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Control of Ion Conduction in L-type Ca²⁺ Channels by the Concerted Action of S5–6 Regions

Department of Pharmacology and Program in Neuroscience, University of Colorado Health Sciences Center, Denver, Colorado

Correspondence: Address reprint requests to William A. Sather, Department of Pharmacology, Box B-138, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262. Tel.: 303-315-3986; Fax: 303-315-2503; E-mail: william.sather{at}uchsc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Voltage-gated L-type Ca²⁺ channels from cardiac (_1C) and skeletal (_1S) muscle differ from one another in ion selectivity and permeation properties, including unitary conductance. In 110 mM Ba²⁺, unitary conductance of _1S is approximately half that of _1C. As a step toward understanding the mechanism of rapid ion flux through these highly selective ion channels, we used chimeras constructed between _1C and _1S to identify structural features responsible for the difference in conductance. Combined replacement of the four pore-lining P-loops in _1C with P-loops from _1S reduced unitary conductance to a value intermediate between those of the two parent channels. Combined replacement of four larger regions that include sequences flanking the P-loops (S5 and S6 segments along with the P-loop-containing linker between these segments (S5–6)) conferred _1S-like conductance on _1C. Likewise, substitution of the four S5–6 regions of _1C into _1S conferred _1C-like conductance on _1S. These results indicate that, comparing _1C with _1S, the differences in structure that are responsible for the difference in ion conduction are housed within the S5–6 regions. Moreover, the pattern of unitary conductance values obtained for chimeras in which a single P-loop or single S5–6 region was replaced suggest a concerted action of pore-lining regions in the control of ion conduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The voltage-gated L-type Ca²⁺ channels from cardiac muscle (_1C) and skeletal muscle (_1S), though closely related in structure, differ from one another in a number of important functional ways. The high degree of sequence conservation between _1C and _1S has facilitated structure-function analysis for these channels. For example, structural elements regulating channel activation (Nakai et al., 1994) and mediating excitation-contraction coupling (Tanabe et al., 1990) have been identified using strategies that rely on this sequence similarity.

A substantial body of work has also been directed toward understanding the structural basis of ion selectivity in Ca²⁺ channels. Earlier work had led to the conclusion that selectivity in ion transport was mediated by preferential binding of Ca²⁺ over Na⁺, the two principal competitors for transport through Ca²⁺ channels under physiological conditions (Almers and McCleskey, 1984; Hess and Tsien, 1984). More recent work using site-directed mutagenesis has identified amino acid residues that form the selectivity filter that binds Ca²⁺ in the pore (Tang et al., 1993; Yang et al., 1993; Ellinor et al., 1995; Cibulsky and Sather, 2000; Koch et al., 2000; Wu et al., 2000).

Despite the fact that tight binding of Ca²⁺ is essential for selection against nonpreferred permeants such as Na⁺, the observed rate of Ca²⁺ conduction through the pore nonetheless requires fast Ca²⁺ unbinding and transit. For highly selective ion channels generally, no simple relationship between selectivity and conduction exists. Thus, for example, all voltage-gated K⁺ channels are highly selective for K⁺, yet their unitary conductance values range over two orders of magnitude (Hille, 2001). Likewise, voltage-gated Ca²⁺ channels are all highly selective for Ca²⁺, and though less extreme than in the case of K⁺ channels, different kinds of Ca²⁺ channels differ among themselves in unitary conductance. In particular, the unitary conductance of _1C Ca²⁺ channels is roughly double that of _1S channels.

