|
|
|
|
|
|
|
||
Original Article |
1C) by Dihydropyridine Agonist and Strong Depolarization Occur via Distinct Mechanisms
kbeam{at}lamar.colostate.edu
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1C) are linked. We found that the mutant L-type channel GFP-1C(TQ
YM), bearing the mutations T1066Y and Q1070M, was able to undergo depolarization-induced potentiation but not potentiation by agonist. Conversely, the chimeric channel GFP-CACC was potentiated by agonist but not by strong depolarization. These data indicate that the mechanisms of agonist- and depolarization-induced potentiation of 1C are distinct. Since neither GFP-CACC nor GFP-CCAA was potentiated significantly by depolarization, no single repeat of 1C appears to be responsible for depolarization-induced potentiation. Surprisingly, GFP-CACC displayed a low estimated open probability similar to that of the 1C, but could not support depolarization-induced potentiation, demonstrating that a relatively low open probability alone is not sufficient for depolarization-induced potentiation to occur. Thus, depolarization-induced potentiation may be a global channel property requiring participation from all four homologous repeats.
Key Words: ion channel modulation • facilitation • muscle
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Hoshi and Smith 1987; Tsien et al. 1988; Tanabe et al. 1993). Thus, modification of Ca2+ influx provides an important mechanism for the regulation of many downstream Ca2+-dependent responses. One source of modification is potentiation, whereby a channel is stabilized in the open state, and intracellular Ca2+ levels are increased as a result.
L-type Ca2+ channels show a shift in gating mode in response to either strong depolarization or 1,4-dihydropyridine (DHP) agonist. After strong depolarization, the channel enters a state of higher open probability (Po) and long open times that can be detected by a number of different pulse paradigms. For example, after a strong, conditioning depolarization (e.g., +120 mV) followed by a 50–150-ms return to the holding potential, a subsequent, moderate depolarization elicits a Ca2+ channel current that is about twofold larger than that measured without the conditioning depolarization (Bourinet et al. 1994; Cens et al. 1996, Cens et al. 1998). This effect, which implies an alteration of gating that persists after channel closing, will be designated "depolarization-induced facilitation." Both L- and non–L-type channels are also able to undergo another form of prepulse facilitation ("Ca2+/CAM-dependent facilitation"; Lee et al. 2000; Zühlke et al. 2000; DeMaria et al. 2001), which differs from depolarization-induced facilitation in that it has a bell-shaped dependence on the prepulse potential that arises from a primary dependence upon Ca2+ entry. Ca2+/CAM-dependent facilitation appears to depend specifically (Lee et al. 2000) upon the β2a subunit, and does not occur in cells expressing β1, the isoform present in the dysgenic myotubes used in our study. Unlike Ca2+/CAM-dependent facilitation, depolarization-induced facilitation may be related to another form of altered gating observed when a strong depolarization is followed immediately by repolarization to an intermediate potential (Hoshi and Smith 1987; Pietrobon and Hess 1990; Kleppisch et al. 1994). This phenomenon, referred to here as depolarization-induced potentiation, results in a mode of gating characterized at the single-channel level by high Po and long open times, which is also referred to as "mode 2" gating (Pietrobon and Hess 1990). As an indication of depolarization-induced entry into mode 2, we have measured whole-cell tail currents upon repolarization to –50 mV immediately after a strong, conditioning depolarization. With this protocol, high Po and long open times are reflected by an increased tail-current amplitude and slower rate of tail-current decay, respectively. Mode 2 gating of L-type channels is also promoted by DHP agonists (Hess et al. 1984; Nowycky et al. 1985; Hoshi and Smith 1987). Given the similarities between agonist- and depolarization-induced potentiation, it is important to know the degree to which the two processes are related.
The cardiac L-type channel, 1C, normally exhibits a low Po < 0.05 (Cachelin et al. 1983; Lew et al. 1991) and can be potentiated by both strong depolarization and DHP agonists. By contrast, the neuronal non–L-type channel, 1A, exhibits a high Po of 
0.6 (Llinas et al. 1989) and lacks both depolarization-induced facilitation (Bourinet et al. 1994), and DHP-induced potentiation (Sather et al. 1993). These observations suggest the possibility that depolarization- and agonist-induced potentiation can only occur in channels like 1C that have an intrinsically low Po.
