its activity accounts for the large chloride conductance of the sarcolemma at rest that is necessary to stabilize the membrane potential .
mutations in the clcn1 gene lead to dominant and recessive myotonia ( koch et al . , 1992 ; george et al . , 1993 ; steinmeyer et al
the channel is a member of the gene family of clc cl channels and cl / h antiporters ( zifarelli and pusch , 2007 ) , which are homodimeric proteins with independent ion permeation pathways in each subunit ( dutzler et al . , 2002 ) .
in addition to the membrane - embedded part , all mammalian clc proteins possess a large cytoplasmic c - terminal domain that bears two so - called cbs ( from cystathionine--synthase ) domains ( ponting , 1997 ) .
the precise role of the cbs domains is still unclear , but several recent reports indicate that cbs domains may be involved in the regulation of protein function by intracellular nucleotides like atp , adp , and amp ( scott et al . , 2004 ; bennetts et al . , 2005 ; wellhauser et al . , 2006 ; meyer et al
scott et al . ( 2004 ) showed that isolated cbs domains from clc proteins are able to bind nucleotides .
( 2006 ) reported low affinity binding of atp at the c terminus of clc-5 that could be competed by 1 m amp .
moreover , nucleotides were found to be bound in the crystal structure of the c - terminal fragment of clc-5 ( meyer et al . , 2007 ) .
( 2005 ) reported that the activity of clc-1 is regulated by intracellular atp and other nucleotides .
they found that the presence of 5 mm atp shifted the open probability ( popen ) of the slow gate by 50 mv toward positive potentials .
moreover , very recently bennetts et al . ( 2007 ) and tseng et al . ( 2007 ) reported that the effect of atp was greatly enhanced by acidification of the intracellular solution .
( 2005 , 2007 ) were performed on human clc-1 expressed in cultured cells and measured using the whole - cell configuration of the patch clamp technique .
first , the effects of different intracellular solutions and their washout are not tested on the same cell .
second , indirect effects of atp in the complex cellular environment can not be excluded .
third , if large currents are measured , as is the case for clc-1 , the whole cell configuration allows only a relatively poor control of the ion concentrations close to the plasma membrane .
for example , a reduction of the intracellular chloride concentration close to the plasma membrane may decrease inward currents after long hyperpolarizing pulses because of a reduced driving force for chloride . to find out if atp has a direct effect on clc-1 and if acidification may eventually modulate such an effect , we tested the nucleotide on inside - out patches .
, we could not detect any significant effect of atp on clc-1 activity even at acidic ph . during the elaboration of this manuscript , tseng et al .
( 2007 ) , in a study performed in inside - out oocytes patches , reported , in rough agreement with the findings of bennetts et al .
( 2007 ) , that intracellular atp modulates clc-1 activity and that intracellular acidification increases this effect .
wt hclc-1 ( koch et al . , 1992 ) was expressed in xenopus oocytes and currents were measured at 18c ( some control experiments were performed at 26c to ensure that the temperature did not influence the results ) , 24 d after injection using the inside - out configuration of the patch clamp technique ( hamill et al . , 1981 ) with an epc-7 ( list ) amplifier and a custom acquisition program ( gepulse , visual c + + , microsoft ) .
data analysis was performed using custom software ( written in visual c + + , microsoft ) and the origin program ( originlab corporation ) .
the control internal solution contained ( in mm ) 100 nmdg - cl , 2 mgcl2 , 10 hepes , 2 egta , ph 7.3 .
the extracellular ( pipette ) solution contained ( in mm ) 100 nmdg - cl , 5 mgcl2 , 10 hepes , ph 7.3 .
some control experiments were conducted with solutions identical to those used by tseng et al .
( 2007 ) ( 120 mm nmdg - cl , 1 mgcl2 , 10 hepes , 1 egta , ph 7.4 or 6.2 ) . in these experiments
solutions containing atp ( adenosine 5-triphosphate bis(tris ) salt dehydrate , sigma - aldrich ) were prepared diluting the appropriate volume of atp from a stock solution of 50 mm atp in the internal solution .
solutions containing mgatp ( sigma - aldrich ) were prepared in a similar way but without supplementation of mg .
solutions were applied by inserting the patch pipette into perfusion tubes of 0.5 mm diameter .
the functionality of the atp solution and the efficiency of the perfusion system were evaluated on the r - cftr construct that was reconstituted from the injection of two separate rnas encoding the n - terminal and c - terminal half of the protein , respectively .