Regions of Ca²⁺ channels that may be involved in specifying ion conduction include the P-loops, four pore-lining structures in each channel molecule that together contribute to formation of the selectivity filter. The P-loops are thought to line the extracellular portion of the pore in members of the voltage-gated ion channel family, which includes Ca²⁺ and K⁺ channels (MacKinnon, 1995). Evidence provided by the crystal structure of a bacterial K⁺ channel, an ancestor of both voltage-gated K⁺ channels and Ca²⁺ channels, has strengthened this view (Doyle et al., 1998). This bacterial K⁺ channel structure also shows that transmembrane segments homologous to S6 contribute to the intracellular portion of the pore, the portion that opens into the cytosol; the S6 segment appears to help form the intracellular portion of the pore in voltage-gated K⁺ channels of higher organisms as well (del Camino et al., 2000). Consonant with this basic structural model, P-loops have been implicated in the control of unitary conductance in many members of the family of voltage-gated ion channels. In some cases, the P-loop or the entire S5–S6 linker that encompasses the P-loop has been suggested as the sole determinant of unitary conductance (Hartmann et al., 1991; Goulding et al., 1993; Yatani et al., 1994; Repunte et al., 1999). In other cases, flanking S5 and S6 segments were additionally shown to influence conduction (Aiyar et al., 1994; Shieh and Kirsch, 1994; Immke et al., 1998). Sequences even farther from the P-loop, including the cytosolically-disposed S4–S5 linker and C-terminal tail, have been implicated as determinants of unitary conductance (Isacoff et al., 1991; Slesinger et al., 1993; Choe et al., 2000). In the present work, we have used a systematic set of chimeras constructed between the _1C and _1S Ca²⁺ channel isoforms to identify domains that determine these channel's characteristic ion transport rates. The aim of work such as this is to understand how ion channels that are very similar in ion selectivity can differ significantly in rate of ion transport.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Ca²⁺ channel chimeras
Three kinds of chimeras were constructed between cDNAs encoding the _1C (Mikami et al., 1989; EMBL/GenBank accession number X15539) and _1S (Tanabe et al., 1987; Kim et al., 1990; accession number X05921) L-type Ca²⁺ channel subunits. In the first kind of chimera, P-loop sequence was substituted from _1S into _1C. Based on the better-known structure of P-loops in voltage-gated K⁺ channels (Yellen et al., 1991), P-loops of Ca²⁺ channels were, in this work, considered to be 18-residue sequences within the linker between the S5 and S6 transmembrane segments. However, in motif IV, 20-residue P-loop sequences were substituted to include one additional difference in sequence between the two parent channels. Numbering the EEEE locus glutamates as position 0 in each motif, the substituted P-loop regions comprised residue positions -13 to +4, amino to carboxy, or in the case of motif IV, positions -13 to +6. In _1S, the P-loop segments for motifs I–IV were bounded by residues G279/D296, P601/S618, L1001/Q1018, and P1310/L1329; for _1C, the P-loops were bounded by A380/D397, P723/S740, L1132/E1149, and P1433/M1452. In the other two kinds of chimeras, the entire sequence from the beginning of the S5 transmembrane segment through the end of the S6 segment (S5–6) was transferred from _1C to _1S, and vice versa. Hydropathy plot analysis has identified the S5 (20 residues) and S6 (25 residues) transmembrane segments in L-type Ca²⁺ channels (Tanabe et al., 1987), and the S5–6 regions of the four motifs range 100–136 residues in length. In _1S, the S5–6 segments for motifs I–IV were bounded by residues I199/S334, L561/V661, I931/I1065, and V1270/M1384; for _1C, the S5–6 segments were bounded by I301/S435, L684/V783, I1062/I1196, and V1393/M1506. The quadruple chimeras and the parent _1C and _1S subunits are diagrammed in Fig. 1. The single-motif chimeras for P-loop and S5–6 regions are not illustrated.

View larger version (36K): [in this window] [in a new window]   FIGURE 1  Schematic representation of the pore-forming subunits of wild-type 1C and 1S Ca2+ channels and some chimeric constructs. 1S sequence is indicated by bold lines and by filled segments representing transmembrane regions, whereas 1C sequence is indicated by thin lines and unfilled transmembrane segments. Only chimeras in which sequence was substituted in all four motifs are illustrated (Quad chimeras).

Chimeras in which S5–6 sequence from _1S was substituted into _1C are, for each of the four single-motif chimeras, denoted as C_IS5–6S, C_IIS5–6S, C_IIIS5–6S, and C_IVS5–6S. A four-motif chimera produced by combining the four S5–6 single-motif chimeras is referred to as C_QuadS5–6S. The S5–6 single-motif chimeras were constructed using a 5-primer strategy. In the first round of PCR, _1S S5–6 sequence fused at either end to a short stretch of _1C sequence was produced using an _1S template and a pair of fusion primers (typically 39-mers; primers 1 and 2) that included 5' overhangs (24 bases in length) corresponding to _1C sequence located either immediately upstream of S5 or downstream of S6. In a second round of PCR, the gel-purified product of the first round, a primer complementary to upstream _1C sequence (primer 3; 30-mer), primer 1, and an _1C template were used to amplify the _1C sequence upstream of S5. To avoid amplifying nonchimeric, WT _1C in the final round of PCR, primer 3 included a 15-base, 5'-terminal, non-sense sequence that was complementary to neither _1C nor _1S. Primer 1 was added to this second-round reaction only after completing five thermocycles. In the third and final round of PCR, the gel-purified second-round product, a downstream primer complementary to _1C sequence (primer 4; 18-mer), an upstream primer complementary to the nonsense sequence of primer 3 (primer 5; 15-mer), and _1C template were used to amplify _1C sequence downstream of S6. Primer 5 was added to the reaction mix after completing five thermocycles. The final PCR product and the pCARDHE vector were digested with a pair of motif-specific restriction enzymes and gel purified. Each S5–6 chimera was completed by ligating the PCR cassette (600–1260 bp) into pCARDHE.

A chimera in which the S5–6 sequences of the four motifs of _1S were replaced by the corresponding sequences in _1C is referred to as S_QuadS5–6C. To make this chimera, the Sac II–Bgl II fragment of _1S, corresponding to most of the coding region, was first subcloned into pGEMHE (Liman et al., 1992) to make use of advantageous restriction sites in this construct. The strategy used to construct the S_QuadS5–6C chimera was conceptually similar to that described for the C_QuadS5–6S chimera.

