l - type ca channels perform essential roles in the cardiovascular system , where they trigger excitation  contraction coupling and contribute to pacemaker and action potentials ( boyett et al . , 1996 ) . inactivation of   l - type channels is induced by membrane depolarization and elevations in intracellular [ ca ] , although these   two types of ca channel inactivation appear to proceed   via distinct and independent mechanisms ( hadley and   lederer , 1991 ; obejero - paz et al . , ca - dependent inactivation is a prominent feature of cardiac l - type ca channels that has important   implications for the function of these channels in cardiovascular physiology . voltage - gated ca channels are heteromultimeric   complexes composed of pore - forming 1 and accessory   2 and  subunits ( hofmann et al . , 1994 ) . transfection of mammalian cell lines or xenopus oocytes with   the cardiac 1 subunit ( 1c ) by itself produces voltage - gated ca channels that exhibit ca - dependent inactivation ( neely et al . , 1994 ; perez - garcia et al . , 1995 ; zong   and hofmann , 1996 ) , suggesting that this type of inactivation is an intrinsic property of the 1c subunit . because ca - dependent inactivation is induced by a rise in   intracellular ca concentration ( haack and rosenberg ,   1994 ) , it is reasonable to postulate the five major putative cytoplasmic domains of 1c include the nh2 and cooh termini and three linkers   ( the i iii , and iii  iv loops ) that connect the four   major transmembrane domains ( mikami et al . , 1989 ) . two previous studies have provided evidence that   one or more of these cytoplasmic domains play important roles in ca - dependent inactivation . thus , ca- dependent inactivation is abolished by simultaneous replacement of all five of the major cytoplasmic domains   of 1c with the corresponding regions from the skeletal   muscle 1s subunit ( zong et al . , ca - dependent   inactivation is also eliminated by replacing all or a portion of the cooh terminus of 1c with the corresponding region of the neuronal 1e subunit ( de leon et al . ,   1995 ) . because 1s and 1e both appear to exhibit only   voltage - dependent inactivation ( donaldson and beam ,   1983 ; beam and knudson , 1988 ; de leon et al . , 1995 ) ,   these results imply that the structural determinants of   ca - dependent inactivation are encoded within the cytoplasmic domains of 1c . the goal of the present study   was to test this hypothesis . toward this end , we have   studied a series of chimeric 1 subunits in which the   major cytoplasmic domains of 1c were individually replaced by their counterpart from 1s . our results suggest that the cytoplasmic cooh terminus , and i  ii and   ii in addition , our   findings indicate that ca - dependent inactivation can be   prevented by the distal cooh terminus from the skeletal muscle 1s . some of these results have appeared previously in abstract form ( adams and tanabe , 1996 ) . human embryonic kidney cells were obtained from the american type culture collection ( crl 1573 ; rockville , md ) and   propagated using standard techniques . the culture medium contained 90% dmem ( 11995 - 065 ; gibco brl , gaithersburg ,   md ) , 10% heat - inactivated horse serum ( 26050 - 13 ; gibco brl )   and 50 g / ml gentamicin ( 15710 - 015 ; gibco brl ) . every 23 d ,   these cells were briefly trypsinized and replated onto the maintenance culture at a fourfold lower density . at the time of replating , additional 35-mm culture dishes ( 3001 ; becton dickinson &   co. , franklin lakes , nj ) were seeded with 10 cells / dish . approximately 16 h later , the capo4 precipitation technique ( cell   phect kit ; pharmacia lkb biotechnology inc . , piscataway , nj )   was used to transfect the seeded cells with a combination ( at 1 g   of each plasmid cdna per dish ) of expression plasmids encoding   the rabbit cardiac 1c ( or chimeras constructed between 1c and   the rabbit 1s ) and the rabbit skeletal muscle 2a and 1a subunits . the transfection mixture also included an expression plasmid ( ebo - pcd - leu2 ; 59565 ; american type culture collection )   encoding the human cd8 protein at a fivefold lower concentration ( 0.2 g per dish ) . 13 d later , paramagnetic beads ( 4.5 m   diameter ) coated with anti  cd8 antibody ( dynal , inc . , great   neck , ny ) were added to each dish . cells expressing cd8 protein   on the surface membrane were visually identified by virtue of being decorated with the beads ( jurman et al . , 1994 ) and were selected for electrophysiological analysis . the amino acid compositions and construction of the expression   plasmids encoding the 3 deletion mutant of 1c ( 1c3del ) and   the chimeric 1 subunits csk1 , csk2 , csk3 , and csk4 have been   previously described ( tanabe et al . the cdnas encoding the chimeras csk5 and csk8 are composed of the following restriction fragments ( the origin of the   fragments is given in parentheses ) . pcsk5 : 4.2-kb pair hindiii - aatii ( pcard1 ; see mikami et al . , 1989 ) , 0.88-kb pair aatii - bglii   ( pcard1 ) , and 0.55-kb pair bglii - hindiii ( pc61 ; see beam et   al . , 1992 ) . pcsk8 : 3.8-kb pair hindiii - bsphi ( pcard1 ) , 0.24-kb   pair bsphi - bstxi ( pcac6 ; see tanabe et al . , 1988 ) , 0.19-kb pair   bstxi - aatii ( pcard1 ) , and 2.9-kb pair aatii - hindiii ( pcard1 ) . the expression plasmids pcsk5 and pcsk8 , carrying the cdnas   encoding the