skeletal muscle dihydropyridine ( dhp ) receptors are l - type ca channels that serve a dual role in muscle , functioning both as ca - conducting pores and as the voltage sensors for excitation  contraction ( e - c ) coupling ( ros and brum 1987 ; tanabe et al .
these channels are heteromeric complexes containing the pore - forming 1s subunit plus the 1 , 2 , and  regulatory subunits ( hofmann et al .
two forms of the 1s subunit have been detected in skeletal muscle : a 212-kd full - length translation product ( 1s ) , present as a minor component , and a more abundant 175-kd form ( 1sc , 90% of 1 subunit recovered from transverse tubule membrane preparations ) created by post - translational cleavage of 175 amino acids from the cooh terminus ( de jongh et al .
while the cooh termini of other cloned ca channel 1 subunits have been shown to play a role in channel regulation ( wei et al .
1999 ) , the functional significance of the cooh - terminal truncation of 1s is unknown .
one long - standing hypothesis holds that some dhp receptors in skeletal muscle are specialized voltage sensors for e - c coupling , while others are capable of both voltage sensing and ca conduction .
comparison of dhp binding to l - type ca current amplitude in intact muscle fibers suggested that only a small percentage of dhp receptors are functional ca channels ( schwartz et al .
the discovery of two size forms of 1s occurring in a 90%:10% ratio led to the hypothesis that the rarer full - length form is capable of both sensing the voltage and passing current , while the more abundant truncated form is a dedicated voltage sensor for e - c coupling ( de jongh et al .
injection of cdnas encoding either 1s or a cooh - terminally truncated form of 1s ( at asn 1662 ) into the nuclei of dysgenic mouse myotubes lacking 1s restores contraction , gating charge movement , and l - type ca current ( tanabe et al . 1988 ; adams et al . 1990 ; beam et al .
1992 ) . while the version of 1sc used in these experiments was artificially truncated slightly upstream of the native cleavage site , these results clearly suggest that 1sc can function as both an ion channel and a voltage sensor for e - c coupling .
however , the role of full - length 1s remains unknown , since it is possible that an undetermined proportion of the 1s subunits were cleaved to 1sc in the myotubes .
one recent study has address this issue . when the green fluorescent protein was fused to the cooh terminus of 1s and
expressed in dysgenic myotubes , the fluorescence was localized to the t tubules , which suggests a significant amount of full - length 1s was inserted into the membrane and little cleavage occurred within the 4872-h duration of the experiment ( flucher et al .
heterologous expression in nonmuscle cells provides an alternative opportunity to distinguish the functional roles of the two 1s forms .
injection of crnas encoding 1sc plus the muscle - associated 1b , 2 , and  subunits into xenopus oocytes was recently found to give rise to robust dhp - sensitive l - type currents , whereas injection of full - length 1s cdna gave little or no current ( ren and hall 1997 ) . while these results suggest a difference in channel function for 1s and 1sc , they leave open the possibility that full - length 1s simply expresses poorly in oocytes , but conducts ca just as well as 1sc
. moreover , measurements of ionic current alone can not address the crucial question of whether 1s and 1sc function differently as voltage sensors . to study the current - carrying and voltage - sensing capabilities of the two size forms of 1s in parallel , we expressed 1s and 1sc with the 1 , 2 , and  auxiliary subunits in xenopus oocytes .
we examined the 1a and 1b splice variants , both of which are expressed in muscle tissue ( hofmann et al .
1994 ; ren and hall 1997 ) , because of a previous report that the less - abundant 1b variant was critical for forming channels capable of conducting ba current ( ren and hall 1997 ) .
the cut - open oocyte voltage clamp technique ( stefani and bezanilla 1998 ) was used to record ionic currents ( in 10 mm ba ) and gating charge movement ( in 2 mm co ) in parallel .
we found that both size forms of the 1s subunit supported gating charge movements in 2 mm co when expressed with auxiliary subunits in oocytes , even though only the truncated form conducted appreciable l - type currents in 10 mm ba .
we conclude that , while 1s and 1sc function almost identically as voltage sensors , 1sc is the form specialized to carry l - type current , while 1s appears to be somehow inhibited from passing substantial ionic current .
the rabbit 1s , rat brain 1b , rabbit 2 , and rabbit  ca channel cdnas were obtained in the pkcrh , pbluescript , pcdna3 , and pcd - x vectors , respectively , as a gift from dr .
the rabbit 1a subunit was obtained in the pnks2 vector as a gift from dr .
emily liman , harvard university ) at the hindiii and bamhi polylinker sites , respectively .
