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Introduction

L-type calcium channels transport Ca2+ into myocytes and are important in the regulation of heart rhythm, excitation-contraction coupling and gene expression (1). Voltage-gated Ca2+ channels (VGCCs) are heteromultimeric complexes composed of α1, β, α2/δ and occasionally γ-subunits. The pore-forming α1 subunit contains all the structural determinants required for ion permeation, voltage-dependent gating and drug binding. The membrane topology of the α1 subunit consists of four homologous domains (I, II, III and IV), with six transmembrane segments (S1–S6) in each (Fig. 1A). Segments connecting with S5 and S6 in each domain contain four negatively charged glutamate residues that shape the pore of the channel and form binding sites for ions, known as the selectivity filter (EEEE locus) (2).

Figure 1 Phe-1144 residue in the pore segment of domain III. (A) The membrane topology of the α1 subunit consists of four homologous domains (domain III is shown), each consisting of six transmembrane segments (S1–S6). The selectivity filter is formed by four conserved glutamate residues (E), each residing on one of the four S5–S6 connecting loops. The intracellular portions of the four S6 transmembrane segments form the inner lining of the permeation pathway and the activation gate. The S4 segment from each repeat is an amphipathic helix consisting of several positively charged lysine or arginine residues and functions as the voltage sensor that couples membrane depolarization to channel activation. (B) Alignment of the domain III pore segment from each of the 10-voltage-gated Ca2+ channel family members. The alignments are highly conserved. At position 1144 (boxed) where located neighboring Glu-1145, all L-type Ca2+ channels (CaV1.1–4) possess a phenylalanine (F). However, all non-L-type high-voltage-activated Ca2+ channels (CaV2.1–3) possess a glycine (G) and all low-voltage-activated Ca2+ channels (CaV3.1–3) possess a lysine (K).

The nature of multi-ion permeation through the pore of voltage-gated Ca2+ channels has previously been investigated. Hess and Tsien (3) and Almers and McCleskey (4) suggested that Ca2+ binds the channel more strongly than other ions and that there is an electrostatic interaction between ions within the channel. Such channel selectivity and ion interactions demonstrate a phenomenon known as the anomalous mole fraction effect (AMFE). The AMFE arises when a mixture of two permeant ions produce less current than either permeant ion alone at the same total ion concentration. The AMFE is a complex phenomenon that depends on the holding voltage, total ion concentration and the intrinsic binding properties of the channel. Thus, AMFE is an important indicator of ion-ion interactions in the pore of voltage-gated Ca2+ channels.

Ba2+ currents through L-type Ca2+ channels, as well as most other high-voltage-activated Ca2+ channels, are approximately twice the size of Ca2+ currents (2,5). The differences between Ba2+ and Ca2+ conductance depend on the binding affinities of single cations to the selectivity filter of the channel (3,4,6,7). The binding affinity of a single Ba2+ ion to the selectivity filter is reported to be 70-fold lower than that of Ca2+(8). Therefore, the repulsive forces exerted by the entry of a second ion promote the exit of a Ba2+ ion more readily than Ca2+, which increases the exit rate and ionic flux. However, how ions permeate through the pore of voltage-gated Ca2+ channels and interact with a second ion remains a controversial topic.

There are several numeric simulation models dependent on the binding-repulsion events in the pore that predict the ion-ion interactions in the pores of Ca2+ channels. The Ca2+ channel model of a pair of high-affinity binding sites and ion interactions with electrostatic repulsion is widely known (3,4). Findings of previous studies indicated that the Ca2+ channel pore contains only a single high-affinity binding site and that ions compete for binding moieties (9,10). Dang and McCleskey (7) revealed the stepwise model, which has a single high-affinity Ca2+ binding site flanked by two low-affinity divalent cation binding sites, each composed of two carboxyl groups from the EEEE locus. Newer structure-based models have aimed to define the volume of the Ca2+ channel pore using structure theory. The ionic diameter of Ba2+ is much larger than that of Ca2+ and the resulting Ba2+ ions show a higher degree of crowding in the pore, causing a faster exit rate and larger conductance (11–13).

Mutagenesis studies indicate that the glutamate residue in the pore of domain III is the important determinant for ion permeation (9,10,14–16). The domain III pore segments of all 10 families of voltage-gated Ca2+ channels (Fig. 1B) are highly conserved. All L-type Ca2+ channels (CaV1.1–4) possess a phenylalanine (F) at position 1144, located next to Glu-1145. However, all non-L-type high-voltage-activated Ca2+ channels (CaV2.1–3) possess a glycine (G) and all low-voltage-activated Ca2+ channels (CaV3.1–3) possess a lysine (K). The physicochemical properties of the amino acid residue at position 1144 are important to the permeation of multiple Ba2+ and Ca2+ ions through the pore (17). Amino acid substitutions at position 1144 of the CaV1.2 channel reduce barium currents without affecting calcium currents. Therefore, we substituted Phe-1144 (F) with G (F/G) and K (F/K) and observed the effects of mutation on the voltage and concentration dependency of AMFE.