individual chimeric ca channels , were constructed   by inserting the corresponding cdnas into the hindiii site of   the plasmid pkcrh2 ( mishina et al . , 1984 ) . the amino acid compositions of csk5 and csk8 are as follows   ( c and sk , cardiac and skeletal muscle ca channel , respectively ;   numbers in parentheses , amino - acid numbers [ tanabe et al . ,   1987 ; mikami et al . , 1989 ] ; the junctional sequences common to   the two ca channels are represented by amino acid numbers of   the cardiac ca channel ) . csk8 : c ( 11204 ) , sk ( 10741129 ) , and c ( 12612171 ) . patch pipettes were fabricated from 100-l borosilicate micropipettes ( 53432 - 921 ; vwr scientific , west chester , pa ) and filled   with a solution containing ( mm ) : 155 cscl , 10 cs2egta , 4   mgatp , 0.38 tris - gtp , and 10 hepes , with ph adjusted to 7.4   using csoh . aliquots of this solution were stored at 80c and   kept on ice after thawing . filled pipettes had dc resistances   of 12 m. pipette tips were coated with paraffin to reduce capacitance , and then fire polished . residual pipette capacitance   was compensated in the cell - attached configuration , using the   negative capacitance circuit of the axopatch 200a amplifier ( axon   instruments , inc . , foster city , ca ) . in earlier experiments , the external solution contained ( mm ) : 145 tetraethylammonium ( tea )   chloride , 40 cacl2 or bacl2 , and 10 hepes , with ph adjusted to   7.4 using teaoh . in later experiments , an external solution   containing ( mm ) 140 nacl , 2 kcl , 40 cacl2 or bacl2 , and 10   hepes ( ph 7.4 with naoh or hcl ) was used because the cells   appeared to remain healthy for longer periods in this solution . because the voltage dependence of ionic currents recorded in   the nacl - based solution was shifted by 10 mv relative to currents recorded in the teacl - based solution , data obtained using   the two different external solutions have been analyzed separately . ca or ba currents were recorded using the whole - cell patch - clamp technique ( hamill et al . , 1981 ) . after establishment of the   whole - cell configuration , electronic compensation was used to   minimize the access resistance and the time required to charge   the cell capacitance . the dc resistance of the whole - cell configuration typically exceeded 1 g , and leakage currents were usually < 50 pa at the steady holding potential of 80 mv . linear   cell capacitance was monitored throughout each experiment and   was calculated from the integral of charges required to clamp the   membrane from 80 to 70 mv . the compensated series resistance ( rs ) was calculated from the time constant for decay of the   capacity transient and the linear cell capacitance . linear membrane capacitance and leakage currents were subtracted from test currents using the p/6 method , and all analyzed and reported   data were obtained from corrected currents . currents were filtered at 0.510 khz using the built - in bessel filter ( 4-pole low   pass ) of the axopatch 200a patch - clamp amplifier , and were sampled at 150 khz using a digidata 1200 analogue - to - digital board   installed in a gateway 486 - 66v personal computer . the pclamp   software programs clampex and clampfit ( version 6.0 ) were   used for data acquisition and analysis , respectively . human embryonic kidney cells were obtained from the american type culture collection ( crl 1573 ; rockville , md ) and   propagated using standard techniques . the culture medium contained 90% dmem ( 11995 - 065 ; gibco brl , gaithersburg ,   md ) , 10% heat - inactivated horse serum ( 26050 - 13 ; gibco brl )   and 50 g / ml gentamicin ( 15710 - 015 ; gibco brl ) . every 23 d ,   these cells were briefly trypsinized and replated onto the maintenance culture at a fourfold lower density . at the time of replating , additional 35-mm culture dishes ( 3001 ; becton dickinson &   co. , franklin lakes , nj ) were seeded with 10 cells / dish . approximately 16 h later , the capo4 precipitation technique ( cell   phect kit ; pharmacia lkb biotechnology inc . , piscataway , nj )   was used to transfect the seeded cells with a combination ( at 1 g   of each plasmid cdna per dish ) of expression plasmids encoding   the rabbit cardiac 1c ( or chimeras constructed between 1c and   the rabbit 1s ) and the rabbit skeletal muscle 2a and 1a subunits . the transfection mixture also included an expression plasmid ( ebo - pcd - leu2 ; 59565 ; american type culture collection )   encoding the human cd8 protein at a fivefold lower concentration ( 0.2 g per dish ) . 13 d later , paramagnetic beads ( 4.5 m   diameter ) coated with anti  cd8 antibody ( dynal , inc . , great   neck , ny ) were added to each dish . cells expressing cd8 protein   on the surface membrane were visually identified by virtue of being decorated with the beads ( jurman et al . , 1994 ) and were selected for electrophysiological analysis . the amino acid compositions and construction of the expression   plasmids encoding the 3 deletion mutant of 1c ( 1c3del ) and   the chimeric 1 subunits csk1 , csk2 , csk3 , and csk4 have been   previously described ( tanabe et al . the cdnas encoding the chimeras csk5 and csk8 are composed of the following restriction fragments ( the origin of the   fragments is given in parentheses ) . pcsk5 : 4.2-kb pair hindiii - aatii ( pcard1 ; see mikami et al . , 1989 ) , 0.88-kb pair aatii - bglii   ( pcard1 ) , and 0.55-kb pair bglii - hindiii ( pc61 ; see beam et   al . , 1992 ) . pcsk8 : 3.8-kb pair hindiii - bsphi ( pcard1 ) , 0.24-kb   pair bsphi - bstxi ( pcac6 ; see tanabe et al . , 1988 ) , 0.19-kb pair   bstxi - aatii ( pcard1 ) , and 2.9-kb pair aatii - hindiii ( pcard1 ) . the expression plasmids pcsk5 and pcsk8 , carrying the cdnas   encoding the individual chimeric ca channels , were constructed   by inserting the corresponding cdnas into the hindiii site of   the plasmid pkcrh2 ( mishina et al . , 1984 ) . the amino acid compositions of csk5 and csk8 are as follows   ( c and sk , cardiac and skeletal muscle ca channel , respectively ;   numbers in parentheses , amino - acid numbers [ tanabe et al . ,   1987 ; mikami et al . , 1989 ] ; the junctional sequences common to   the two ca channels are represented by amino acid numbers of   the cardiac ca channel ) . csk8 : c ( 11204 ) , sk ( 10741129 ) , and c ( 12612171 ) . patch pipettes were fabricated from 100-l borosilicate micropipettes ( 53432 - 921 ; vwr scientific , west chester , pa ) and filled   with a solution containing ( mm ) : 155 cscl , 10 cs2egta , 4   mgatp , 0.38 tris - gtp , and 10 hepes , with ph adjusted to 7.4   using csoh . aliquots of this solution were stored at 80c and   kept on ice after thawing . filled pipettes had dc resistances   of 12 m. pipette tips were coated with paraffin to reduce capacitance , and then fire polished . residual pipette capacitance   was compensated in the cell - attached configuration , using the   negative capacitance circuit of the axopatch 200a amplifier ( axon   instruments , inc . , foster city , ca ) . in earlier experiments , the external solution contained ( mm ) : 145 tetraethylammonium ( tea )   chloride , 40 cacl2 or bacl2 , and 10 hepes , with ph adjusted to   7.4 using teaoh . in later experiments , an external solution   containing ( mm ) 140 nacl , 2 kcl , 40 cacl2 or bacl2 , and 10   hepes ( ph 7.4 with naoh or hcl ) was used because the cells   appeared to remain healthy for longer periods in this solution .   because the voltage dependence of ionic currents recorded in   the nacl - based solution was shifted by 10 mv relative to currents recorded in the teacl - based solution , ca or ba currents were recorded using the whole - cell patch - clamp technique ( hamill et al . , 1981 ) . after establishment of the   whole - cell configuration , electronic compensation was used to   minimize the access resistance and the time required to charge   the cell capacitance . the dc resistance of the whole - cell configuration typically exceeded 1 g , and leakage currents were usually < 50 pa at the steady holding potential of 80 mv . linear   cell capacitance was monitored throughout each experiment and   was calculated from the integral of charges required to clamp the   membrane from 80 to 70 mv . the compensated series resistance ( rs ) was calculated from the time constant for decay of the   capacity transient and the linear cell capacitance . linear membrane capacitance and leakage currents were subtracted from test currents using the p/6 method , and all analyzed and reported   data were obtained from corrected currents . currents were filtered at 0.510 khz using the built - in bessel filter ( 4-pole low   pass ) of the axopatch 200a patch - clamp amplifier , and were sampled at 150 khz using a digidata 1200 analogue - to - digital board   installed in a gateway 486 - 66v personal computer . the pclamp   software programs clampex and clampfit ( version 6.0 ) were   used for data acquisition and analysis , respectively . fig . 1 shows whole - cell ca or ba currents recorded   from a human embryonic kidney cell expressing the   wild - type 1c subunits . as demonstrated by previous   studies ( perez - reyes et al . , 1994 ; zong et al . , 1994 ; de   leon et al . , 1995 ; perez - garcia et al . , 1995 ; ferreira et   al . , 1997 ) , the heterologously expressed cardiac 1c exhibits prominent ca - dependent inactivation , as evidenced by the faster inactivation of ca than ba currents . the inactivation rate of currents mediated by the   subunit combination expressed here ( 1c , 2a , and   1a ) closely approximates the inactivation rate of natively expressed cardiac channels ( see imredy and yue ,   1994 ) , and is considerably faster than the inactivation   rate of currents mediated by expression of 1c and 2a   in the absence of 2a ( perez - reyes et al . , 1994 ; de   leon et al . , 1995 ; perez - garcia et al . , 1995 ; ferreira et   al . , 1997 ) .   to quantify the time course of inactivation , the decaying phase of ca or ba currents was fit with a single   or double exponential function . most ca currents required two exponentials for a good fit , whereas ba currents evoked by relatively short test pulses ( 250 ms )   could usually be well fit by a single exponential . however , some ba currents displayed two distinct components of inactivation . 