1b was subcloned into pgemhe between the sacii and hindiii sites , while 2 and 1a were left in pcdna3 and pnks2 , respectively .
1sc - pgemhe was made from 1s - pgemhe using the clontech transformer site - directed mutagenesis kit ( clontech laboratories , inc . ) .
the mutagenic primer caused a loop - out deletion of base pairs 53205844 ( amino acids 16981893 ) of the rabbit 1s sequence , while the selection primer mutated the mfei site at position 2788 of the pgemhe sequence to a unique ndei site .
for in vitro synthesis of rna , the plasmids containing the ca channel subunits were linearized using the following restriction enzymes : sse8387i for 1s and 1sc , xbai for 1a , noti for 1b , pvuii for 2 , and nhei for . capped crna was synthesized using the mmessage mmachine t7 kit ( for 1s , 1sc , 1b , 2 , and  ) or sp6 kit ( for 1a ; ambion corp . ) and extracted using the rnaid purification kit ( bio101 ) .
stage v and vi oocytes were harvested from egg - bearing female xenopus laevis frogs under anesthesia with 3-aminobenzoic acid ethyl ester ( 1 mg ml in a cold water bath for 25 min ; sigma - aldrich ) , in accordance with the guidelines of the subcommittee on research animal care at the massachusetts general hospital .
oocytes were removed into ca - free or-2 solution containing ( mm ) : 82.5 nacl , 2.5 kcl , 1 mgcl2 , and 5 hepes , ph 7.6 .
the egg sacs were manually torn open using forceps , and the oocytes were incubated in or-2 containing 2 mg ml collagenase ( gibco brl ) for 2.5 h in a room temperature shaker ( 60 rpm ) to remove the follicular membrane .
the oocytes were then washed four times in or-2 solution and transferred for storage to nd-96 solution containing ( mm ) : 96 nacl , 2 kcl , 1.8 cacl2 , 1 mgcl2 , 2.5 pyruvate , 5 hepes , and 50 g ml gentamicin ( gibco brl ) , ph 7.6 .
oocytes were injected with 50100 nl of a 1:1:1:1 mixture of 1s ( or 1sc ) , 1a ( or 1b ) , 2 , and  crna using injection pipettes pulled from thin - wall capillary glass ( world precision instruments ) and were stored at 18c for 57 d before recording .
oocytes were voltage clamped in the cut - open configuration , with active clamp of the upper and guard compartments , using an oocyte clamp ( ca-1b ; dagan corp . ) under control of a custom stimulation / recording program written in axobasic and running on an ibm - compatible computer .
voltage - sensing electrodes , fabricated from borosilicate capillary glass ( 1.65-mm outer diameter ; vwr scientific ) using a multi - stage puller ( sutter instrument co. ) , were filled with 3 m kcl and had resistances of 0.21 m. the leads of the amplifier headstage , attached to ag / agcl pellets in plastic wells containing 2 m nacl solution , were connected to the upper , guard , and lower oocyte compartments using glass agar bridges containing 110 mm na - methanesulfonic acid and 10 mm hepes , in 3% agarose , ph 7.0 , and threaded with a platinum wire .
current traces , evoked by depolarizing test pulses from a holding potential of 90 mv , were corrected for linear leak and capacity currents ( minimized by the analog compensation of the amplifier ) by addition of control currents evoked by hyperpolarizing pulses of equal amplitude from the holding potential ( p/1 protocol ) . since the charge movement was not noticeably altered by changing the holding potential to 110 or 120 mv ( data not shown ) , we judged our leak subtraction protocol to be sufficient to selectively subtract linear components .
signals were filtered at 1 khz and sampled at 20 khz ( for gating currents ) or 2 khz ( for ionic currents ) .
analysis and curve fitting was performed using a combination of custom axobasic routines and sigmaplot ( jandel scientific ) .
student 's t test was used to make pairwise comparisons between sets of data ( p values are reported in the text ; p = 0.05 was taken as the limit of statistical significance ) .
solutions for recording ba currents and ca channel gating currents were prepared following olcese et al .