In an attempt to determine whether potentiation of 1C by DHP agonist and strong depolarization occurs via a common pathway, we have characterized wild-type and mutant 1C channels and chimeric channels composed of 1C and 1A sequence. The channels were fused at their amino termini to green fluorescent protein (GFP), expressed in dysgenic myotubes and examined using whole-cell patch clamp. For an 1C in which the agonist binding site was mutated (GFP-1C(TQYM)), 10 µM Bay K 8644 had no significant effect on whole-cell currents, whereas depolarization-induced potentiation remained intact. A chimeric channel containing repeat II and the I-II linker of 1A sequence embedded in L-type background (GFP-CACC) could not support depolarization-induced potentiation but was potentiated by DHP agonist. Channels containing three (CACC), two (CCAA), or no (1A) repeats of L-type sequence were not potentiated significantly by depolarization, suggesting that depolarization-induced potentiation cannot be localized to any single channel repeat. Interestingly, despite having a relatively low estimated Po comparable to that of 1C, GFP-CACC was not potentiated by depolarization, indicating that depolarization-induced potentiation must not be dependent solely on a low Po. Our results demonstrate that the mechanisms of DHP agonist- and depolarization-induced potentiation of 1C are distinct, and that depolarization-induced potentiation may be a global channel property requiring the participation of all four homology repeats.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 cDNAs1A (Mori et al. 1991) or 1C (Mikami et al. 1989), respectively. An asterisk indicates a restriction site introduced by PCR. The wild-type clones GFP-1C and GFP-1A were produced by fusing the 1 subunit of either the cardiac L-type channel (1C) or neuronal P/Q-type channel (1A) at the amino terminus to GFP as previously described (Grabner et al. 1998). The GFP tag has been shown not to alter any of the functional properties of the 1C and 1A subunits (Grabner et al. 1998). The clone GFP-1C(TQYM) was created using overlapping PCR mutagenesis (Horton et al. 1989) to replace two residues in the IIIS5 transmembrane domain of 1C (Thr 1066 and Gln 1070), which were previously identified as essential components of the DHP binding site (Mitterdorfer et al. 1996; He et al. 1997), with the corresponding residues of 1A (Tyr 1393 and Met 1397). In brief, the SalI*-FspI fragment of 1C (nt –12C–5054C) was subcloned into the SalI and SmaI sites of the pSP72 (Promega) polylinker. Overlapping PCR using GFP-1C as the template yielded an 816-bp amplification product (nt 2638C–3453C) carrying the point mutations, which was then cut with AflII (nt 2689) and AspI (nt 3385) and ligated into the AflII-AspI restriction sites of the pSP72 subclone. Finally, the SalI*-EcoRV fragment (nt –12C–4348C) of the subclone was ligated into GFP-1C at the corresponding restriction sites to yield the clone GFP-1C(TQYM). The 1C/1A chimera GFP-CACC consisted of repeat II and the I-II linker of 1A contained an 1C background (amino acids 1–426C/352–671A/740–2171C). To produce GFP-CACC, the SalI*-EcoRV fragment of 1C (nt –12C–4348C) was ligated into the corresponding sites of the plasmid pSP72. PCR mutagenesis was used to amplify a 980-bp product of 1A sequence, containing the introduced 5' BamHI* and 3' EcoRI* restriction sites at nt 1039 and 2015, respectively. The amplification product was first digested with BamHI, and then partially digested with EcoRI (to avoid cutting an internal EcoRI site), and the resulting fragment was ligated into the corresponding restriction sites of the pSP72/cardiac subclone. Finally, the SalI*-EcoRV fragment from this subclone was ligated into the SalI*-EcoRV restriction sites of GFP-1C. The chimera GFP-CCAA was composed of repeats I and II (including the II-III linker) of 1C and repeats III and IV of 1A (amino acids 1-920C/1244-2424A). To create GFP-CCAA, the HindIII*-PvuII fragment (nt 3730A–4216A) of chimera AL2 (Grabner et al. 1996) was coligated with the PvuII-BglII fragment of 1A (nt 4216A–5891A) into the corresponding restriction sites of the plasmid pSP72. Subsequently, the XhoI-HindIII* fragment (nt 1395A–2758C) of clone AL5 (Grabner et al. 1996) was ligated into the corresponding restriction sites of the subclone. The entire XhoI-BglII insert (nt 1395A–5891A) of the final subclone was ligated into the corresponding restriction sites of GFP-1A to produce the intermediate clone GFP-ALC. The SalI*-AvrII fragment (nt –12C–759C) of GFP-1C was coligated with the AvrII-AocI fragment (nt 759C–1752A) of AL5 into the SalI-AocI (5' polylinker-1752A) restriction sites of GFP-ALC, yielding the subclone GFP-C/3A. Finally, the ClaI-AflII fragment (nt 256C–2689C) of 1C was ligated into the corresponding restriction sites of subclone GFP-C/3A to yield the final chimera GFP-CCAA. The integrity of all channel constructs was confirmed using automated sequence analysis (Macromolecular Resources).
