages . from the shallow voltage dependency of kvsynth1 , it can be speculated that the movement of a single vsd is probably sufficient for opening of the channel . because the typical pore gating features of kcv are still maintained in kvsynth1 , the movement of the vsd is most likely operating a cytosolic gate of the channel ; the latter is operated by salt bridge interactions at the cytosolic entrance of kcv ( tayefeh et al . , 2009 ) .
previous data implied that the pore and the voltage - sensor domains of kv channels presumably required a tight coevolution at their interacting surfaces to achieve voltage - dependent gating ( long et al .
in contrast , the present data show that a naive electromechanical coupling between an unrelated vsd and a pore is already sufficient for a voltage - dependent gating of the channel pore .
the distinct coevolution between the sensor and the pore in modern kv channels is therefore more likely a means to a fine - tuning of channel gating and not a basic structural requirement .
another remarkable finding is that the effective voltage control of the vsd over the pore is a function of the linker length .
it is evident that the linker must be longer than 4 amino acids and performs best with a length between 6 and 12 amino acids .
this is in the range of linker length in kv channels ( long et al . , 2005 ) .
efficient coupling can be achieved in kvsynth1 with completely unrelated linker sequences , such as the n terminus of kcv and the c terminus of ciona vsd ( see fig .
3 ) . even though we can not exclude that both linkers generate a similar fold
, the data suggest that the length of the linker is more important for coupling of the two domains than its particular sequence . in this respect ,
the data are in good agreement with the idea that a rigid connection , which is presumably better provided by a short linker , favors the mechanical coupling between voltage sensor and pore ( long et al . , 2005 ; lee et al . ,
third and lastly , the data show that a primitive channel with poor control on gating can acquire sophisticated voltage regulation via a naive fusion with a regulatory element from another source .
this finding makes it likely that the appearance of kv channels indeed occurred as a result of a singular evolutionary step , as suggested previously ( chanda and bezanilla , 2008 ) .
 the modular architecture of voltage - gated k+ ( kv ) channels suggests that they resulted from the fusion of a voltage - sensing domain ( vsd ) to a pore module . here 
 , we show that the vsd of ciona intestinalis phosphatase ( ci - vsp ) fused to the viral channel kcv creates kvsynth1 , a functional voltage - gated , outwardly rectifying k+ channel . 
 kvsynth1 displays the summed features of its individual components : pore properties of kcv ( selectivity and filter gating ) and voltage dependence of ci - vsp ( v1/2 = + 56 mv ; z of 1 ) , including the depolarization - induced mode shift . 
 the degree of outward rectification of the channel is critically dependent on the length of the linker more than on its amino acid composition . 
 this highlights a mechanistic role of the linker in transmitting the movement of the sensor to the pore and shows that electromechanical coupling can occur without coevolution of the two domains . 
INTRODUCTION
MATERIALS AND METHODS
Chimeric constructs
Electrophysiological measurements
RESULTS
DISCUSSION