DNA sequences for all chimeras were confirmed by restriction digests and dideoxy chain termination sequencing of both strands of all PCR-amplified regions.

Ca²⁺ channel expression in Xenopus oocytes
cRNAs encoding ₁ subunits were synthesized using vectors for _1C- and _1S-based constructs that yielded high functional expression in Xenopus oocytes. Before construction of _1C-based chimeras, the _1C insert was subcloned into a modified version of pGEMHE, a vector that incorporates the 5' and 3' untranslated regions of the Xenopus ß-globin gene (Liman et al., 1992). In the subcloning process, several in-frame start- and stop-codons in the 5' untranslated region of the original _1C clone were deleted, and a Kozak consensus sequence for initiation of translation was inserted immediately upstream of the true _1C start codon. The resulting high-expression construct, termed pCARDHE, was used in the fabrication of all _1C-based chimeras.

To enhance expression of _1S in Xenopus oocytes, the 3' coding region was truncated (Ren and Hall, 1997; Morrill and Cannon, 2000). One _1S construct was truncated after the codon specifying amino acid 1662 (Beam et al., 1992) and another construct was truncated after codon 1698 (DeJongh et al., 1991; Ren and Hall, 1997). However, when subcloned into pGEMHE, neither the full-length _1S cDNA nor the two 3'-truncated forms of _1S yielded highly-expressed cRNAs (100–500 nA whole-oocyte Ba²⁺ currents when coexpressed with _21a and ß_1b). When subcloned into pAGA2 (Ren and Hall, 1997), the version of _1S truncated after codon 1698 produced significantly larger currents. Therefore, after the S_QuadS5–6C chimera was constructed in pGEMHE, the Sac II–Bgl II fragment of the chimera was subcloned into the pAGA2 vector to enhance chimera expression.

To further enhance functional expression of Ca²⁺ channels, cDNAs for the ancillary subunits _21a (rabbit; Mikami et al., 1989; the 3' noncoding region was truncated), ß_2b (rabbit; Hullin et al., 1992; EMBL/GenBank accession number X64298), and ß_1b (rat; Pragnell et al., 1991; accession number X61394) were subcloned into the modified version of pGEMHE that was used for _1C. Ca²⁺ channel subunit cRNAs were transcribed in vitro using the mMESSAGE mMACHINE T7 RNA synthesis kit (Ambion, Austin, TX). Equimolar concentrations of ₁-, ₂- and ß-subunit cRNAs were injected into Xenopus laevis oocytes. _1C- and _1C-based chimeras were coexpressed with _21a and ß_2b, whereas _1S- and _1S-based chimeras were coexpressed with _21a and ß_1b, except where noted. According to the most recently proposed systematic nomenclature, the subunit makeup of these channels is written Ca_v1.2a/ß_2b/_21a for _1C-based channels, and Ca_v1.1a/ß_1b/_21a for _1S-based channels (Ertel et al., 2000). Oocytes were dissociated from ovarian tissue by shaking in a Ca²⁺-free OR-2 solution (in mM: 82.5 NaCl, 2 KCl, 1 MgCl₂, 5 n-(2-hydroxyethyl)piperazine-n'-(2-ethanesulfonic acid) (HEPES), pH 7.5 with NaOH) containing 2 mg/ml collagenase B (Boehringer-Mannheim) for 60–90 min. Injected oocytes were incubated in ND-96 solution (in mM: 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, 5 HEPES, pH 7.6 with NaOH) supplemented with 2.5 mM sodium pyruvate (Sigma, St. Louis, MO), 100 U/ml penicillin (Sigma) and 0.1 mg/ml streptomycin (Sigma). Injected oocytes were maintained at 18°C, and were studied 3–14 days postinjection.

Two-electrode voltage clamp recording
Whole-oocyte currents were recorded as described previously (Sather et al., 1993). The bath was continuously perfused with a Cl^--free, nominally 40 mM Ba²⁺ solution (in mM: 40 Ba(OH)₂, 52 TEA-OH, 5 HEPES, pH 7.4 with methanesulfonic acid). Owing to precipitation, Ba²⁺ concentration was substantially lower than the nominal value, and was measured as 10 mM (Williamson and Sather, 1999). To test the Mg²⁺ permeability of _1S channels, 40 mM or 100 mM Mg(OH)₂ solutions were used (in mM: 40 Mg(OH)₂, 52 TEA-OH, 5 HEPES, pH 7.4 with methanesulfonic acid, or 100 mM Mg(OH)₂, 5 HEPES, pH 7.4 with methanesulfonic acid). Currents were measured with a model OC-725C amplifier (Warner Instruments), filtered at 500 Hz (4-pole Bessel filter, Warner Instruments) and sampled at 1 kHz. Data were acquired and analyzed using software custom-written in AxoBASIC (Axon Instruments, Foster City, CA). For voltage pulses of size P, peak currents were subtracted using the average of 10 pulses to -P/4. For the Cd²⁺ block experiments, a 1 mM CdCl₂ stock solution was diluted to a final concentration of 1 µM in the Ba²⁺ solution.