1 d plots the time constants   for inactivation of 1c currents as a function of test potential . for ca currents , the time constants for inactivation had a u - shaped dependence on test potential ,   whereas time constants for inactivation of ba currents   decreased progressively with increasing test potential . these results are consistent with the expectation that   1c undergoes ca- but not ba - dependent inactivation . however , in some cells the availability of ba currents   ( measured using a double - pulse protocol ) displayed a   weak u - shaped dependence on test potential ( data not   shown ) , consistent with the idea that ba can also trigger ion - dependent inactivation , although less effectively than ca . to investigate the structural basis for ca - dependent   inactivation , we expressed a series of chimeric 1 subunits in which one of the major intracellular domains   of 1c was replaced by the corresponding region from   the skeletal muscle 1s subunit . previous studies have shown that deletion of the distal cooh terminus of 1c or 1s produces a fully functional ca channel ( beam et al . , 1992 ; 3 a confirms this result for   the deletion mutant 1c3del in which amino acids   18132166 have been removed from the cooh terminus of 1c . as shown in table i , inactivation of 1c3del   proceeded at the same rate as the wild - type 1c , confirming that deletion of the distal cooh terminus of   1c does not appreciably alter the process of ca - dependent inactivation . we next examined the potential role of the nh2 terminus in ca - dependent inactivation . the nh2 terminus of 1c is 103 amino acids longer than that of 1s   ( mikami et al . , 1989 ) and contains four consensus sites   for potential phosphorylation by pkc , whereas the nh2   terminus of 1s lacks predicted pkc sites . in chimera csk1 , the nh2 terminus of 1c has been replaced by its   counterpart from 1s ( fig . currents mediated by   csk1 closely resembled those produced by 1c and   1c3del ( fig . thus , ca currents inactivated faster   than ba currents , and time constants for inactivation of   ca currents were indistinguishable between csk1 and   1c ( table i ) . however , inactivation of ba currents was   slightly faster for csk1 than for 1c . overall , these results suggest that ca - dependent inactivation was not   significantly altered by replacing the nh2 terminus of   1c with the corresponding region from 1s . the ca channel 1 subunit is highly homologous to   the  subunit of voltage - gated na channels , and the cytoplasmic linker between transmembrane domains iii   and iv ( the iii iv loop ) of na channels is a critical   structural determinant of fast inactivation ( vassilev et   al . , 1988 ; sthmer et al . , 1989 ) . to examine the possibility that the homologous iii  iv loop of ca channels is   involved in ca - dependent inactivation , we constructed   chimera csk8 in which the iii  iv loop and the first   half of ivs1 of 1c were replaced by the corresponding   region from 1s ( fig . 3 c , ca currents mediated by csk8 inactivated faster than ba currents and in general closely resembled currents produced by expression of 1c3del or csk1 . furthermore ,   the time constants for inactivation of currents mediated by csk8 were comparable to those obtained for   1c , 1c3del , and csk1 ( table i ) , suggesting that ca - dependent inactivation is not altered in csk8 . in both 1c and 1s , the i ii loop contains a number   of negatively charged aspartate and glutamate residues   that could potentially form a ca - coordination site or   sites . there are 25 negatively charged residues within   the i  ii loop of 1c and 19 such residues within the i  ii   loop of 1s . to examine the possibility that the extra   acidic residues within the i  ii loop of 1c might play a   functional role in ca - dependent inactivation , we expressed chimera csk2 in which the i  ii loop and most   of the iis1 segment of 1c were replaced by the corresponding region from 1s ( fig . 4 b. ca currents produced by csk2 inactivated significantly faster than ba currents , indicating the presence of ca - dependent inactivation . furthermore , when   relatively long test pulses ( 1.25 s ) were used , both ca   and ba currents exhibited two distinct components of   inactivation . ( 1997 )   for 1c , the fast component of ba current inactivation   likely represents an ion - dependent process because it   parallels ba influx , whereas the slow component of inactivation likely represents a voltage - dependent process   because it parallels the immobilization of gating charge . the presence of two components for inactivation of   currents mediated by csk2 is consistent with the thesis   of ferreira et al . ( 1997 ) that ba as well as ca can trigger   inactivation . in the present work , we have analyzed only   the faster time constants for inactivation of ca and ba   currents . the fast time constants for inactivation of ca currents mediated by csk2 exhibited a u - shaped dependence on test potential , whereas those for ba currents   progressively decreased with increasing test potential   ( fig . the time constants for inactivation of ca   currents were similar for csk2 and 1c currents of comparable densities ( table i ) . in marked contrast , the inactivation of ba currents mediated by csk2 was dramatically slowed ( fig . 