the gating of this channel does not require phosphorylation and depends only on atp ( bompadre et al . , 2005 ) .
the r - cftr was injected in the same batch of oocytes and measured before clc-1 .
the voltage protocol used to study cftr - mediated currents consists of voltage steps ranging from 80 to 80 mv in 20-mv increments . to study hclc-1mediated currents we used a tail protocol consisting of a 100-ms test pulse to a variable potential ranging from 100 to 160 mv followed by a repolarization to 140 mv for 100 ms . to study in isolation slow gating transitions
the test pulses were prolonged to 200 ms , and a 200-s pulse to 160 mv , to fully activate the fast gating , was applied before the 140-mv repolarization . for measurements at ph 6.2 the length of the test pulse was prolonged to 300 ms as in tseng et al .
the presence of this prepulse did not alter the gating parameters derived from the boltzmann fits .
the open probability was derived from tail currents fitting the latter with a monoexponential function and extrapolating the current values to the beginning of the tail pulse .
open probabilities were derived from the tail currents by fitting a modified boltzmann function ( pusch et al . ,
1995):(1)\documentclass[10pt]{article } 
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 \begin{equation*}i(v)=i_{{\mathrm{max}}}(p_{{\mathrm{min}}}+(1-p_{{\mathrm{min}}})/(1+{\mathrm{exp}}[(v_{1/2}-v)/k)])),\end{equation*}\end{document}where imax is the ( fitted ) maximal current , pmin the residual open probability at negative voltages , v1/2 is the midpoint voltage of the open probability , and k the slope factor of the voltage dependence of the open probability . to estimate the apparent kd for atp activation of the r - cftr channel
, we perfused inside - out patches with different concentrations of atp and measured the current at 80 mv .
not all patches allowed the application of many concentrations and because most patches showed a slow rundown over time we bracketed all measurements by recordings with 1 mm atp and normalized the response to the current measured with 1 mm atp . assuming that , for a single ideal patch without rundown , the atp dependence can be described by\documentclass[10pt]{article } 
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 \begin{equation*}i(c)=i_{{\mathrm{max}}}\frac{(c / k_{d})^{n}}{1+(c / k_{d})^{n}},\end{equation*}\end{document}where c is the concentration of atp
, kd is the apparent dissociation constant , and n is the hill coefficient , the normalized response , inorm , is given by(2)\documentclass[10pt]{article } 
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 \begin{equation*}i_{{\mathrm{norm}}}=\frac{i(c)}{i(1\hspace{.167em}{\mathrm{mm}})}=\frac{(c / k_{d})^{n}}{1+(c / k_{d})^{n}}\frac{1+(1\hspace{.167em}{\mathrm{mm}}/k_{d})^{n}}{(1\hspace{.167em}{\mathrm{mm}}/k_{d})^{n}}\end{equation*}\end{document}this equation was used to fit the data shown in fig .
we provide supplemental online information on the application of intracellular atp on clc-1 at 26 c ( figs . s1 and s2 ) , demonstrating the lack of a significant effect also at this elevated temperature .
furthermore , we show the result of sequencing the hclc-1 insert in the expression vector with four primers ( fig .
the online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1 . we provide supplemental online information on the application of intracellular atp on clc-1 at 26 c ( figs . s1 and s2 ) , demonstrating the lack of a significant effect also at this elevated temperature .
furthermore , we show the result of sequencing the hclc-1 insert in the expression vector with four primers ( fig .
in this work we wanted to study the effect of intracellular atp on hclc-1 expressed in xenopus oocytes using inside - out patches . to test
whether our perfusion system and the atp containing solutions were appropriate for this task , we used as a positive control a cftr construct lacking the regulatory domain ( r - cftr ) ( bompadre et al . , 2005 ) .
in this construct , activation of the cl conductance depends only on the presence of atp on the cytoplasmic side and does not require activation by protein kinase .
we expressed r - cftr in xenopus oocytes and performed inside - out patch experiments , perfusing with control solution and with solutions containing 0.01 ( n = 11 ) , 0.1 ( n = 14 ) , 1 mm ( n = 25 ) , and 5 mm atp ( n = 17 ) . in the absence of atp , we observed only very small background currents ( fig .
1 a ) progressively activated larger cl currents , with 1 mm being already a saturating concentration . switching back to the control solution , devoid of atp , currents returned to background levels ( not depicted ) .
( a ) current recordings from an inside - out patch upon perfusion with control internal solution ( left ) , and with solutions containing atp as indicated in the panels .