Single-channel recording
The vitelline membrane was manually stripped from an oocyte after soaking in a hyperosmotic solution (Sather et al., 1993). Single-channel currents were recorded in cell-attached patches while the stripped oocyte was bathed in a high K⁺ solution that zeroed the membrane potential (in mM: 100 KCl, 10 HEPES, 10 ethylene glycol-bis(beta-aminoethyl ether)-n,n,n',n'-tetraacetic acid) (EGTA), pH 7.4 with KOH). The L-type Ca²⁺ channel agonist FPL 64176 (RBI, Natick, MA) was included in the bath solution at a concentration of 2 µM to prolong channel openings. Pipettes were pulled from borosilicate glass (Warner Instruments, Hamden, CT), coated with Sylgard (Dow Corning, Midland, MI) and heat-polished. Pipettes typically had resistances of 25–40 M when filled with the recording solution of (in mM) 110 BaCl₂, 10 HEPES (pH 7.4 with TEA-OH). Single-channel records were obtained using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The amplifier's internal filter was set to 10 kHz and an external filter (8-pole Bessel filter, Frequency Devices, Haverhill, MA) was set to 2 kHz, yielding a -3 dB frequency for the cascaded filters of 1.96 kHz. The data were sampled at 10 kHz using a Digidata 1200A (Axon Instruments) A/D converter and Pulse software (HEKA, distributed by Instrutech Corp., Great Neck, NY). Single-channel current amplitudes were determined by cursor analysis of long-duration openings (Pulse, HEKA).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
In two-electrode voltage-clamp recordings, channels containing ₁ subunits of predominantly _1S-based or _1C-based origin carried currents of roughly similar size, with peak inward currents of typically 1–3 µA in the 40 mM Ba²⁺ solution. The resulting similarity of voltage clamp quality and of single-channel event frequency facilitated comparisons among channel constructs.

The chimeric constructs were designed to study ion permeation. However, as an indicator of the specificity in effect of the structural manipulations, we examined whether channel gating might have been altered in the chimeras. We found that wild-type and chimeric channels containing _1C-based subunits exhibited the fast activation kinetics expected for _1C channels, whereas channels containing _1S-based subunits exhibited the slow activation kinetics characteristic of the skeletal muscle Ca²⁺ channel (Fig. 2 A) (Tanabe et al., 1991). During a test pulse to +20 mV, _act for WT _1C channels was 3.2 ± 0.1 ms (mean ± SE; n = 6), whereas _act for _1S channels was 21.8 ± 1.0 ms (n = 6). The _1C-based chimeras C_QuadPS (_act = 2.4 ± 0.3 ms, n = 6) and C_QuadS5–6S (_act = 1.3 ± 0.1 ms, n = 6) activated with time courses like that of wild-type _1C, and the _1S-based chimera S_QuadS5–6C activated with a time course like that of _1S (21.6 ± 3.7 ms, n = 6). Thus as judged by the general similarity of chimeras to their parents in regard to activation gating, these manipulations of pore structure appear to have had restricted effects on the behavior of the channels.

View larger version (15K): [in this window] [in a new window]   FIGURE 2  Whole-oocyte currents for 1C, 1S, and the three Quad chimeras (CQuadPS, CQuadS5–6S, and SQuadS5–6C) with 40 mM Ba2+ solution in the bath. 1C and 1C-based chimeras were coexpressed with 21a- and ß2b-subunits. 1S and the 1S-based chimera were coexpressed with 21a- and ß1b-subunits. (A) Normalized currents elicited by test pulses to +20 mV. (B) Representative current-voltage relationships. Peak current is plotted versus test pulse voltage. Holding potential was -80 mV. (C) Percent block by 1 µM Cd2+ of inward Ba2+ current at +20 mV (mean ± SE; n = 3–10 oocytes).

Percent block of Ba²⁺ current by 1 µM Cd²⁺ (Fig. 2 C) was also different between wild-type _1C (56.9 ± 3.2%, n = 4) and _1S (68.9 ± 6.7%, n = 3). Block of C_QuadPS (62.4 ± 2.2%, n = 10) was intermediate between that of the two wild-type channels and block of C_QuadS5–6S (78.4 ± 0.7%, n = 6) was somewhat greater than that of _1S. S_QuadS5–6C, however, was significantly more sensitive to Cd²⁺ block than was either parent (96.0 ± 2.5%, n = 6). Based on the 1:1 binding that describes Cd²⁺ block of Ca²⁺ channels, these percent block values correspond to calculated half-block (IC₅₀) values of 757 nM and 451 nM for _1C and _1S; to 603 nM and 276 nM for the C_QuadPS and C_QuadS5–6S chimeras; and to 42 nM for the S_QuadS5–6C chimera. Thus in all three cases, chimeric substitution increased the channel's affinity for Cd²⁺ relative to the parents. This systematic enhancement of Cd²⁺ affinity in chimeras relative to the parent channels suggests that, as for the reversal potential measurements, interactions between the transferred amino acid sequence and the bulk of the channel protein are likely to be important in determining the structure and selectivity behavior of the pore.