Expression and Electrophysiological Analysis of Channels in Dysgenic Myotubes
1 wk after plating, primary cultures of mouse dysgenic myotubes (Adams and Beam 1989), which lack an endogenous 1S subunit (Knudson et al. 1989), were microinjected in single nuclei with cDNAs (200–600 ng/µl) encoding GFP-tagged 1 subunits. 36–52 h after injection, expressing myotubes were identified by green fluorescence and used for electrophysiology. Macroscopic Ca2+ currents were measured using the whole-cell patch-clamp method (Hamill et al. 1981). Whole-cell patch pipettes of borosilicate glass had resistances of 1.5–2.0 M
when filled with an internal solution containing 140 mM cesium aspartate, 10 mM Cs2EGTA, 5 mM MgCl2, and 10 mM HEPES, pH 7.4 with CsOH. The external bath solution contained 10 mM CaCl2, 145 mM TEA-Cl, and 10 mM HEPES, pH 7.4 with TEA-OH, plus 3 µM tetrodotoxin. Test currents were obtained by stepping from a holding potential of –80 to –30 mV for 1 s (to inactivate endogenous T-type Ca2+ current; Adams et al. 1990), to –50 mV for 30–50 ms, to the test potential for 200 ms, to –50 mV for 125 ms, and back to –80 mV. Test currents were corrected for linear components of leak and capacitative currents by digitally scaling and subtracting the average of 10 preceding control currents elicited by hyperpolarizing steps (20–40 mV in amplitude) applied from the holding potential. Data were included only for cells in which the maximum voltage error (calculated by the product of peak inward current and compensated series resistance) was 
10 mV. Except for tail currents, data were sampled at 1 kHz. Tail currents, elicited by repolarizing to –50 mV for 125 ms, were recorded with fast sampling (10 kHz). Tail-current amplitude (Itail) was measured 0.5 ms after the onset of the repolarization from the test pulse to –50 mV. The rate of tail-current decay (
deact) was measured by fitting tail currents with a single exponential function. Maximal Ca2+ conductance (Gmax) and half-maximal activation potential (V1/2) were calculated by fitting peak inward current values with the equation:
 | (1) |
|
and 
according to the equation:
 | (2) |
= - average dysgenic charge (2.5 nC/µF; Adams et al. 1990), 
is the single-channel conductance in 10 mM Ca2+, assumed to be 4 pS for 1A (Adams et al. 1994), 5.8 pS for 1C (Gollasch et al. 1992), or an average of the two (4.9 pS) for chimeras CACC and CCAA, q is the assumed single-channel gating charge (9 e–; Noceti et al. 1996), F is Faraday's constant (96,487 C/mol) and A is Avogadro's number (6.023 x 1023 e–/mol).