( b ) atp dependence of currents measured at 80 mv normalized to currents measured in 1 mm atp .
error bars indicate sd ( the data point at 1 mm has no error bar because it was used for the normalization ) .
2 with kd = 38 m and n = 0.63 . in particular , to make sure that the atp was prepared and delivered at the appropriate concentrations , we performed a dose  response curve for atp activation of r - cftr . fig . 1
b shows the atp dependence of currents at 80 mv normalized to the current measured in 1 mm atp .
2 , resulting in an apparent dissociation constant of ( 38  4 ) m and a hill coefficient of 0.63  0.03 .
( 2005 ) who reported a kd value of 89 m for the atp dependence of the single channel open probability .
these results prove that we have appropriate control of the amount of atp delivered to the patch .
using the same recording condition , we then investigated the effect of atp on currents mediated by hclc-1 .
2 a. as can be seen , the macroscopic currents do not seem to be largely affected by the presence of 10 mm atp .
this was the case also for measurements performed after perfusion lasting up to 5 min .
2 b shows that the apparent open probability , derived from the analysis of the tail currents reported in fig .
2 a , follows a modified boltzmann distribution whose midpoint ( v1/2 ) and minimal open probability ( pmin ) are only marginally affected by the presence of 10 mm atp as compared with control solution ( see legend of fig .
( a ) macroscopic current recordings from an inside - out patch upon perfusion with control internal solution ( left ) , during application of 10 mm atp ( middle ) , and after washout ( right ) .
( b ) voltage dependence of the overall open probability obtained from the macroscopic currents in a as described in materials and methods .
symbols are : square , control ; circle , 10 mm atp ; triangle , wash .
1 with the following parameters : control , v1/2 = 89 mv , pmin = 0.14 ; 10 mm atp , v1/2 = 88 mv , pmin = 0.13 ; wash v1/2 = 87 mv , pmin = 0.14 .
data are from the same patch and were obtained from tail currents preceded by a 200-s pulse to 160 mv .
1 with the following parameters : control , v1/2 = 82 mv , pmin = 0.59 ; 10 mm atp , v1/2 = 81 mv , pmin = 0.58 ; wash v1/2 = 78 mv , pmin = 0.63 .
( 2005 ) reported that the effect of atp was mostly on the slow ( or common ) gate , which acts simultaneously on both pores of the double - barreled channel ( miller and white , 1984 ; accardi and pusch , 2000 ) . to isolate the voltage dependence of the common gate
, we adopted the voltage protocol devised by accardi and pusch ( accardi et al . , 2001 )
also in this case , we did not observe a significant difference between currents recorded in the presence and in the absence of atp ( traces not shown ) .
in fact , the voltage dependence of the open probability of the slow gate is virtually unaffected by the presence of 10 mm atp as shown for a representative patch ( fig .
3 displays the statistical evaluation of the parameters v1/2 and pmin derived as described for the representative measurements in fig .
the mean values of v1/2 and pmin are not significantly changed by the presence of atp when the overall gating of clc-1 is considered ( fig .
the same conclusion holds when only the slow gating mechanism is taken into account ( c and d ) .
statistical evaluation of the gating parameters v1/2 and pmin at ph 7.3 obtained in control solution ( n = 10 ) and in solutions containing 1 ( n = 3 ) , 5 ( n = 5 ) , or 10 mm atp ( n = 5 ) .
mean values of v1/2 ( a ) and pmin ( b ) obtained from the analysis of the overall gating mechanism of hclc-1 .
mean values of v1/2 ( c ) and pmin ( d ) obtained from the analysis of isolated slow gating mechanism of hclc-1 . the values are not significantly different ( p > 0.06 using student 's unpaired t test ) .
( 2007 ) recently suggested that the atp effect was greatly enhanced by decreasing phint . in particular at ph 6.2 , atp was reported to dramatically shift the open probability 
voltage curve by > 100 mv to more positive voltages , with 1 mm atp being sufficient to reach a maximal effect . to test whether acidification of the intracellular solution could unmask an effect of atp that was not evident at the neutral ph used in the previous experiments
, we decreased the phint from 7.3 to 6.2 and assessed the effect of 1 mm atp .
4 a ) , ph 6.2 ( fig . 4 b ) , and ph 6.2 in the presence of 1 mm atp ( fig . 4
c ) . in agreement with previous observations ( rychkov et al . , 1996 ; accardi and pusch , 2000 ) , switching from ph 7.3 to 6.2 slows down the deactivation kinetics and increases the steady - state value of the currents at negative potentials .
surprisingly , addition of 1 mm atp had no detectable further effect on the kinetics and voltage dependence of the currents .
the changes produced by acidification are quickly reversed upon wash with solution at ph 7.3 ( fig .