Although Cd²⁺ block of Ba²⁺ current clearly differed between _1C and _1S, the differences were not so large that chimeras could be readily used to identify pore features responsible for differences in this property of the parent channels. And because Cd²⁺ sensitivity of the chimeras did not fall between that of the parents, Cd²⁺ block of Ba²⁺ current was not used for comparative structure-function analysis of _1C and _1S channels.

Previous work on native Ca²⁺ channels in skeletal muscle indicated that monovalent cation current carried by _1S would be orders-of-magnitude less sensitive to block by Cd²⁺ than monovalent current carried by _1C (compare Almers et al., 1984 with Yang et al., 1993), but we found no large difference between _1S and _1C in potency of Cd²⁺ block of monovalent current: Cd²⁺ blocked current carried by 100 mM Li⁺ through these two channels with roughly similar potency when the channels were expressed in oocytes (data not shown). It has also been reported that native skeletal muscle L-type Ca²⁺ channels can carry Mg²⁺ current (Almers and Palade, 1981; McCleskey and Almers, 1985), in contrast to the case for cardiac L-type channels (Hess et al., 1986; Lansman et al., 1986). For wild-type _1S channels expressed in oocytes, however, we were unable to detect inward Mg²⁺ (40 mM or 100 mM) current. Thus because _1C and _1S differed only modestly or not at all in reversal potential, Cd²⁺ block, and Mg²⁺ permeability, we have focused our investigation of structural determinants of Ca²⁺ channel permeation upon the robust difference in unitary conductance between _1C and _1S channels, as described below.

Unitary conductance: P-loop transfer from _1S to _1C
Unitary current-voltage relationships in 110 mM Ba²⁺ for _1C and _1S are plotted in Fig. 3. The relationships for both wild-type channels as well as all of the chimeras are slightly curvilinear. They were, however, reasonably well fit with linear regressions. We used such fits to estimate unitary conductance (slope of the fit to data over the range -100 to +20 mV), which allows comparisons to be made with work by others. _1C had a unitary conductance of 28.9 pS, which is in close agreement with the value of 29.1 pS measured from ventricular myocytes by Yue and Marban (1990). Conductance for _1S was 16.3 pS, which is also similar to that measured from native channels, in this case, in skeletal myotubes (14.3 pS; Dirksen et al., 1997). The small difference between the two values for _1S may be due to the difference in voltage range over which unitary current amplitude was measured: Dirksen et al. (1997) used -20 to +20 mV, whereas we used -100 to +20 mV, and curvature in the current-voltage relationship results in steepening of the slope at more negative voltages. The transfers of P-loop sequences from _1S to _1C, one motif at a time, each had a small effect on unitary conductance (Fig. 3). P-loop replacement in motif II reduced _1C conductance to 27.2 pS (C_IIPS), in motif III to 27.4 pS (C_IIIPS), in motif IV to 27.8 pS (C_IVPS), and in motif I to 28.6 pS (C_IPS). When all four P-loops were transferred together, conductance was reduced to a level intermediate between those for _1C and _1S (C_QuadPS; 22.9 pS). This observation, that replacement of all four _1C P-loops with the corresponding _1S P-loops did not fully transfer an _1S-like conductance to _1C, suggests that additional parts of the channel influence unitary conductance.

View larger version (18K): [in this window] [in a new window]   FIGURE 3  Unitary current-voltage relationships for 1C, 1S, and mutants in which P-loop sequence from 1S was substituted into 1C. 1C and chimeras were coexpressed in oocytes with 21a- and ß2b-subunits, whereas 1S was coexpressed with 21a- and ß1b-subunits. Currents in cell-attached patches were measured with 110 mM Ba2+ in the pipette. From the holding potential of -80 mV, a 25 or 50 ms prepulse of +20 to +80 mV was usually applied immediately before the 300 ms test pulse, with no interval between the prepulse and test pulse. The prepulse facilitated channel activation, and 1S generally required stronger facilitation (+80 mV for 50 ms). Mean unitary current amplitude ± SE (n = 3–7 patches at each potential) is plotted versus test pulse voltage for 1S (•), 1C (), CIPS (), CIIPS(), CIIIPS (), CIVPS (), and CQuadPS (). Solid lines represent linear regression fits to the data. Representative single-channel currents recorded during a test pulse to -40 mV are displayed in the lower part of the figure.