Several different measures were used to quantify potentiation. For the DHP agonist (±)Bay K 8644, one measure was the ratio 
, where the numerator and denominator represent the peak currents elicited by depolarizing test pulses in the presence or absence of drug, respectively. 
was usually elicited by a Vtest of approximately +20–30 mV, and 
for a Vtest 20–30 mV more hyperpolarized. Agonist-induced potentiation was also measured by means of the ratios 
and 
, where the tail current was produced by repolarizing to –50 mV from a Vtest of +40 mV. Depolarization-induced potentiation was quantified by the ratios 
and 
, where the numerator and denominator were determined from tail currents produced by repolarization to –50 mV after a Vtest of +90–110 mV or +40 mV, respectively.
Statistical Analysis
Statistical significance was assessed using one-way analysis of variance (ANOVA) and SAS software (version 8). All data are presented as mean ± SEM.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1C Is Potentiated by DHP Agonist and Strong Depolarization while 1A Is Potentiated by Neither1C or GFP-1A to the indicated potentials, followed by repolarization to –50 mV. Based upon steady-state activation calculated from peak currents during the test depolarizations (Fig. 1 B), both channels were fully activated by test pulses to +40 mV and above. Consistent with this, the tail currents for GFP-1A had a similar amplitude and time course after the depolarizations to either +40 or +60 mV (Fig. 1 A). However, for GFP-1C, the tail current after the +60-mV depolarization was larger and decayed more slowly than the tail after the +40-mV step. This behavior is an indication that strong depolarization caused 1C channels to enter a mode of gating having longer open times and increased Po.
Fig. 2 compares currents produced by GFP-1C and GFP-1A before and after exposure to 10 µM Bay K 8644. For GFP-1A, application of Bay K 8644 had little effect on either the current elicited by a test depolarization to +20 mV or on the tail current after repolarization (Fig. 2 A). Likewise, the average, peak current versus voltage relationship for GFP-1A was not significantly (P > 0.1) affected by the agonist (Fig. 2 B). By contrast, Bay K 8644 caused a hyperpolarizing shift in the test potential evoking maximum inward current for GFP-1C, together with a substantial increase in the magnitude of this current. In addition to affecting the peak current, Bay K 8644 also caused an approximately threefold increase in tail-current amplitude (Itail) and in the time constant of tail-current deactivation (deact) for GFP-1C (Fig. 2 A and Table ). Overall, the effects of Bay K 8644 on GFP-1C tail currents qualitatively resemble those of strong depolarization (compare Fig. 1 A and 2 A).
|
|
1C or GFP-1A. Fig. 3 A shows the standard protocol for quantifying depolarization-induced potentiation, which was determined as the ratio of either Itail or deact for a tail current after a Vtest of +90 mV, to the corresponding values for a tail current after a Vtest of +40 mV. By both measures, GFP-1C showed substantial depolarization-induced potentiation, whereas GFP-1A did not (Fig. 3 A and Table ). Fig. 3 B illustrates the dependence of Itail on prior test potentials ranging from –40 to +80 mV. For GFP-1A, Itail reached a maximum after a Vtest of +30 mV, in good agreement with the conductance versus voltage curve calculated from peak currents (Fig. 1 B). For still stronger depolarizations, Itail became smaller for GFP-1A, as expected for a channel undergoing voltage-dependent inactivation that becomes faster with stronger depolarization. In contrast to GFP-1A, Itail for GFP-1C increased monotonically over the entire range of test potentials. This monotonic increase differs from the saturating conductance versus voltage relationship (Fig. 1 B) and is consistent with entry into a potentiated state having high Po. This monotonic voltage dependence also suggests that depolarization-induced potentiation is not dependent upon Ca2+ entry during the prepulse. The application of 10 µM Bay K 8644 caused a still further increase in Po, which is indicated by a substantial increase in Itail for GFP-1C at any given test potential (Fig. 3 B). In the presence of agonist, Itail was still increased by stronger test depolarizations, up to at least +70 mV. Table summarizes the effects of DHP agonist and strong depolarization on tail currents for GFP-1C and GFP-1A.