4 d ) . the voltage dependence of the popen derived for the representative patch shown in fig .
4 ( a  d ) is practically unaffected by the presence of 1 mm atp ( fig . 4 e ) .
this is confirmed by the statistical evaluation of the v1/2 and pmin of the boltzmann distribution describing the popen ( fig . 5 , a and b ) , where average values of these parameters are shown .
top panel , macroscopic current recordings from an inside - out patch upon perfusion with control internal solution at ph 7.3 ( a ) , ph 6.2 ( b ) , solution at ph 6.2 with 1 mm atp ( c ) , and after washout ( d ) .
( e ) voltage dependence of the overall open probability obtained from the macroscopic currents in the top panel as described in materials and methods .
symbols are : square , internal solution at ph 6.2 ; circle , solution at ph 6.2 with 1 mm atp .
1 with the following parameters : solution at ph 6.2 , v1/2 = 94 mv , pmin = 0.50 ; solution at ph 6.2 with 1 mm atp , v1/2 = 95 mv , pmin = 0.43 .
( f ) effect of atp at ph 6.2 on the slow gating mechanism of hclc-1 .
data are from the same patch and were obtained from tail currents preceded by a 200-s pulse to 160 mv .
1 with the following parameters : solution at ph 6.2 , v1/2 = 98 mv , pmin = 0.74 ; solution at ph 6.2 with 1 mm atp , v1/2 = 93 mv , pmin = 0.74 . statistical evaluation of the gating parameters v1/2 and pmin obtained with internal solution at ph 6.2 without atp and with 1 mm atp .
mean values of v1/2 in the absence and in the presence of 1 mm atp ( n = 36 and n = 21 , respectively ) ( a ) and pmin ( n = 28 and n = 9 , respectively ) ( b ) obtained from the analysis of the overall gating mechanism of hclc-1 .
mean values of v1/2 in the absence and in the presence of 1 mm atp ( n = 8 and n = 5 , respectively ) ( c ) and pmin ( n = 19 and n = 13 , respectively ) ( d ) obtained from the analysis of isolated slow gating mechanism of hclc-1 . the values in the absence and in the presence of 1 mm atp are not significantly different ( p > 0.06 using student 's unpaired t test ) .
the lack of any effect of atp at acidic ph was confirmed by isolating the voltage dependence of the slow gate as already described for ph 7.3 ( fig . 4 f ) and by the statistical evaluation of the v1/2 and pmin of the boltzmann distribution ( fig . 5 , c and d ) . in the experiments described above
we had used tris - atp and have added mgso4 at the same concentration , whereas tseng et al . ( 2007 ) and bennetts et al .
whether the discrepancies between our results and theirs could be due to this different source of atp , we performed additional experiments with mgatp . also with mgatp , r - cftr
could be reliably activated and no effect on clc-1 currents could be detected ( unpublished data ) , showing that the source of the nucleotide has no influence on our conclusions .
we also performed some experiments at 26c to assess whether higher temperature could uncover an effect of atp . in these experiments we employed recording solutions identical to those of tseng et al .
the voltage dependence of opening is slightly shifted by 30 mv to more positive voltages , compared with the recordings at 18c , ph 7.3 ( see figs . s1 and s2 , available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1 ) .
this shift is probably caused by the combined effect of the higher temperature ( bennetts et al .
interestingly , at ph 6.2 almost no effect of temperature on the gating parameters could be observed ( fig .
most importantly , however , we could not observe any significant effect of 1 mm atp on current magnitude or voltage dependence of gating at ph 7.4 or ph 6.2 ( see figs . s1 and s2 ) .
in skeletal muscle , cl conductance ( gcl ) accounts for 80% of the overall membrane conductance at rest ( aromataris and rychkov , 2006 ) and clc-1 is the main determinant of this conductance ( lueck et al . , 2007 ) .
the cl conductance is required to keep an appropriate resting potential and to repolarize the voltage following an action potential ( aromataris and rychkov , 2006 ) .
this critical role is underscored by the fact that mutations in the gene coding for clc-1 ( pusch , 2002 ) or aberrant splicing of clc-1 ( charlet et al .