Materials and methods

Amino acid residue substitution

Site-directed mutagenesis was used to substitute amino acid residues. Mutant fragments of the channel were generated by polymerase chain reaction (PCR) using mutagenic primers. The mutant PCR products were gel-purified, digested using restriction endonucleases and subcloned into a digested CaV1.2 vector. The presence of the mutation and the integrity of each mutant were confirmed by qualitative restriction map analysis and directional DNA sequence analysis of the entire subcloned region. Functional expression of the mutant cDNAs was confirmed by western blot analysis and whole-cell patch-clamp electrophysiology.

Cell culture and transfections

HEK293 cells were grown at 37°C in 6% CO2 and DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% penn-strep antibiotics. cDNAs encoding wild-type and mutant CaV1.2 channels were co-transfected with α2δ (18), β2a (19) and CaM1234(20) into HEK293 cells by calcium phosphate precipitation, as previously described (17,20,21). CaM1234 was used to eliminate complications that could arise from Ca2+/CaM-dependent changes in channel gating, as it encodes an inactive form of calmodulin that has been shown to eliminate Ca2+-dependent inactivation and facilitation (17,20–24). All cDNAs were expressed using pcDNA3 mammalian expression plasmids (Invitrogen, Carlsbad, CA, USA).

Patch-clamp electrophysiology

Whole-cell patch-clamp recordings were obtained as previously described (17,21). Briefly, whole-cell currents were recorded at room temperature 2–3 days following transfection. Pipettes were pulled from borosilicate glass using a P-97 Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA, USA) and fire-polished on an MF200 microforge (World Precision Instruments, Sarasota, FL, USA). External solutions contained: N-methyl-D-glutamine (NMG)-aspartate, 130 mM; HEPES, 10 mM; 4-aminopyridine, 10 mM. BaCl2 and CaCl2 were used to give the desired molar ratio of the two ions and the total divalent cation concentration was maintained at 2, 10 or 20 mM. The osmolarity was adjusted to 300 mmol/kg with glucose and the pH was adjusted to 7.4 using 1 mM NMG base solution. Pipettes had resistances of 2.5–3.0 MΩ when filled with internal solution. The peak tail currents were evoked at potentials from −90 to +50 mV of all channels at each ion concentration.

Data acquisition and analysis

Data were acquired using a HEKA EPC-9/2 amplifier and PULSE/PULSEFIT software (HEKA Electronik, Lambrecht, Germany). Leak and capacitive transients were corrected by -P/4 compensation. Series resistance was <6 MΩ and compensated to 70%. Tail currents were sampled at 50 kHz and filtered at 5.0 kHz. Pulse protocols are described in each figure legend. Data analysis was performed using FitMaster (HEKA Electronik) and Origin 7 (OriginLab, Northampton, MA, USA). The significance of differences for the smallest normalized current and IBa/ICa among wild-type, F/G and F/K channels were analyzed using one-way ANOVA with Tukey’s test. The significance of differences between wild-type and F/K was analyzed using two-sample independent t-tests. Data were reported as the means ± SE. Statistically significant results (P<0.05) are indicated in the figures by an asterisk or a pound sign.

Results

F/G does not promote AMFE

Unlike wild-type channels (Fig. 2), the tail current amplitudes for F/G were reduced almost monotonically as Ba2+/(Ba2+ + Ca2+) progressed to 0.7, but failed to increase as Ba2+/(Ba2+ + Ca2+) approached 1.0 in 2 and 10 mM external solutions (Fig. 3A and B). The relative current amplitudes for F/G were indistinguishable when Ba2+/(Ba2+ + Ca2+) = 1.0. However, as shown in Fig. 3C, the tail current amplitudes for F/G increased as Ba2+/(Ba2+ + Ca2+) approached 1.0 in 20 mM external solution. Compared with a previous report (25), we found that the conditions at 20 mM for F/G did not detect AMFE, as the maximum AMFE appeared when the total divalent cation concentrations were kept low (i.e., 2 mM).