1996 and stefani and bezanilla 1998 and were cl free to avoid contamination with oocyte cl currents ( including ba - activated cl currents )
. for gating current measurements , the external solution ( applied in the external and guard compartments ) contained ( mm ) : 2 co(oh)2 , 96 naoh , and 10 hepes , adjusted to ph 7.0 with methanesulfonic acid . for ionic current measurements , the external solution contained 10 mm ba(oh)2 instead of 2 mm co(oh)2 , but was otherwise identical .
the lower chamber in contact with the part of the oocyte permeabilized with 0.1% saponin ( sigma - aldrich ) contained 110 mm potassium glutamate and 10 mm hepes , adjusted to ph 7.0 with naoh . to avoid possible contamination with nonlinear charge movement related to the oocyte na 
k pump ( neely et al . 1993 ; olcese et al . 1996 ; stefani and bezanilla 1998 ) , 0.1 mm ouabain ( sigma - aldrich ) was present in all external solutions .
oocytes injected with either 1s or 1sc plus the auxiliary subunit crnas showed transient currents at the beginning and end of short depolarizing pulses , while uninjected oocytes and oocytes injected with auxiliary subunit crnas alone lacked these currents ( fig .
1 a ) . these transient currents had the properties expected of gating currents arising from the movement of charged elements in the ion channel protein : ( a ) they depended on the presence of the pore - forming  subunit ( fig .
1 a ) ; ( b ) they always followed the direction of the voltage step ( a ) ; ( c ) the integral of the current at the onset of a voltage step ( qon ) matched the integral of the current at the offset of the step ( qoff ) ( b ) ; and ( d ) qon and qoff approached limiting values at both positive and negative potentials , with a voltage dependence that followed a boltzmann distribution ( c ) .
2 , which compares the magnitude and voltage dependence of the charge movement measured in oocytes expressing 1s and 1sc , shows that both constructs were clearly expressed abundantly at the surface membrane regardless of which 1 subunit isoform was coexpressed .
qoff was used for the comparison instead of qon because of the faster kinetics ( and hence greater single - to - noise ratio at small test depolarizations ) of the off response .
the maximum qoff appeared larger in oocytes expressing 1sc than in oocytes expressing 1s ( fig .
b ) . while this effect on qoff , max was not statistically significant in the presence of 1a ( fig .
2 a ; p = 0.13 ) , it was statistically significant in the presence of 1b ( b ; p = 0.008 ) .
the normalized qoff versus voltage ( q - v ) curves for 1s and 1sc had roughly the same midpoints and slopes ( fig .
as has been observed for other voltage - dependent ion channels and for native l - type ca channels in skeletal muscle , the q - v curve started at significantly more negative potentials and was less steep than the g - v curve for l - type current ( fig .
this is consistent with the idea that skeletal muscle ca channels undergo rapid voltage - dependent transitions among several closed conformations before opening , some of which are presumably involved in excitation 
contraction coupling ( feldmeyer et al . 1990 ; ma et al . 1996 ) . to compare directly the voltage - sensing and current - carrying properties of 1s and 1sc
, we measured both ionic and gating currents in individual oocytes expressing these constructs ( fig .
ionic currents were measured in the presence of 10 mm ba , the extracellular ba was then replaced with 2 mm co using a manual pipet , and gating charge movements were measured ( see fig .
, qon was measured instead of qoff , despite the lower signal - to - noise ratio of the on response , to avoid the possibility of contamination by small ba tail currents remaining after the solution exchange .
these experiments confirmed that the full - length form of the channel conducts ba current very poorly compared with the truncated form ( ren and hall 1997 ) , but that injecting crna coding for either full - length or truncated 1s resulted in large voltage - driven gating charge movements not found in uninjected eggs .
the maximal amplitude of the gating currents measured in each egg was comparable with that of the largest ionic current , consistent with the idea that channel expression was high but the channel open probability was low . indeed , while the ionic currents , even those carried by 1sc , were small compared with those measured for other cloned ca channels expressed in oocytes , the gating currents were as large as or larger than those of other ca channels ( neely et al .
this suggests that oocyte expression of the skeletal muscle ca channels is considerably more robust than has been previously believed .
oocytes injected with crna coding for the auxiliary subunits alone had dhp - insensitive inward ba currents comparable in size with the l - type current , but with rapid kinetics , presumably carried by an endogenous ca channel ( fig .