|
1C Eliminates Agonist- but Not Depolarization-induced Potentiation1C is potentiated by both agonist and strong depolarization, whereas GFP-1A is potentiated by neither, raises the possibility that agonist- and depolarization-induced potentiation are linked. As one test of this hypothesis, we created GFP-1C(TQYM), in which two residues of IIIS5 that are critical for the DHP sensitivity of 1C (Mitterdorfer et al. 1996; He et al. 1997) were converted to the corresponding residues of 1A. Currents produced by GFP-1C(TQYM) were not affected by the addition of 10 µM Bay K 8644 (Fig. 4 A), whereas depolarization-induced potentiation was intact (Fig. 4 B). In particular, tail currents were larger and decayed more slowly after a Vtest of +90 mV compared with a Vtest of +40 mV (Fig. 4 B, top) and the tail-current amplitude increased monotonically as a function of test potential (Fig. 4 B, bottom). On average, GFP-1C(TQYM) was quantitatively similar to GFP-1C with respect to depolarization-induced potentiation, but was indistinguishable from GFP-1A in the lack of agonist-induced potentiation (Table ). Interestingly, mutation of T1066 and Q1070 in the IIIS5 transmembrane segment of 1C resulted in a decreased steepness, and positive shift, of the steady-state activation curve in comparison to GFP-1C (Fig. 4 C), indicating that these residues can affect activation gating. Taken together, the data of Fig. 4 demonstrate that depolarization-induced potentiation still occurs in a mutant 1C lacking a response to DHP agonist.
|
1C is sufficient to allow depolarization-induced potentiation, we constructed the chimeras GFP-CACC and GFP-CCAA and tested them for agonist- and depolarization-induced potentiation (Fig. 5). GFP-CACC consists of repeat II and the I-II linker of 1A in an otherwise 1C background (Fig. 5A and Fig. C), and thus contains an intact DHP agonist binding site (Grabner et al. 1996; Hockerman et al. 1997; Ito et al. 1997; Sinnegger et al. 1997). As shown in Fig. 5 A, 10 µM Bay K 8644 potentiated maximum inward current in cells expressing GFP-CACC and caused a leftward shift in the peak current versus voltage relationship. Quantitatively, both effects were similar to those of 10 µM Bay K 8644 on GFP-1C (Table ). However, this chimera failed to show the large depolarization-induced potentiation characteristic of either GFP-1C or GFP-1C(TQYM) (Table ; also, compare Fig. 5 C with Fig. 3 B and 4 B). Thus agonist-induced potentiation can be present in a channel construct that lacks significant depolarization-induced potentiation. The chimera GFP-CCAA (Fig. 5B and Fig. D) consists of the first two repeats and the II-III linker of 1C fused to repeats III and IV of 1A. GFP-CCAA lacked both agonist- and depolarization-induced potentiation (Fig. 5B and Fig. D, and Table ). Fig. 6 summarizes the effects of strong depolarization and DHP agonist on the constructs GFP-1C, GFP-1C(TQYM), GFP-CACC, and GFP-1A. The asterisks indicate a significant (P < 0.05) difference from onefold (where onefold indicates a lack of potentiation). Fig. 6 demonstrates that depolarization-induced potentiation can persist in the absence of potentiation by agonist (i.e., GFP-1C(TQYM)), and potentiation by agonist can occur in the absence of depolarization-induced potentiation (i.e., GFP-CACC); therefore, the two processes likely occur via distinct mechanisms.
|
|
1C Is Sufficient for Depolarization-induced Potentiationdeact (B), the chimeras GFP-CACC and GFP-CCAA, like 1A, lacked depolarization-induced potentiation. Because GFP-CACC lacked depolarization-induced potentiation, none of the three cardiac repeats contained in this construct (i.e., I, III, and IV) appears to be sufficient, individually or in concert, to mediate this process. In addition, because GFP-CCAA contains a cardiac repeat II and lacked depolarization-induced potentiation, a cardiac repeat II does not appear to be sufficient, either alone or in combination with repeat I. In conclusion, no single repeat of 1C seems to be sufficient for depolarization-induced potentiation, which may instead represent a more global property. Several combinations of multiple repeats are tested by the chimeras examined in this paper, and others cannot be tested because not all possible combinations of repeats of L-type and non–L-type sequence produce functional channels (Grabner et al. 1996; Spaetgens and Zamponi 1999).