Figure 2 Voltage and concentration dependency of IBa/ICa and the anomalous mole fraction effect (AMFE) for wild-type channels. Cells were stepped from a holding potential of −90 to +50 mV for 100 msec, and peak tail currents were measured at molar ratios of external Ba2+ to total divalent cation concentrations [Ba2+/(Ba2+ + Ca2+)] of 0.0, 0.3, 0.5, 0.7, 0.9 and 1.0. The combined divalent concentration (Ba2+ + Ca2+) at each data point was kept at (A) 2, (B) 10 and (C) 20 mM, respectively. AMFE is observed when peak tail currents, normalized to 1.0 when Ba2+/(Ba2+ + Ca2+) = 0, are plotted against Ba2+/(Ba2+ + Ca2+) at holding potentials of −80, −60, −40 and −20 mV. The dashed line corresponds to peak tail currents measured in an external solution containing 0.0 mM Ba2+. (A) AMFE is evident at 2 mM combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.576±0.018 [Ba2+/(Ba2+ + Ca2+) = 0.9] and IBa/ICa is 1.082±0.035, n=6. (B) AMFE is evident at 10 mM combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.831±0.033 [Ba2+/(Ba2+ + Ca2+) = 0.7] and IBa/ICa is 1.547±0.052, n=9. Each is different from that at 2 mM combined divalent concentration and shown as ‘*’ and ‘#’, respectively. (C) AMFE is evident at 20 mM combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.769±0.022 [Ba2+/(Ba2+ + Ca2+) = 0.5] and IBa/ICa is 1.445±0.027, n=9. Each is different from that at 2 mM combined divalent concentration and shown as ‘*’ and ‘#’, respectively.

Figure 3 Voltage and concentration dependency of IBa/ICa and the anomalous mole fraction effect (AMFE) for F/G mutant channels. Cells were stepped from a holding potential of −90 to +50 mV for 100 msec, and peak tail currents were measured at molar ratios of external Ba2+ to total divalent cation concentrations [Ba2+/(Ba2+ + Ca2+)] of 0.0, 0.3, 0.5, 0.7, 0.9 and 1.0. The combined divalent concentration (Ba2+ + Ca2+) at each data point was kept at 2 (A; n=8), 10 (B; n=9) and 20 mM (C; n=6), respectively. The dashed line corresponds to peak tail currents measured in an external solution containing 0.0 mM Ba2+.

Voltage and concentration dependency of IBa/ICa

The experimental conditions were established to detect changes in AMFE at a varied series of recording holding voltages and combined divalent concentrations. The AMFE was determined by measuring peak tail currents in the presence of various molar ratios of Ca2+ and Ba2+. Under conditions that promote AMFE, a difference was detected for wild-type and F/K channels, IBa/ICa determined at the concentration of 2 mM compared with IBa/ICa determined at higher concentrations (i.e., 10 and 20 mM) (Figs. 2 and 4). Furthermore, we plotted the voltage range of −80 to −20 mV at which we could record the clear peak tail current of all three types of channels at each ion concentration. For each category of wild-type and F/K channels, IBa/ICa values were similar at each ion concentration, while tail currents were evoked at potentials from −80 to −20 mV. These observations suggest that the extracellular divalent ion concentration is a critical parameter in determining the selectivity properties of the pore.

Figure 4 Voltage and concentration dependency of IBa/ICa and the anomalous mole fraction effect (AMFE) for F/K mutant channels. Cells were stepped from a holding potential of −90 to +50 mV for 100 msec, and peak tail currents were measured at molar ratios of external Ba2+ to total divalent cation concentrations [Ba2+/(Ba2+ + Ca2+)] of 0.0, 0.3, 0.5, 0.7, 0.9 and 1.0. The combined divalent concentration (Ba2+ + Ca2+) at each data point was kept at (A) 2, (B) 10 and (C) 20 mM, respectively. AMFE is observed when peak tail currents, normalized to 1.0 when Ba2+/(Ba2+ + Ca2+) = 0, are plotted against Ba2+/(Ba2+ + Ca2+) at holding potentials of −80, −60, −40 and −20 mV. The dashed line corresponds to peak tail currents measured in an external solution containing 0.0 mM Ba2+. (A) AMFE is evident at 2 mM combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.491±0.021 [Ba2+/(Ba2+ + Ca2+) = 0.9] and IBa/ICa is 0.966±0.440, n=5. The smallest normalized current of F/K is distinguished from that of wild-type (Δ). (B) AMFE is evident at 10 mM combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.590±0.019 [Ba2+/(Ba2+ + Ca2+) = 0.9], which is different from that at 2 mM combined divalent concentration and shown as an asterisk and IBa/ICa is 0.908±0.044, n=8. (C) AMFE is evident at 20 mM of the combined divalent concentration. The most pronounced AMFE is at a holding potential of −20 mV. The smallest normalized current is 0.716±0.010 [Ba2+/(Ba2+ + Ca2+) = 0.7], which is different from that at 2 mM combined divalent concentration and shown as ‘*’ and IBa/ICa is 0.990±0.027, n=9.