Unitary conductance: S5–6 transfer from _1S to _1C

The size of the effect of transfer of S5–6 from _1S to _1C was motif-specific (Fig. 4). Replacement of S5–6 in motif I or in motif II had larger effects, lowering unitary conductance from the wild-type _1C value of 28.9 pS to 24.4 pS in the C_IS5–6S chimera or to 24.9 pS in the C_IIS5–6S chimera. Transfer of S5–6 in either motif III or IV had almost negligible effect on unitary conductance (28.3 pS in C_IIIS5–6S and 30.0 pS in C_IVS5–6S). The effect of single motif S5–6 transfers was in no case as large as the combined transfer of all four P-loops (C_QuadPS). However, replacement of all four S5–6 regions in _1C produced a channel with an _1S-like conductance: in fact, the conductance of C_QuadS5–6S (14.1 pS) was slightly smaller than that of wild-type _1S (16.3 pS). The similarity in conductance between wild-type _1S and the C_QuadS5–6S chimera suggests that, for wild-type _1C versus wild-type _1S, the differences in pore structure that are responsible for differences in unitary conductance are contained within the S5–6 regions.

View larger version (19K): [in this window] [in a new window]   FIGURE 4  Unitary current-voltage relationships for chimeras in which S5 through S6 sequence from 1S was substituted into 1C. Chimeras were coexpressed in oocytes with 21a- and ß2b-subunits. Currents in cell-attached patches were recorded with 110 mM Ba2+ in the pipette. Holding potential was -80 mV, and a 25 or 50 ms prepulse to +20 or +40 mV was usually applied to facilitate channel activation (no interval between prepulse in test pulse). Unitary current amplitude (mean ± SE, n = 3–7 patches at each potential) is plotted versus test potential for CIS5–6S (), CIIS5–6S (), CIIIS5–6S (), CIVS5–6S () and CQuadS5–6S (). Solid lines are linear regression fits to the data. For comparison, the linear regression fits to the i-V relationships for 1S and 1C from Fig. 3 are shown as dotted lines. Representative single-channel records at a test potential of -40 mV are illustrated in the lower part of the figure.

Reciprocality of chimeric effects on unitary conductance: S5–6 transfer from _1C to _1S
The diminishment of unitary conductance produced by chimeric manipulation of the _1C pore can be interpreted in competing ways. It might reflect the straightforward transfer of _1S-like ion transport behavior along with _1S pore sequence, or it might arise from incompatibility of the transferred _1S sequence with the host _1C sequence, resulting in misfolding in the pore region and retarded ion flux. To discriminate between these alternatives, we examined the unitary conductance of an _1S-based chimera in which the four S5–6 regions were replaced with the corresponding sequences from _1C. For this S_QuadS5–6C chimera, complementary to C_QuadS5–6S, we specifically tested whether transfer of _1C sequence into the _1S host would yield a chimera with _1C-like unitary conductance. Indeed, as illustrated in Fig. 5, the unitary conductance of the S_QuadS5–6C (30.0 pS) chimera closely approximated that of the wild-type _1C channel.

View larger version (15K): [in this window] [in a new window]   FIGURE 5  Unitary current-voltage relationship for the chimera in which S5 through S6 sequence from 1C was substituted into 1S, SQuadS5–6C (), (mean ± SE, n = 3–5 patches at each potential). The chimera was coexpressed in oocytes with 21a- and ß1b-subunits. Currents were recorded in cell-attached patches with 110 mM Ba2+ in the pipette and with a holding potential of -80 mV. A 25- or 50-ms prepulse to +20 or +40 mV was given immediately before the test pulse to facilitate channel activation. The solid line is a linear regression fit to the data. For comparison, the linear regression fits to the i-V data for 1S and 1C are represented as dotted lines. Representative unitary currents recorded during a test pulse to -50 mV are illustrated below the i-V plot.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

S5–6 regions control ion flux through _1C and _1S Ca²⁺ channels
Unitary conductance and unitary current results for all the chimeras studied are compared in Fig. 6. The results are scaled relative to the normalized difference between _1C and _1S in either conductance (Fig. 6 A) or current (Fig. 6 B). Dotted lines mark values for _1C (upper level in both panels) and _1S (lower level in both panels). In general, the pattern of results is similar for unitary conductance and unitary current. Thus whether comparing conductance or current results for the quadruple chimeras (black or white bars), the quadruple P-loop substitution shifted the ion transport rate only about halfway toward the donor rate whereas quadruple S5–6 substitution more or less completely transferred the ion transport rate of the donor. Roles for non-P-loop regions in controlling ion conduction have previously been suggested for voltage-gated K⁺ channels (Lopez et al., 1994; Aiyar et al., 1994; Shieh and Kirsch, 1994; Taglialatela et al., 1994; Immke et al., 1998) and for inward rectifier K⁺ channels (Choe et al., 2000), and the full S5–6 region has been specifically implicated in cyclic nucleotide-gated channels (Siefert et al., 1999).