3 e , bottom ) , suggesting that the endogenous channel had a much smaller ratio of gating to ionic current than the skeletal muscle channel . based on its kinetic properties
, we judged the small ionic current seen in oocytes injected with crna coding for full - length 1s to be mostly , though not completely , endogenous in origin ( a conclusion confirmed by separate experiments in which 5 m nimodipine was found to block < 10% of the current expressed in these cells ; data not shown ) .
these currents were consistently smaller than the endogenous current observed in oocytes injected with the auxiliary subunit crnas alone , most likely because of competition between 1s subunits and endogenous ca channel subunits for the auxiliary subunits .
comparison of the mean qon - v and i - v curves measured in oocytes expressing 1sc and 1s with either the 1a subunit ( fig .
b ) or the 1b subunit ( c and d ) emphasizes that while the full - length and truncated subunits are comparable voltage sensors , the truncated version is a much better conductor of ionic current .
we quantified this functional distinction by calculating the ratio of maximum ionic current to maximum charge movement ( imax / qon , max ) for each oocyte ( fig .
imax / qon , max was sixfold greater for oocytes expressing 1sc than for oocytes expressing 1s when either the 1a or the 1b subunit was used ; a difference that was highly statistically significant in each case ( p = 4.8  10 and 6.2  10 , respectively ) .
this difference is a conservative estimate , since at least 90% of the current observed in 1s - injected oocytes was conducted by a dhp - insensitive  subunit , as mentioned above ( data not shown ) .
imax / qon , max was smaller for channels containing 1a than for channels containing 1b , reflecting both the somewhat smaller ionic currents and larger gating currents observed , on average , when 1a was injected . as was also seen in the separate experiments of fig .
2 , truncation of the 1s increased the size of gating currents significantly in the presence of the 1b subunit ( p = 0.02 ) , but not in the presence of the 1a subunit ( p = 0.63 ) .
the primary goal of this study was to determine whether the different magnitudes of l - type ba current observed previously when the full - length 1s and truncated 1sc subunits were expressed in xenopus oocytes ( ren and hall 1997 ) represents a function specialization of these two size forms of the skeletal muscle l - type ca channel . using the cut - open oocyte voltage clamp method , we measured large transient currents from oocytes expressing 1s or 1sc with the 1 ( 1a or 1b ) , 2 , and  subunits that represented gating charge movements in the 1 subunit molecule , demonstrating that both size forms of the channel are expressed in oocytes and constitute working voltage sensors .
the range and steepness of the voltage dependence of the charge movement mediated by 1s and 1sc were essentially the same , independent of which 1 subunit was coexpressed . the maximum amount of charge moved at depolarized voltages , qmax , tended to be larger for 1sc than for 1s .
however , this difference in qmax was statistically significant ( both qon , max and qoff , max ) only when the 1b subunit , rather than the 1a subunit , was coexpressed . measurements of both charge movement and ionic current from the same oocyte showed that : ( a ) oocytes expressing the truncated subunit plus auxiliary subunits supported both charge movements and l - type ba current ; ( b ) oocytes expressing the full - length subunit plus auxiliary subunits had charge movements but very little l - type ba current ; and ( c ) oocytes expressing the auxiliary subunits alone had neither detectable charge movements nor l - type ba current .
the stark differences observed between cells expressing 1s versus 1sc suggest there was no appreciable cooh - terminal truncation of 1s in the oocyte .
we conclude that the two naturally occurring size forms of the skeletal muscle dhp receptor  subunit are functionally specialized when expressed with the auxiliary subunits found in muscle . 1s and 1sc are both functional voltage sensors , but 1sc is a much more effective ca channel .
only tiny ionic currents were seen when the 1s subunit was coexpressed with either the 1a or 1b subunit ( fig . 4a and fig .
c ) , and the currents observed when the 1s/1b/2/ combination was injected were 90% insensitive to 5 m nimodipine ( data not shown ) .
this differs from the data of ren and hall 1997 , who observed what appeared to be l - type current when 1s was expressed with 1b , 2 , and  ( although this conclusion was based on current kinetics rather than on dihydropyridine block ) .
we observed robust ionic currents when either 1a or 1b crnas were coinjected with 1sc crna , in contrast to the original report of a requirement for 1b ( ren and hall 1997 ) .
moreover , separate experiments in which 5 m nimodipine was applied to oocytes expressing the 1sc/1a/2/ combination confirmed that the inward current seen in this case was 8090% l - type ( data not shown ) .
our results therefore support the hypothesis that the predominant form of native dhp receptors in skeletal muscle , those containing 1sc and 1a , can function both as voltage sensors and as l - type calcium channels . given the strong similarity of the kinetics and voltage dependence of the ionic and gating currents measured here in oocytes to those measured previously in mammalian muscle cells ( dulhunty and gage 1983 ; simon and beam 1985 ; lamb 1987 ; delbono 1992 ; garcia et al .