Voltage and concentration dependence of AMFE

The AMFE of the indicated holding voltages and external solution concentrations for wild-type and mutant channels were recorded, respectively. While the holding potential declined from −80 to −20 mV, AMFEs for wild-type and F/K channels became more pronounced (Figs. 2 and 4). The smallest normalized currents exhibited differently when the total divalent cation concentrations remained at 2 mM compared to 10 or 20 mM (Fig. 2). Consistent with a previous report (25), AMFE was greatest when tail currents were evoked at relatively positive potentials (−20 mV) and when the total divalent cation concentrations were kept low (i.e., 2 mM).

The AMFE can be attenuated or accentuated by pore substitution

The normalized current for wild-type in 2 mM external solution decreased to ~0.58 when Ba2+/(Ba2+ + Ca2+) = 0.9 before rapidly climbing to ~1.08 as Ba2+/(Ba2+ + Ca2+), approaching 1.0 (Fig. 2A). In contrast to F/G (Fig. 3), F/K (Fig. 4) exhibited robust AMFE, similar to wild-type. The relative current amplitudes for F/K are indistinguishable when Ba2+/(Ba2+ + Ca2+) = 1, yet F/K presents an AMFE, indicating that the IBa/ICa and AMFE may be altered independently. In 2 mM external solution the AMFE for F/K (Fig. 4A) was greater than that for wild-type (Fig. 2A) and the normalized current for F/K reached a minimum value of 0.49 compared with 0.58 for wild-type.

Discussion

General

Mutagenesis was applied in the pore segment to assess the effects on the voltage and concentration dependency of IBa/ICa and the AMFE of CaV1.2 channels. Of the substitutions investigated in this study, F/G does not promote AMFE. However, F/K exhibits the voltage and concentration dependence of AMFE, which is more pronounced than that of wild-type channels.

Glycine and lysine substitutions of Phe-1144 affect AMFE through different mechanisms

The multi-ion interaction within the pore of the Ca2+ channel has long been a topic of research. Traditional theories (3,4) have indicated that an electrostatic interaction occurs between one ion bound to the pore and a second entering the pore. The electrostatic interaction causes an intricate adjustment of the conductance and activity relationship between the ions. The phenomenon of AMFE is an important demonstration of this interaction. AMFE appears when a mixture of two classes of permeant ions generates less current than either class of permeant ion alone. AMFE is an intricate phenomenon that is determined by the holding voltage and total ion concentration, as well as the inherent binding characteristics of the channel. Thus, AMFE is a significant biophysical probe of ion-ion interactions in the open pore of VGCCs.

Furthermore, Williamson and Sather (26) suggested that the unitary channel conductance is proportional to the volume of the side chain introduced by the amino acid residue at position 1144 adjacent to the residue of the selectivity filter. As the van der Waals volume of the positively charged lysine is similar to that of phenylalanine, F/K would be expected to demonstrate conductance resembling that of wild-type. However, a previous finding (17) that revealed the Ba2+ conductance of F/K is similar to that of F/G but not wild-type, is not compatible with this hypothesis. We suggest that Glu-1145 may produce a more restricted interaction with its neighboring residue as a positively charged lysine but a less restricted interaction with its neighboring residue as a neutral glycine. Moreover, one would predict that there are extra subtle differences between the conductance properties of the two mutants as both F/G and F/K reduce Ba2+ currents (17). Such differences may be further revealed by observing their respective AMFEs.

We studied AMFE at a variety of voltages and concentrations to examine how the mutations would affect ion-ion interactions in the open pore of Ca2+ channels. The results indicate that AMFE for F/G is lower than that for wild-type, while for F/K it is more pronounced than that for wild-type, although the voltage and concentration dependency remain the same. These results suggest that the characteristics of the amino acid residue at position 1144 are capable of accentuating or attenuating the AMFE. Thus, the finding that glycine and lysine substitutions at position 1144 are important determinants in modifying the AMFE suggests that F/G and F/K reduce Ba2+ conductance through different mechanisms.

Phe-1144 substitutions confer to structure-based models for Ca2+ channel permeation

The pore selectivity filter, the EEEE locus, is the critical structure for high Ca2+ selectivity while simultaneously promoting the high rates of Ca2+ flux of the voltage-gated calcium channel, as it forms the Ca2+ binding site (27). However, our findings are consistent with an increasing number of studies indicating that residues near or even far from the EEEE locus also participate in channel permeation (21,26,28,29). The traditional two-site, three-barrier models for the fundamental properties of ion selectivity and permeation through Ca2+ channels are based on measurable forces and binding energies. Therefore, these models cannot be used for channel structural studies, as the forces and binding energies cannot be measured for realistic channel structures. There are several later models based on structures to simulate many of the biophysical characteristics of ion permeation for the Ca2+ channel pore (11–13,30–32).