View larger version (24K): [in this window] [in a new window]   FIGURE 6  Summary of relative differences in unitary conductance () and in unitary current (i) at -80 mV among 1C, 1S, and chimeras. (A) Differences in conductance between chimeras and 1S (chim – S) are plotted relative to the difference in unitary conductance between 1C and 1S (C – S). Dotted lines represent the relative difference values for 1C (1.0; = 28.9 pS) and 1S (0; = 16.3 pS). For 1C-based chimeras, bars representing single-motif substitutions (I, II, III, IV) are shaded in gray and bars representing Quad chimeras (Q; CQuadPS or CQuadS5–6S) are filled in black. The 1S-based Quad chimera (Q; SQuadS5–6C) is represented by a white bar. Unitary conductance was determined from linear regression fits to unitary current amplitudes measured over the range -100 to +20 mV (n = 3–7 patches; 110 mM Ba2+), as illustrated in Figs. 3–5. (B) Differences in unitary current at -80 mV between chimeras and 1S, denoted (ichim – iS), are plotted relative to the difference in unitary current at -80 mV between 1C and 1S, denoted (iC – iS). Dotted lines represent the relative difference values for 1C (1.0; i = 3.83 ± 0.03 pA, n = 4) and 1S (0; i = 1.91 ± 0.11 pA, n = 4). For 1C-based chimeras, bars representing single-motif substitutions (I, II, III, IV) are shaded in gray and bars representing Quad chimeras (Q; CQuadPS or CQuadS5–6S) are filled in black. The 1S-based Quad chimera (Q; SQuadS5–6C) is represented by a white bar.

Second, the effects on conductance produced by single-motif sequence transfers are not in every instance additive: the magnitude of the change in ion transport rate produced by a quadruple transfer is not necessarily predicted by summing the magnitudes of the four corresponding single-motif transfers. In the most striking case, substituting all four _1S S5–6 regions into _1C (C_QuadS5–6S) reduced unitary conductance by about twice that of the summed reductions produced by the four individual S5–6 transfers. When considering instead unitary currents or the results for P-loops, the evidence for non-additivity was much weaker. Nonetheless, the absence of additivity of conductance for the S5–6 transfers raises the possibility of cooperative or synergistic interaction among the four motifs.

Third, interactions between P-loop sequence and other parts of the S5–6 region seem to be complex. The data summarized in Fig. 6 show that although individual S5–6 substitutions caused distinctive decrements in ion conduction, individual P-loop substitutions produced approximately similar, small decrements in ion conduction. Regarding motif IV, for example, P-loop substitution produced bigger changes in ion conduction than did S5–6 substitution, as though the effects of P-loop transfer could be reversed by transfer of structural features contained in the non-P-loop components of the S5–6 region. Alternatively, this finding might indicate that "improper" interactions of transplanted P-loop residues with host channel residues led to local protein misfolding and diminished ion conduction. This view may also account for our finding that Na⁺ channel P-loops not only fail to confer Na⁺ selectivity on Ca²⁺ channels, but the resulting Na⁺ channel/Ca²⁺ channel chimera also failed to carry Ca²⁺ or Ba²⁺ current (unpublished data).

Our findings generally agree with previous work by Dirksen et al. (1997) in that the motif I P-loop and S5–6 region house the key determinants of ion conduction, but our results using systematic sets of P-loop and S5–6 region chimeric constructs reveal significant participation of other motifs as well. In the study by Dirksen and colleagues (1997), substitution of _1S sequence into the motif I S5–S6 linker of _1C, which left the flanking S5 and S6 segments of _1C in place, displaced unitary conductance 75% of the way toward the _1S value. In contrast, we have found that substitution of a larger region in motif I, encompassing the S5–S6 linker but also including the flanking S5 and S6 segments, displaced unitary conductance only 35% (C_IS5–6S, Fig. 6 A) of the way toward the _1S value. In our work, we found that full transfer of _1S-like conductance required substitution of all four S5–6 regions. Various explanations for the apparent discrepancy between the two studies can be proposed, but a likely one stems from the fact that different chimeras were studied. As discussed above, interactions between the S5–S6 linker and surrounding parts of the S5–6 region may be important in determining conductance, and in the absence of appropriate interactions between these parts of the conductance-determining S5–6 regions, unitary conductance might consequently be reduced. The fact that swapping the four S5–6 regions reciprocally transferred unitary conductance between _1C and _1S confirms the idea that the S5–6 regions contain the structural features responsible for the difference in ion conduction between _1C and _1S. Comparison of results with our chimeras and those of Dirksen and colleagues (1997) also supports the idea, discussed above, that structural features contained within the S5–6 regions but outside the S5–S6 linker specify unitary conductance in these two L-type Ca²⁺ channels.