|
(MATERIALS AND METHODS) to estimate Po for GFP-1C, GFP-CACC, GFP-CCAA, and GFP-1A from measured values of Gmax and Qmax. The values estimated by this approach for both GFP-1C and GFP-1A (Table ) are in reasonable agreement with values determined from single-channel measurements for 1C (<0.05; Cachelin et al. 1983; Lew et al. 1991) or 1A (0.6; Llinas et al. 1989). Moreover, as shown in Fig. 7 C, the estimated Po for GFP-1A was about 30-fold higher than for GFP-1C. The estimated Po for GFP-CCAA was similar to that of GFP-1A, but estimated Po for GFP-CACC was much closer to that of GFP-1C (Fig. 7 C and Table ). The absence of depolarization-induced potentiation for GFP-CACC indicates that a low Po alone is not a sufficient condition for this process to occur. The lack of depolarization-induced potentiation for GFP-CACC is even more striking given that this construct can be strongly potentiated by agonist.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1C) and neuronal (1A) 1 subunits in dysgenic myotubes. For GFP-1C, both strong depolarization and agonist (10 µM Bay K 8644) caused tail currents to become larger and to decay more slowly, whereas tail currents for GFP-1A were not affected by either manipulation. Introduction of two point mutations (T1066Y and Q1070M) into GFP-1C abolished potentiation by agonist without any evident effect on potentiation by depolarization. Conversely, agonist but not depolarization caused potentiation of a chimera of 1C and 1A (GFP-CACC). Because depolarization-induced potentiation was absent for both GFP-CACC and the chimera GFP-CCAA, it appears that no single repeat of 1C can be responsible for this process. GFP-CACC displayed a relatively low estimated Po, quite similar to that of GFP-1C, whereas the estimated Po for both GFP-CCAA and GFP-1A was much higher. Therefore, a channel that displays a low Po (and is potentiated by agonist) can fail to be potentiated by depolarization.
Independent Pathways for Potentiation by DHP Agonist and Depolarization
Unitary records of L-type Ca2+ channels have been described as having three modes of gating upon depolarization: mode 0 (null sweeps); mode 1 characterized by brief openings (<1 ms) in bursts; and mode 2 defined by longer openings and high Po (Hess et al. 1984). Mode 1 is the predominant mode accessed during moderate depolarizations from the holding potential in the absence of DHP agonist, whereas mode 2 is promoted by the presence of agonist (Hess et al. 1984). Strong depolarization also promotes long openings of L-type channels in both chromaffin (Hoshi and Smith 1987) and cardiac cells (Pietrobon and Hess 1990). Because we have found that potentiation by either agonist or depolarization can be eliminated without a quantitative reduction in the effect of the other, it appears that these two processes occur via distinct pathways. In addition, we have used a concentration of agonist (10 µM Bay K 8644) that is supramaximal (Kokubun and Reuter 1984); therefore, the additional potentiation of tail currents by depolarization in the presence of the agonist also strongly suggests the presence of two independent pathways leading to a potentiated open state. Several other labs have likewise concluded from the additivity of the effects of depolarization and agonist, that these two stimuli cause an increased Po by distinct pathways (Bourinet et al. 1994; Parri and Lansman 1996). Moreover, single-channel measurements show both different open times and first latencies depending on whether potentiation is induced by depolarization or agonist (Hoshi and Smith 1987). In combination, these data suggest not only that mode 2 gating can be accessed by multiple pathways, but also that mode 2 consists of more than one potentiated open state.
Bay K 8644 is well-known to shift activation in the hyperpolarizing direction (Fig. 2; Hess et al. 1984; Sanguinetti et al. 1986), indicating that it shifts equilibrium towards the open state of the channel. We have shown here that mutation of residues T1066 and Q1070 in the IIIS5 transmembrane domain of 1C not only ablates the response to agonist, but also shifts the voltage dependence of activation oppositely, in the depolarizing direction. On this basis, one could hypothesize that Bay K 8644 promotes a conformation of these two residues that stabilizes open states of the channel, and mutation of these residues destabilizes this conformation.