Lipkind and Fozzard (12) suggested a structural model that may be considered as a structural correlate to the stepwise model of Dang and McCleskey (7). The former model concludes that there are three binding sites formed by the eight carboxyl groups from the EEEE locus: a central, single high-affinity divalent cation binding site composed of four of the carboxyl groups flanked by two low-affinity divalent cation binding sites on either side, each composed of two carboxyl groups. On the basis of this model, we conclude that substitutions at position 1144 change the interactions between one of the two low-affinity binding sites and Ba2+.

The early barrier models (3,4) proposed that the selectivity filter has a defined volume. Furthermore, newer models (10,11,13,31) proposed that the volume is formed by the eight carbonyl oxygen atoms from the EEEE locus. Wang et al(17) used the ‘volume exclusion/charge neutralization’ model to explain the reduced conductance of Ba2+ but not Ca2+ in the pores of the F/G mutant channels. The crystal diameters of Ca2+ and Na+ ions are almost equal (2.00 vs. 2.04 Å, respectively), but each Ca2+ ion carries twice as much charge as an Na+ ion. Thus, Ca2+ is capable of neutralizing the highly negatively charged EEEE to bind tightly to the selectivity filter without the overcrowding suspected for monovalent cations such as Na+. Ba2+ and Ca2+ ions carry the same charge, but the ionic diameter of Ba2+ is ~36% larger than that of Ca2+ (2.72 vs. 2.00 Å, respectively). Thus, for wild-type channels, Ba2+ ions are expected to exhibit a higher degree of crowding than Ca2+ ions, as multiple ions attempt to bind and neutralize the negatively charged EEEE, resulting in an increasing exit rate, lower binding affinity and higher conductance relative to Ca2+. Our results resemble this ‘volume exclusion/charge neutralization’ model. The substitutions at position 1144 are assumed to alter the geometry and/or electrostatic context of the selectivity filter, in order to lessen the capability of overcrowding by Ba2+ ions, allowing it to contain multiple larger Ba2+ ions; however, its ability to contain multiple Ca2+ ions changes little.

Residues at position 1144 participate in the permeation of several classes of voltage-gated Ca2+ channels

Results of the present study have shown that non-glutamate residues in the pore of L-type Ca2+ channels change the characteristics of ion permeation. The CaV3-like mutant channel tested, F/K, promoted the AMFE phenomenon that was more comparable with CaV1.2 channels than CaV2 channels. Thus, the non-glutamate residues are critical for determining the permeability characteristics of voltage-gated Ca2+ channels. There are an increasing number of other studies also supporting this conclusion (17,21,26,28,29,33). Our previous study substituted an outer vestibule amino acid, Glu-1126, of CaV1.2 channels and revealed that the preference of the channel is approximately halved for passing Ba2+, but remains the same for passing Ca2+(21). Even substitutions as far as 100 residues upstream of EEEE in CaV2.2 channels reduce the preference of the channel for passing Ba2+ over Ca2+ currents (33). Regardless of the precise mechanism and simulation, our results provide further support for how the nonglutamate residues participate in an electrostatic and chemical environment contributing to ion permeation. More specifically, we have investigated the voltage and concentration dependence of the AMFE phenomenon of substituting mutant channels. The consequences of this study are likely to expand the understanding of the molecular mechanisms contributing to ion permeation of VGCCs.

Clinical implications

VGCCs are important in physiological functions of the cardiovascular system. They are critical factors of pathological changes in common or frequently encountered diseases of the cardiovascular system, such as arrhythmia, hypertension and coronary heart disease. Furthermore, they are functional targets of multiple commonly used drugs, such as calcium channel blockers. Ion permeation, as an essential biophysical property of VGCCs, is under close attention from investigators worldwide; however, its functional mechanisms have not been clearly elucidated. Explorations of the functional mechanisms of calcium channels to determine the essential qualities at the ion channel level of physiological and pathological phenomenon as well as investigating the theoretical basis at a molecular level of the pathogenesis and therapeutics of diseases are underway.

Acknowledgements

We are grateful for the financial support from the Superior Group Grant of Hubei Province Funding no. 2007ABC011 to C.-X.H. and the Special Grant for Basic Scientific Research Expenses of the Central Universities no. 302274994 to Z.L.

References