4 a ) , with the fast time constants for inactivation being   approximately threefold larger than for 1c ( table i ) . if the fast phase of ba current inactivation primarily reflects an ion - dependent process as proposed by ferreira et al . ( 1997 ) , then the slower inactivation of csk2   may indicate that ba is less effective in triggering ion - dependent inactivation when the i  ii loop has skeletal   muscle as opposed to cardiac sequence . such an interpretation would imply that the i  ii loop of 1c has a   functional role in ca - dependent inactivation if ba- and   ca - dependent inactivation are equivalent . it is also possible that the process of voltage - dependent inactivation is affected somewhat by replacement of the i iii loop   of 1s performs a critical function in skeletal muscle type excitation  contraction coupling ( tanabe et al . iii loops of 1c and 1s also contain numerous ( 35 and 27 , respectively ) negatively   charged aspartate or glutamate residues that could potentially be involved in ca coordination ( fujita et al . ,   1993 ) . in addition , the ii  iii loop of 1s contains a consensus site for phosphorylation by pka ( tanabe et al . ,   1987 ) , whereas the ii  iii loop of 1c lacks predicted   pka sites ( mikami et al . , 1989 ) . to test whether the ii iii loop of 1c performs a unique function in ca - dependent inactivation , we expressed chimera csk3 in which   the ii  iii loop of 1c was replaced by its counterpart   from 1s ( fig . csk3 undergoes ca - dependent inactivation because ca currents inactivated much faster   than ba currents ( fig . the fast time constants for inactivation of ca currents exhibited a u - shaped dependence on test potential ; in contrast , such a relationship was not apparent   for ba currents ( fig . similar to the results obtained for csk2 , the fast component of ba current inactivation was slightly slower for csk3 than for 1c ( table   i ) , raising the possibility that the ii iii loop may perform a functional role in ca- or voltage - dependent inactivation . several previous studies have identified the cooh   terminus of 1c as an important structural determinant   of ca - dependent inactivation ( de leon et al . , 1995 ; , we expressed chimera   csk4 in which the distal 80% of the cooh terminus of   1c was replaced by the corresponding region from 1s   ( fig . in contrast to the other chimeras examined ,   ca and ba currents mediated by csk4 inactivated at   equivalent rates ( fig . 6 , a and b ) . both ca and ba currrents exhibited two distinct components of inactivation . however , there was no difference between the fast   time constants for inactivation of ca currents and those   for ba currents over a wide range of test potentials and   time constants for inactivation of ca currents did not   have a u - shaped dependence upon test potential ( fig . as shown in table i , the fast   time constants for inactivation of ca currents were   three- to fourfold larger for csk4 than for 1c . this   slow inactivation of ca currents can not be explained by   low expression of csk4 , because inactivation was also   slow compared with low density currents mediated by   1c ( table i ) . interestingly , the fast time constants for   inactivation of ba currents were not different between   csk4 and 1c , suggesting that the fast component of ba   current inactivation is unaltered in csk4 .   the results obtained with 1c-3del ( fig . 3 ) confirm   that a large segment of the distal cooh terminus is   not required for ca - dependent inactivation ( zong et   al . 6 ) suggest that the cooh terminus of 1s can somehow prevent ca - dependent inactivation . 7 ) reveals substantial conservation of their   sequences for 200 amino acids after the end of the   last predicted transmembrane segment ( ivs6 ) . beyond   this point , however , the cooh termini of 1c and 1s   diverge significantly . to test whether the distal , nonconserved portion of the cooh terminus from 1s   could be responsible for the inability of csk4 to undergo ca - dependent inactivation this construct is a truncation mutant of csk4 in which the final 211 amino acids of the   cooh terminus have been deleted , effectively removing the majority of the sequence that is not conserved   between 1c and 1s ( fig . csk5 undergoes ca - dependent inactivation , as evidenced by the significantly faster inactivation of ca   than of ba currents ( fig . two distinct components of inactivation were present in both   ca and ba currents mediated by csk5 . the fast time   constants for inactivation of ca currents exhibited a   u - shaped voltage dependence , whereas those for ba   currents did not ( fig . 8 d ) . at test potentials of + 10 ,   + 20 , and + 30 mv , the fast time constants for inactivation of ca currents were significantly smaller than for   ba currents ( fig . 8 d ) . the fast component of ca current inactivation was slower for csk5 than for 1c , suggesting that ca - dependent inactivation proceeds at a   slower rate than in 1c . as was found for csk4 , the fast   time constant for inactivation of ba currents was not   different between csk5 and 1c ( table i ) . the goal of the present study was to gain new insights   into the molecular mechanism of ca - dependent inactivation by identifying structural regions of 1c involved   in this phenomenon . toward this end , we compared   inactivation of ca and ba currents mediated by chimeras constructed between the cardiac 1c , which exhibits   prominent ca - dependent inactivation , and the skeletal   muscle 1s , which lacks this property ( donaldson and   beam , 1983 ; beam and knudson , 1988 ) . the protein sequence homology between 1c and 1s   is 66% , with the majority of the amino acid differences occurring within the major cytoplasmic domains   ( mikami et al . , 1989 ) . for example , the nh2 terminus   of 1c contains 154 amino acids , whereas that of 1s   contains only 50 amino acids . at the other extreme , the   iii  iv loops of 1c and 1s are highly conserved , both   being 53 amino acids in length with only 7 amino acid   differences . our results with chimeras csk1 and csk8   suggest that neither the nh2 terminus nor the iii  iv   loop of 1c performs an essential function in ca - dependent inactivation , because ca and ba currents mediated by these constructs are nearly identical to those   mediated by 1c ( fig . iv   loop is so highly conserved between 1c and 1s , this region may function interchangeably in ca - dependent inactivation . we have also found that inactivation of ba current is   dramatically slowed for chimera csk2 , in which the i ii   loop of 1c is replaced by the corresponding region   from 1s ( fig . 