several recent reports indicate that intracellular cbs domains may be involved in the regulation of clc proteins by intracellular nucleotides like atp , adp , and amp ( scott et al . , 2004 ; bennetts et al . , 2005 ; wellhauser et al
in particular , bennetts et al . reported that the activity of clc-1 expressed in hek cells and studied with the whole - cell configuration of the patch - clamp technique is regulated by intracellular atp and other nucleotides . at physiological ph ( 7.2 ) , atp shifted the open probability of the slow gate of clc-1 by 50 mv toward positive voltages with a half - maximal concentration of around 1 mm ( bennetts et al . , 2005 ) .
moreover , the presence of 5 mm atp produced a sixfold reduction in the number of channels that remained open at negative voltages ( bennetts et al . , 2005 ) .
on the basis of an in silico docking study of atp binding , using the crystal structure of impdh ( inosine - monophosphate dehydrogenase ) ( zhang et al . , 1999 ) to model the cytoplasmic region of clc-1 , they also suggested residues that modified the effect of atp .
in particular , mutating l848 , predicted from the model to interact with the adenine base of atp , to alanine , abrogated the effect of atp .
very recently , two different publications reported that the effect of atp was greatly enhanced by intracellular acidification , finding that at ph 6.2 , addition of 1 mm atp further shifted the open probability toward positive voltages ( bennetts et al .
in contrast with previous findings ( rychkov et al . , 1996 ; accardi and pusch , 2000 ) , bennetts et al .
( 2007 ) reported also a slight shift toward positive potentials of the popen at acidic ph even in the absence of atp .
in stark contrast to these conclusions , we have demonstrated here that direct application of atp to the intracellular side of excised inside - out patches does not exert a significant effect on the current amplitude or on the voltage dependence of the open probability of hclc-1 expressed in xenopus oocytes at neither neutral nor slightly acidic ph ( 7.3 and 6.2 ) . in particular , we were not able to detect any significant shift in the midpoint of the voltage dependence of the open probability , or on the residual open probability at negative voltages for both fast and slow gate .
we observed practically no effect for concentrations of atp up to 10 mm , even after perfusion for up to 5 min .
( 2007 ) and in agreement with previous studies ( rychkov et al . , 1996 ; accardi and pusch , 2000 )
, we find that acidic ph has at most a small effect on the v1/2 of the popen , and that inward currents are drastically increased at low ph . as already mentioned , bennetts et al .
( 2007 ) reported a slight shift toward positive potentials of the popen at acidic ph even in the absence of atp .
the estimated pk for the effect of protons was found to be compatible with the pk of histidine residues . on the basis of this observation and of a structural model
, they suggested that a histidine residue ( h847 ) is important to mediate the effect of ph - dependent atp modulation .
residue h847 was suggested to interact electrostatically with the phosphate of atp , however the mutational analysis of bennetts et al . failed to support this hypothesis as changing this residue to either a or to r similarly affected atp dependence .
finally , the mutant l848a that was reported in the first work of bennetts et al .
( 2005 ) to have the largest impact on atp effect was not investigated in their latest work , nor in the work of tseng et al .
we proved that we efficiently apply active atp because we could robustly and reversibly activate currents mediated by r - cftr , a channel that is gated by intracellular atp ( bompadre et al . , 2005 ) .
moreover , we quantitatively controlled the concentration of atp delivered to the patches by performing a dose  response curve of the atp activation of r - cftr , obtaining a kd in agreement with published results ( bompadre et al . , 2005 ) .
to exclude possible sequence variations of the cdna construct we fully sequenced the open reading frame of our hclc-1 clone and found it to be in complete agreement with the published hclc-1 cdna ( see fig .
s3 , available at http://www.jgp.org/cgi/content/full/jgp.200709899/dc1 ) . to exclude any influence of the type of atp salt used , we tested both tris salt and mg salt without observing any difference .
moreover , we used pulse protocols of variable length ( see materials and methods ) to ensure that the current reached a steady state during our experiments .
finally , we proved that temperature in the range between 18 and 26c did not influence the lack of effect of atp on clc-1induced currents .
hence , we conclude that intracellular atp does not directly affect the function of clc-1 at both neutral and acidic ph
( 2005 , 2007 ) might result from the different electrophysiological methods used and/or the different expression system adopted . in this respect , it is important to note that the whole - cell configuration used by bennetts et al .
most importantly , in the whole - cell configuration it is normally not possible to change the internal solution , implying that the comparison between different atp concentrations can not be performed directly on the same cell .
furthermore , the interior of the cell , even after internal access has been established by the patch pipette , remains probably a complex and biochemically active milieu in which atp can potentially activate signaling cascades that may indirectly modulate channel function .
for example , clc-1 activity could be regulated by protein kinases ( chen and jockusch , 1999 ; rosenbohm et al . , 1999 ) . to assess whether the difference in the apparent atp sensitivity of clc-1 , is caused by the different expression systems , we attempted to measure clc-1 currents from inside - out patches of transfected hek cells . unfortunately , as also reported by bennetts et al .