Reversal potential, Cd²⁺ block and unitary conductance
Whereas the unitary conductance results are interpretable in a straightforward manner, the effects of chimeric substitution on two other measures of ion permeability, reversal potential, and Cd²⁺ block, are not as readily rationalized. The parent _1C and _1S channels differed little from one another in E_rev and in estimated IC₅₀ for Cd²⁺ block, but as a general trend, the three quadruple chimeras (C_QuadPS, C_QuadS5–6S, S_QuadS5–6C) differed from their parents: in the quadruple chimeras, E_rev was as much as 20 mV less positive (S_QuadS5–6C) and Cd²⁺ IC₅₀ was as much as 10-fold lower (S_QuadS5–6C) relative to the parent channels. Thus quadruple chimeragenesis seemingly reduced ion selectivity if judged from bi-ionic reversal potential, but increased ion selectivity if judged from Cd²⁺ binding affinity. The S_QuadS5–6C chimera represents the most striking case, with the lowest preference for Ba²⁺ over K⁺ (E_rev) but the highest preference for Cd²⁺ over Ba²⁺ (IC₅₀). Part of the explanation for this situation may be that these two measures of ion selectivity differ in the ions compared and in the direction of ion flow, so that inward Ba²⁺ competes with outward K⁺ in one case but inward Cd²⁺ competes with inward Ba²⁺ in the other.

It is noteworthy that E_rev and IC₅₀ were altered in the quadruple chimeras despite the fact that all four selectivity filter glutamates (EEEE locus) were present in these chimeras. One explanation is that the EEEE locus is very sensitive to structural context, so that incompatibility between _1C and _1S sequence in the chimeras results in altered EEEE locus configuration and altered selectivity. Alternatively, non-EEEE locus mutations have previously been found to affect Ca²⁺ channel selectivity, suggesting the possibility that altered pore structure elsewhere in the transplanted region might account for the changes in selectivity (Williamson and Sather, 1999; Feng et al., 2001).

Differences between _1C and _1S in S5–6 sequence and ion conduction
Sequence comparison suggests ways that the S5–6 regions might potentially control ion conduction in Ca²⁺ channels. In motif III, previous work comparing _1C with _1A (P/Q-type Ca²⁺ channel) sequence led to the finding that the side-chain volume of a residue neighboring the EEEE locus influenced unitary conductance (Williamson and Sather, 1999). The residue at this neighbor position is conserved between _1C and _1S, however, and in general, there are few remarkable differences in P-loop sequence between _1C and _1S, which may account for the inability of quadruple P-loop substitution to fully transfer conduction behavior. In the regions flanking the P-loops, the S5–S6 linkers differ between _1C and _1S at several positions. Examining these differences in motif I, _1C has a net charge of -5 relative to _1S, which has previously been suggested to attract permeant cations into the extracellular pore entrance and thereby impart higher conductance on _1C channels (Dirksen et al., 1997). Considering this idea in light of the evidence that surrounding parts of S5–6 are important in specifying conduction rate, electrostatic enhancement of permeant ion entry rate may not be a dominant factor in conduction. Indeed, evidence against electrostatic focusing of permeant divalent metal cations at the mouth of L-type Ca²⁺ channels under the experimental conditions used here has been obtained (Kuo and Hess, 1992).

Regarding our evidence that the S5–6 region is crucial in controlling flux through Ca²⁺ channels, in the homologous K⁺ channels the cytosolically-disposed part of S6 and possibly S5 is thought to contribute to the pore lining (Aiyar et al., 1994; Lopez et al., 1994; Shieh and Kirsch, 1994; Liu et al., 1997; Doyle et al., 1998; del Camino et al., 2000). The cytosolic halves of S5 and S6 in _1C and _1S are highly hydrophobic, which is consistent with the hydrophobicity of homologous sequences lining the central pool and inner pore of the KcsA K⁺ channel (Doyle et al., 1998). In Ca²⁺ channels, differences in these hydrophobic sequences may therefore help to determine conduction rate through the cytoplasmic part of the pore, as has been proposed for the KcsA channel. Additionally, the more extracellularly-disposed parts of the S5 and S6 segments may be involved, based on the fact that the number of differences in sequence between _1C and _1S is greater in the extracellular halves of S5 and S6 than in the intracellular halves. Whether amino acid residues in S5 or S6 contribute directly to pore formation in Ca²⁺ channels, for example at the extracellular entrance, is unknown. However, S5 and S6 may act by exerting indirect effects that influence the conformation of the more external portion of the pore, particularly the P-loop.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank Tsutomu Tanabe, Veit Flockerzi, Franz Hofmann, Kevin P. Campbell, and Linda M. Hall for gifts of Ca²⁺ channel cDNAs, and Emily Liman for the gift of the pGEMHE vector.

This work was supported by grant NS35245 (to W.A.S.) and fellowship MH11717 (to S.M.C.) from the National Institutes of Health.

	FOOTNOTES

 
S. M. Cibulsky's present address is Dept. of Neuroscience, Univ. of Pennsylvania, 223 Stemmler Hall, Philadelphia, PA 19104.

Submitted on June 27, 2002; accepted for publication November 18, 2002.

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