4 ; table i ) . this result is consistent with   the relative inactivation rates of 1c and 1s , when these   two different l - type ca channels are expressed in dysgenic myotubes ( tanabe et al . , 1990a ) . our results with   csk2 are also consistent with the finding of page et al . ii loop of the relatively fast inactivating 1e with   the i  ii loop from the more slowly inactivating 1b . it is   also interesting to compare our results for csk2 with   those of zhang et al . ( 1994 ) , who identified transmembrane segment is6 and its immediately flanking regions as important determinants of voltage - dependent   inactivation . a comparison of the i  ii loops of 1c and   1s reveals that most amino acid differences occur   within the cooh - terminal half , whereas the nh2-terminal half of the i  ii loop is comparatively well conserved ( mikami et al . , 1989 ) . the slowed inactivation of   csk2 may thus indicate an important role for the   cooh - terminal portion of the i  ii loop in ca channel   inactivation . however , because the nh2-terminal portion of the i  ii loop contains an interaction site for the   ca channel  subunit ( pragnell et al . , 1994 ) , and different  subunit isoforms can modulate the rate of ca   channel inactivation ( hullin et al . , 1992 ) , it is also possible that altered interactions between csk2 and the    subunit are partially responsible for its slower inactivation . no consensus sites for phosphorylation by pka or   pkc are present within the i  ii loop of either 1c or   1s ; thus , it seems unlikely that differential phosphorylation could account for the slower inactivation of csk2 . ca - dependent inactivation is usually defined as the   faster inactivation of ca than ba currents and by a   u - shaped voltage dependence of the time constants for   ca current inactivation . inactivation of ba currents is   usually assumed to proceed through a voltage - dependent process . however , ferreira et al . ( 1997 ) have recently demonstrated that ba can trigger the ion - dependent inactivation of 1c . they found that ba currents   inactivate with two distinct components , and that the   rate and extent of the fast component parallels ba influx , whereas the rate and extent of the slow component parallels immobilization of gating charge ( ferreira et al . , 1997 ) . if the fast component of ba current   inactivation measured in our experiments reflects an   ion - dependent process , then this process is significantly   slowed in chimera csk2 , and to a lesser extent in chimera csk3 . in this view , our results with csk2 suggest   that the i  ii loop of 1c may be an important structural   determinant of ion- rather than voltage - dependent inactivation . such inactivation could be triggered ( physiologically ) by ca or ( experimentally ) by ba binding to   the i  ii and ii  iii loops of 1c but not to the homologous regions of 1s . if this interpretation is correct ,   then the i  ii and ii  iii loops of 1c are structural determinants of ca - dependent inactivation . we have demonstrated that csk4 , a chimera in which   the cooh terminus of 1c has been replaced by the   corresponding region from 1s , lacks ca - dependent inactivation . this result is not an artifact stemming from   low channel expression because the current density in   cells expressing csk4 was not significantly different   from that in cells expressing csk2 or csk3 ( table i ) ,   which both displayed prominent ca - dependent inactivation . furthermore , ca - dependent inactivation was   absent even from relatively high density csk4 currents   ( not shown ) , whereas it was present in relatively low   density 1c , csk1 , csk2 , or csk3 currents ( e.g. , figs . 3   and 4 ) . the lack of ca - dependent inactivation by csk4   may explain why this property is not exhibited by the   skeletal muscle l - type ca channel . in this regard , it   would be interesting to know whether the property of   ca - dependent inactivation was gained or retained by   1c during the course of ca channel evolution . a recent report that the neuronal 1d ( an l - type ca channel ) also exhibits ca - dependent inactivation ( hans et   al . , 1997 ) suggests that this property has been retained   by 1c and 1d and lost by 1s . the mechanism of ca - dependent inactivation is not   known , but it has been proposed that a putative ef   hand motif located within the proximal cooh terminus of 1c functions as the essential ca - binding site responsible for triggering ca - dependent inactivation ( de   leon et al . , 1995 ) . however , recent evidence from   other laboratories suggests that the putative ef hand   motif is not important in the mechanism of ca - dependent inactivation . thus , transfer of the putative ef   hand from 1c into 1e fails to confer ca - dependent inactivation and , conversely , transfer of the ef hand from   1e into 1c fails to disrupt it ( zhou et al . , 1997 ) . additionally , ca - dependent inactivation is not abolished by   point mutations within 1c that eliminate the ca - coordination site from the putative ef hand motif but leave   the remainder of the cooh terminus intact ( zhou et   al . , 1997 ) . these results strongly suggest that the exact   site or sites of ca binding remain to be identified . because csk5 undergoes ca - dependent inactivation   ( fig . 