( 2007 ) , we were not able to obtain a sufficient expression level to allow reliable measurements .
it is more difficult to explain the difference with the study of tseng et al .
( 2007 ) in which the same expression system ( xenopus oocytes ) and excised inside - out patch clamp recordings were used .
we are not able to provide any reasonable explanation for the discrepancy between our study and the one of tseng et al .
our results have implications for the interpretation of recent results from x - ray crystallography of clc proteins and for the physiological role of clc-1 . in accordance with the lack of functional effects on clc-1 , meyer and dutzler ( 2006 ) could not detect atp binding to the cytoplasmic domain of the highly homologous clc-0 channel , and no nucleotides could be detected in the crystal structure of the c terminus of the clc - ka channel ( markovic and dutzler , 2007 ) .
interestingly however , the same group found that nucleotides bind with 100 m affinity to the cbs domains of c terminus of the cl / h antiporter clc-5 ( meyer et al . , 2007 ) , in agreement with biochemical data on isolated c - terminal protein fragments ( scott et al .
, 2004 ; wellhauser et al . , 2006 ) . of course our results do not exclude that nucleotides can bind to the c terminus of clc-1 .
however , the lack of direct functional alteration of the channel by atp shows that if nucleotides have any effect in the physiological regulation of the skeletal muscle chloride conductance , it is of indirect nature . regarding the physiological role of clc-1 , an important finding is that macroscopic gcl is reduced by intracellular acidification ( pedersen et al . , 2004 , 2005 ) , an effect that , in principle , could be mediated by a direct ph dependence of clc-1 .
; accardi and pusch , 2000 ) have shown that low phi activates rather than inhibits clc-1 , a finding that we plainly reproduce ( see current traces in fig .
in contrast to these findings , from apparent shifts of activation curves , bennetts et al .
( 2007 ) deduced that clc-1 currents are inhibited by low phi , and speculated that acidosis directly inhibits clc-1 and leads to hyperexcitability .
moreover , they speculated that their finding of increased effect of atp on clc-1 upon intracellular acidification ( bennetts et al . , 2007 ) would explain the so far elusive link between acidification , decrease in gcl , and recovery of muscle excitability ( nielsen et al .
, 2001 ; pedersen et al . , 2004 ; pedersen et al . , 2005 )
because acidification , potentiating the effect of atp , which is to shift the popen toward positive potentials , would greatly decrease gcl facilitating muscle excitation ( bennetts et al . , 2007 )
. our measurements do not support any of these speculations and point to an indirect role of clc-1 and a more complicated mechanism of regulation of gcl that can probably be thoroughly understood only in the context of active skeletal muscle tissues .
 clc-1 belongs to the gene family of clc cl channels and cl/h+ antiporters . 
 it is the major skeletal muscle chloride channel and is mutated in dominant and recessive myotonia . 
 in addition to the membrane - embedded part , all mammalian clc proteins possess a large cytoplasmic c - terminal domain that bears two so - called cbs ( from cystathionine--synthase ) domains . 
 several studies indicate that these domains might be involved in nucleotide binding and regulation . 
 in particular , bennetts et al . 
 ( j. biol . 
 chem . 
 2005 . 
 280:3245232458 ) reported that the voltage dependence of hclc-1 expressed in hek cells is regulated by intracellular atp and other nucleotides . 
 moreover , very recently , bennetts et al . 
 ( j. biol . 
 chem . 2007 . 
 282:3278032791 ) and tseng et al . 
 ( j. gen . 
 physiol . 2007 . 
 130:217221 ) reported that the atp effect was enhanced by intracellular acidification . here , we show that in striking contrast with these findings , human clc-1 , expressed in xenopus oocytes and studied with the inside - out configuration of the patch - clamp technique , is completely insensitive to intracellular atp at concentrations up to 10 mm , at neutral ph ( ph 7.3 ) as well as at slightly acidic ph ( ph 6.2 ) . 
 these results have implications for a general understanding of nucleotide regulation of clc proteins and for the physiological role of clc-1 in muscle excitation . 
INTRODUCTION
METHODS
Online Supplemental Material
RESULTS
DISCUSSION
Supplemental Material