Role of Accessory Subunits and of Phosphorylation in Depolarization-induced Potentiation
The accessory β subunit has been shown to influence modal gating of 1C. In particular, comparison of 1C expressed with or without the β2a subunit showed that β2a increased both open times and the proportion of long openings (Costantin et al. 1998). β subunits also have been reported to affect depolarization-induced facilitation, which may be mechanistically related to depolarization-induced potentiation (INTRODUCTION). Specifically, depolarization-induced facilitation was found to occur when 1C was coexpressed with the β1, β3, or β4 subunits (Bourinet et al. 1994; Cens et al. 1998), but not with β2a (Cens et al. 1996), raising the possibility that the β subunit plays a direct role in depolarization-induced facilitation of 1C, and perhaps in potentiation as well. However, others have found that depolarization-induced potentiation of the smooth muscle 1C occurs in the absence of any β subunit (Kleppisch et al. 1994). Whatever the exact role of the β subunit, our results demonstrate that depolarization-induced potentiation is strongly influenced by the 1 subunit itself, because all of the 1 constructs examined in this study have a conserved "alpha interaction domain" (site of β subunit binding; Pragnell et al. 1994) and were expressed with a common β subunit (β1a, which is endogenous to skeletal muscle; Ruth et al. 1989).
Evidence has been presented that PKA-dependent phosphorylation occurring during depolarizing prepulses is necessary for depolarization-induced facilitation of 1S (Sculptoreanu et al. 1993b; Johnson et al. 1994), the cardiac 1C (Sculptoreanu et al. 1993a), and the neuronal 1C (Sculptoreanu et al. 1995). Evidence also has been presented that phosphorylation during depolarization is not involved in depolarization-induced facilitation of the neuronal 1C, although basal phosphorylation may be required (Bourinet et al. 1994). If phosphorylation is required (either basal or voltage-dependent), then it seems unlikely to involve phosphorylation of 1C directly because truncation of the consensus PKA sites (Gao et al. 1997) of the 1C carboxyl tail does not eliminate depolarization-induced facilitation (Cens et al. 1998). Consistent with this result, we found that depolarization-induced potentiation does not occur for GFP-CACC even though it contains all the consensus PKA sites of 1C.
Structural Determinants of Depolarization-induced Potentiation and Low Po
As discussed above, brief openings predominate during activation of 1C by modest depolarizations applied from a negative holding potential (mode 1 gating). The conformational changes responsible for activation of these brief openings occur rapidly (macroscopic activation occurs with a time constant of several ms at +30 mV; Tanabe et al. 1991). Depolarization-induced entry into mode 2 occurs on a significantly slower time scale (with a time constant of several hundred ms at +30 mV) and over a much more positive voltage range (Pietrobon and Hess 1990). Despite these differences, depolarization-induced potentiation resembles mode 1 activation in being strongly voltage-dependent: based on two-state Boltzmann fits, the effective gating charge is 2.5 for depolarization-induced potentiation and 3.2 for mode 1 activation (Pietrobon and Hess 1990). Thus, the question arises as to the identity of the voltage sensor for depolarization-induced potentiation. One possibility is that, after undergoing the relatively rapid movements leading to mode 1 openings, the S4 segments can undergo subsequent, slower movements in response to still stronger depolarization. It is equally possible that structures other than S4 serve as voltage sensors for depolarization-induced potentiation. Because we found that neither GFP-CACC nor GFP-CCAA undergo depolarization-induced potentiation, it seems unlikely that the voltage-sensing structures for depolarization-induced potentiation are localized within a single repeat. Rather, depolarization-induced potentiation of 1C appears to require large movements of charge distributed throughout the protein.
L-type channels like 1S and 1C differ from 1A channels in that the L-type channels display agonist- and depolarization-induced potentiation, and also have a much lower Po, raising the possibility that the structural determinants of potentiation and low Po reside in similar structures. However, the chimera GFP-CACC had a relatively low Po, yet did not display significant depolarization-induced potentiation. Because the chimera GFP-CCAA displayed a high Po, the amino-terminal half of 1C (repeats I and II) does not appear to be an important determinant of low Po; instead, structural requirements for low Po may reside in the carboxyl half of the protein. Certainly, it is attractive to hypothesize that repeats III and IV are important for the intrinsic, low Po of L-type channels since these same two repeats play an essential role in agonist binding, which increases Po. A role for the carboxyl tail in determining Po is suggested by previous work showing that Po of 1C is markedly increased by partial truncation of the carboxyl tail (Wei et al. 1994).