8) , it is reasonable to suppose that it contains one   or more ca binding sites . it follows that csk4 contains   the same site or sites , because it encompasses the entire   sequence of csk5 ( fig . 6 ) , which leads to the conclusion that ca binding to the 1 subunit is only a prerequisite for ca - dependent inactivation and is by itself   insufficient . presumably , ca - dependent inactivation requires both ca binding and a subsequent conformational shift of the channel protein(s ) . perhaps the most significant result of the present   study is that ca - dependent inactivation was restored in   chimera csk5 by deletion of the nonconserved , distal   region of the cooh terminus present in csk4 ( figs . the cooh terminus of csk5 is similar in   length and composition to that of 1c3del ( fig . the   behavior of 1c3del clearly demonstrates that the most   distal 350 amino acids of the cooh terminus of 1c   are not required for ca channel inactivation ( fig . 3 a ;   zong et al . , 1994 ; de leon et al . , 1995 ) . in contrast , ca - dependent inactivation is conferred upon the 1e backbone by replacing a 134amino acid segment immediately downstream from the putative ef hand region   with the homologous 142amino acid segment from   1c ( zhou et al . , 1997 ) . furthermore , ca - dependent inactivation is profoundly influenced by splice variations   within the proximal cooh terminus of 1c immediately downstream from the putative ef hand region   ( soldatov et al . , 1997 ) . our results with csk5 suggests   that the proximal cooh termini of 1c and 1s ( which   are mostly conserved ) can function interchangeably in   ca - dependent inactivation . when considered altogether , our results and those of other studies indicate that the   proximal cooh terminus downstream from the putative ef hand region is an important structural determinant of ca - dependent inactivation . however , de leon   et al . ( 1995 ) showed that ca - dependent inactivation   was only partially conferred upon the neuronal 1e subunit by replacing its entire cooh terminus with 217   amino acids from the corresponding region of 1c . thus ,   while the proximal cooh terminus appears to be important for ca - dependent inactivation , the participation   of additional channel regions may also be required . our findings that csk4 lacks ca - dependent inactivation , whereas this property is restored in csk5 , suggests   that the distal cooh terminus of 1s ( which is not well   conserved between 1c and 1s ) can somehow block   ca - dependent inactivation . the mechanism by which   this block occurs is , at present , purely speculative . however , because ion channels appear to associate with   many other proteins in vivo ( sheng and kim , 1996 ) , it   seems plausible that csk4 might be tethered through   its distal cooh terminus to other proteins ( such as ryanodine receptors , the cytoskeleton , kinases , phosphatases , or other ion channels ) , and that such interactions might prevent the conformational shift underlying ca - dependent inactivation . ca - dependent inactivation of wild - type 1c ca channels coexpressed in hek293 cells   with skeletal muscle 2a and 1a subunits . ( a ) the compensated series resistance ( rs ) was 4.2 m. linear cell   capacitance ( c ) = 41 pf . ( c ) average i  v relations for   ca and ba currents mediated by 1c . each plotted   point represents the mean ( sem ) of 13 ( ca )   and 6 ( ba ) different cells . ( d ) time constants for   inactivation of ca or ba currents mediated by 1c .   currents were evoked by 250-ms test pulses and   were fit with either a single or double exponential   function . for currents requiring two exponentials , each plotted point represents the mean   ( sem ) of 214 ( ca ) and 37 ( ba ) different   cells . experiments summarized in this figure were   done using the teacl - based external solution . thinner lines denote regions having amino acid sequence   corresponding to the rabbit cardiac 1c subunit ( mikami et al . ,   1989 ) ; thicker lines denote regions corresponding to the rabbit   skeletal muscle 1s subunit ( tanabe et al . , 1987 ) . the amino acid   composition of 1c3del and construction of its cdna has been   previously described ; the final five amino acid residues from the   cooh terminus of 1c ( gvssl ) are retained in 1c3del ( zong   et al . , 1994 ) . the amino acid composition and cdna construction   of csk1 , csk2 , csk3 , and csk4 the compositions of csk5 and csk8 and the constructions of their cdnas are described in materials and methods . ( a ) representative   ca ( top ) and ba ( bottom ) currents mediated by   1c3del , a deletion mutant of 1c lacking amino   acid residues 18132166 from the cooh terminus . test pulses from 0 to + 40 mv ( ca ) and 10   to + 60 mv ( ba ) . rs = 3.8 m. ( b ) representative ca and ba   currents mediated by csk1 , a chimera in which   the nh2 terminus of 1c was replaced by the corresponding region from 1s . test pulses from 0 to   + 40 mv ( ca ) and 0 to + 60 mv ( ba ) . rs = 6.3 m.   ( c ) representative ca and ba currents mediated   by chimera csk8 , in which the iii  iv loop of 1c   was replaced by the corresponding region from   1s . test pulses from + 30 to + 50 mv ( ca ) and   + 20 to + 40 mv ( ba ) . rs = 4.9 m. experiments summarized in this figure were done using the teacl - based external solution . data from hek293 cells cotransfected with expression plasmids encoding 1 , 2a , and 1a ca channel subunits the external solution was based upon teac1 or nacl ( see materials and methods for compositions ) . time constants for inactivation were derived by   fitting maximal ca or ba currents ( i.e. , at the peak of the i  v relationship ) with one or two exponential functions . currents were evoked by test pulses of 250 or 500 ms , or 1.25 or 5 s , as dictated by the inactivation rate . mean  sem , with the number of   cells ( n ) in parentheses . csk2 retains ca - dependent inactivation , but ba current inactivation is dramatically   slowed . test pulses from 10 to + 60   mv ( ca ) and 10 to + 50 mv ( ba ) . files   97428010 , 97428014 . rs = 2.2 m. ( b )   representative ca and ba currents mediated by   1c . test pulses from 10 to + 40 mv ( ca ) and   10 to + 50 mv ( ba ) . rs = 2.3 m. ( c ) i  v relations for   csk2 currents . plotted points represent the mean   ( sem ) of 410 different cells . same cell as   in a. ( c ) i  v relations for csk3 currents . plotted   points represent the mean ( sem ) of three to   eight different cells . experiments summarized in this   figure were done using the nacl - based external   solution . ( a ) representative ca currents mediated by   csk4 , in which the distal 80% of the cooh terminus of 1c has been replaced by the corresponding region from 1s . file 97501003 . same cell as in   a. for the illustrated currents , inactivation appears to be slightly slower for ba than for ca currents , but this is only apparent : the ba currents   are larger and therefore less contaminated by outward currents . ( d ) voltage dependence of   fast time constants for inactivation of csk4 currents . experiments summarized in   this figure were done using the nacl - based external solution . sequence alignment of the cooh - terminal regions of the rabbit cardiac 1c ( mikami et al . , 1989 ) and rabbit skeletal muscle 1s   subunits ( tanabe et al . , 1987 ) . the sequences begin just before the last predicted transmembrane   segment ( s6 ) of domain iv ( first bracket ) . the putative ef hand motif is indicated ( second bracket ) ,   as is the junctional site within csk4 and csk5   where the sequence changes from 1c to 1s   ( white arrow ) . the termination of mutant   1c3del is indicated by a filled triangle ; this construct retains the final five residues ( gvssl ) of   the wild - type cooh terminus . shaded amino acids are identical between 1c   and 1s ; dashes indicate gaps in the alignment . ( a ) representative ca currents mediated   by csk5 , which is identical to csk4 except that   the nonconserved region ( 211 amino acids ) has   been deleted from the end of the cooh terminus . rs = 3.0 m. ( b )   representative ba currents mediated by csk5 .   file 97522021 . same cell as in a. ( c ) i  v relations   for csk5 currents . ( d )   voltage dependence of fast time constants for inactivation of csk5 currents . each plotted point   represents the mean ( sem ) of five to six different cells . experiments summarized in this figure   were done using the nacl - based external solution .
we investigated the molecular basis for ca - dependent inactivation of the cardiac l - type ca channel . 
 transfection of hek293 cells with the wild - type 1c or its 3 deletion mutant ( 1c3del ) produced channels that   exhibited prominent ca - dependent inactivation . to identify structural regions of 1c involved in this process , we   analyzed chimeric 1 subunits in which one of the major intracellular domains of 1c was replaced by the corresponding region from the skeletal muscle 1s subunit ( which lacks ca - dependent inactivation ) . replacing the   nh2 terminus or the iii 
 iv loop of 1c with its counterpart from 1s had no appreciable effect on ca channel inactivation . 
 in contrast , replacing the i  ii loop of 1c with the corresponding region from 1s dramatically slowed   the inactivation of ba currents while preserving ca - dependent inactivation . 
 a similar but less pronounced result   was obtained with a ii 
 iii loop chimera . 
 these results suggest that the i  ii and ii  iii loops of 1c may participate   in the mechanism of ca - dependent inactivation . replacing the final 80% of the cooh terminus of 1c with the   corresponding region from 1s completely eliminated ca - dependent inactivation without affecting inactivation of   ba currents . 
 significantly , ca - dependent inactivation was restored to this chimera by deleting a nonconserved ,   211amino acid segment from the end of the cooh terminus . 
 these results suggest that the distal cooh terminus of 1s can block ca - dependent inactivation , possibly by interacting with other proteins or other regions of the   ca channel . 
 our findings suggest that structural determinants of ca - dependent inactivation are distributed   among several major cytoplasmic domains of 1c .