1992 ) , we believe that the functional differences we observed for 1s and 1sc in oocytes are likely to occur in native dhp receptors .
nevertheless , experimental confirmation of the distinct functional roles of the 1s and 1sc subunits must ultimately be delineated in native cells , especially given the mounting evidence from dyspedic myotubes that both functions of the dhp receptor are strongly influenced by interacting ryanodine receptors ( nakai et al .
definitive proof of distinct roles for the two size forms of the 1s subunit may ultimately require the identification and selective inhibition of the enzyme responsible for cooh - terminal truncation . the mechanism by which the carboxyl terminus modulates the coupling between voltage - dependent gating and channel opening remains unknown .
deletion of amino acids 307472 from the 665-amino acid carboxyl terminus of the 1c subunit increased ba currents four- to sixfold in the oocyte expression system ( wei et al .
the increase in ionic current did not appear to be accompanied by an increase in gating charge , consistent with the notion that the coupling was enhanced , rather than an increase in the number of channels .
the confidence in this conclusion is somewhat limited , however , by the fact that charge movements were measured in the presence of large , unblocked inward ba currents .
several motifs important for modulation of ca currents have been identified in the carboxyl tails of  subunits .
the carboxyl terminus deletion of 1s studied herein removes several consensus sites for pkc and camp - mediated phosphorylation , but leaves intact an ef hand and calmodulin - binding iq consensus motifs , both of which have been implicated in ca - dependent modulation of inactivation and facilitation of ionic current ( lee et al .
it remains to be determined whether the enhanced coupling of gating charge movement to channel opening we observed in 1sc is dependent on any of these previously identified modulatory roles of the carboxyl terminus .
our observation that truncation of the 1s subunit significantly increased the magnitude of gating charge movements ( an effect not seen with cooh - terminal truncation of 1c ) suggests that the cooh terminus of 1s may play a role in channel expression in the membrane as well as in the coupling of gating to pore opening .
our experiments give the unexpected result that both size forms of the 1s subunit express as well as or better than other cloned ca channel isoforms in xenopus oocytes , as judged by gating current amplitudes ( neely et al .
this finding contradicts the common belief that the small ionic currents seen by us and others trying to express 1s in heterologous systems ( perez - reyes et al .
1997 ) reflect an inability of this channel to express stably outside the muscle environment . on the other hand , the local environment of the triadic junction
does enhance the efficacy of dhp receptors to function as ca channels , as shown by a decrease in the ratio of ionic to gating current in dyspedic mice lacking the skeletal muscle isoform of the ryanodine receptor ( nakai et al .
the large gating currents we observed relative to the small ionic currents underscore the idea that the coupling of charge movement to pore opening is poor in dhp receptors ( flockerzi et al .
1986 ; ma et al . 1991 ; dirksen and beam 1995 ) , even for channels containing the truncated 1s subunit .
robust expression of the skeletal muscle l - type ca channel in a heterologous system will be useful in elucidating the functional consequences of muscle - specific modulatory mechanisms , which may include modifications to the channel protein ( such as phosphorylation or the cooh - terminal truncation studied here ) or interactions with other proteins ( such as the auxiliary ca channel subunits or other components of the triad junction ) .
in addition , the ability to record skeletal muscle ca channel gating currents in an expression system lacking the electrically complex features of muscle cells , such as t tubules and additional voltage - gated channels , may allow for unprecedented precision in the study of the channel voltage sensor .
 skeletal muscle dihydropyridine ( dhp ) receptors function both as voltage - activated ca2 + channels and as voltage sensors for coupling membrane depolarization to release of ca2 + from the sarcoplasmic reticulum . in skeletal muscle 
 , the principal or 1s subunit occurs in full - length ( 10% of total ) and post - transcriptionally truncated ( 90% ) forms , which has raised the possibility that the two functional roles are subserved by dhp receptors comprised of different sized 1s subunits . 
 we tested the functional properties of each form by injecting oocytes with crnas coding for full - length ( 1s ) or truncated ( 1sc )  subunits . 
 both translation products were expressed in the membrane , as evidenced by increases in the gating charge ( qmax 80150 pc ) . 
 thus , oocytes provide a robust expression system for the study of gating charge movement in 1s , unencumbered by contributions from other voltage - gated channels or the complexities of the transverse tubules . as in recordings from skeletal muscle , for heterologously 
 expressed channels the peak inward ba2 + currents were small relative to qmax . the truncated 1sc protein , however , supported much larger ionic currents than the full - length product . 
 these data raise the possibility that dhp receptors containing the more abundant , truncated form of the 1s subunit conduct the majority of the l - type ca2 + current in skeletal muscle . 
 our data also suggest that the carboxyl terminus of the 1s subunit modulates the coupling between charge movement and channel opening . 
INTRODUCTION
METHODS
RESULTS
DISCUSSION