As stated earlier, the Po of the L-type channels containing 1C (<0.05; Cachelin et al. 1983; Lew et al. 1991) is much lower than that of the neuronal channels containing 1A (0.6; Llinas et al. 1989) or 1B (0.5; Delcour and Tsien 1993). Because single-channel conductance varies less than twofold amongst these channels ([1C] Kokobun and Reuter, 1984; [1A] Zhang et al. 1993; [1B] Rittenhouse and Hess 1994), the production of an equivalent macroscopic current would require a much higher density of the L-type channels. A primary role of L-type Ca2+ channels in muscle is to regulate Ca2+ movements through ryanodine receptors. For this control to be relatively tight, it may be useful to have an 1:1 correspondence between the plasmalemmal L-type channels and the intracellular ryanodine receptors. Perhaps this correspondence is best served by a relatively high density of low Po channels. Conversely, a high Po and relatively low channel density would be advantageous when it is critical that a cellular response be triggered by the activation of only a few channels. Important goals for future work will be to better define the structures determining the differences in Po between 1C and neuronal channels like 1A and 1B, and to identify the conformational rearrangements that occur during potentiation of L-type channels.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by the National Institutes of Health grant NS24444 to K.G. Beam, an NIH predoctoral fellowship MH12512 to C.M. Wilkens, and by the Fonds zur Förderung der Wissenschaftlichen Forschung, Austria (J01242-GEN) to M. Grabner.
Submitted: 26 June 2001
Revised: 24 September 2001
Accepted: 26 September 2001
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1C L-type calcium channel, EMBO J., 13, 1994, 5032–5039.[Medline]1 subunit, J. Biol. Chem., 272, 1997, 18759–18765.1 subunit Ca2+ channels expressed in CHO cells, EMBO J., 13, 1994, 2502–2507.[Medline]1 subunit of the dihydropyridine receptor in mice with muscular dysgenesis, J. Biol. Chem., 264, 1989, 1345–1348.1 subunit, Nature., 368, 1994, 67–70.[Medline]1 subunits, Neuron., 11, 1993, 291–303.[Medline]1 subunits due to phosphorylation by cAMP-dependent protein kinase, Proc. Natl. Acad. Sci. USA., 90, 1993, 10135–10139a.1A subunitrole of L-type Met-1188, J. Biol. Chem., 272, 1997, 27686–27693.1E Ca2+channels, J. Biol. Chem., 274, 1999, 22428–22436.1 subunit, J. Biol. Chem., 269, 1994, 1635–1640.1C subunit, J. Biol. Chem, 275, 2000, 21121–21129.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  R. A. Bannister, I. N. Pessah, and K. G. Beam The Skeletal L-type Ca2+ Current Is a Major Contributor to Excitation-coupled Ca2+ entry J. Gen. Physiol., December 29, 2008; 133(1): 79 - 91. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Bannister, M. Grabner, and K. G. Beam The {alpha}1S III-IV Loop Influences 1,4-Dihydropyridine Receptor Gating but Is Not Directly Involved in Excitation-Contraction Coupling Interactions with the Type 1 Ryanodine Receptor J. Biol. Chem., August 22, 2008; 283(34): 23217 - 23223. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Tavalin, D. Shepherd, R. K. Cloues, S. E. H. Bowden, and N. V. Marrion Modulation of Single Channels Underlying Hippocampal L-Type Current Enhancement by Agonists Depends on the Permeant Ion J Neurophysiol, August 1, 2004; 92(2): 824 - 837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Leuranguer, R. T. Dirksen, and K. G. Beam Potentiated L-type Ca2+ Channels Rectify J. Gen. Physiol., May 27, 2003; 121(6): 541 - 550. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
