The EEEE locus is the sole high-affinity Ca(2+) binding structure in the pore of a voltage-gated Ca(2+) channel: block by ca(2+) entering from the intracellular pore entrance

Selective permeability in voltage-gated Ca(2+) channels is dependent upon a quartet of pore-localized glutamate residues (EEEE locus). The EEEE locus is widely believed to comprise the sole high-affinity Ca(2+) binding site in the pore, which represents an overturning of earlier models that had postulated two high-affinity Ca(2+) binding sites. The current view is based on site-directed mutagenesis work in which Ca(2+) binding affinity was attenuated by single and double substitutions in the EEEE locus, and eliminated by quadruple alanine (AAAA), glutamine (QQQQ), or aspartate (DDDD) substitutions. However, interpretation of the mutagenesis work can be criticized on the grounds that EEEE locus mutations may have additionally disrupted the integrity of a second, non-EEEE locus high-affinity site, and that such a second site may have remained undetected because the mutated pore was probed only from the extracellular pore entrance. Here, we describe the results of experiments designed to test the strength of these criticisms of the single high-affinity locus model of selective permeability in Ca(2+) channels. First, substituted-cysteine accessibility experiments indicate that pore structure in the vicinity of the EEEE locus is not extensively disrupted as a consequence of the quadruple AAAA mutations, suggesting in turn that the quadruple mutations do not distort pore structure to such an extent that a second high affinity site would likely be destroyed. Second, the postulated second high-affinity site was not detected by probing from the intracellularly oriented pore entrance of AAAA and QQQQ mutants. Using inside-out patches, we found that, whereas micromolar Ca(2+) produced substantial block of outward Li(+) current in wild-type channels, internal Ca(2+) concentrations up to 1 mM did not produce detectable block of outward Li(+) current in the AAAA or QQQQ mutants. These results indicate that the EEEE locus is indeed the sole high-affinity Ca(2+) binding locus in the pore of voltage-gated Ca(2+) channels.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Calcium channel block by (-)devapamil is affected by the sequence environment and composition of the phenylalkylamine receptor site.

The pore-forming alpha 1 subunit of L-type calcium (Ca2+) channels is the molecular target of Ca2+ channel blockers such as phenylalkylamines (PAAs). Association and dissociation rates of (-)devapamil were compared for a highly PAA-sensitive L-type Ca2+ channel chimera (Lh) and various class A Ca2+ channel mutants. These mutants carry the high-affinity determinants of the PAA receptor site in a class A sequence environment. Apparent drug association and dissociation rate constants were significantly affected by the sequence environment (class A or L-type) of the PAA receptor site. Single point mutations affecting the high-affinity determinants in segments IVS6 of the PAA receptor site, introduced into a class A environment, reduced the apparent drug association rates. Mutation I1811M in transmembrane segment IVS6 (mutant AL25/-I) had the highest impact and decreased the apparent association rate for (-)devapamil by approximately 30-fold, suggesting that this pore-lining isoleucine in transmembrane segment IVS6 plays a key role in the formation of the PAA receptor site. In contrast, apparent drug dissociation rates of Ca2+ channels in the resting state were almost unaffected by point mutations of the PAA receptor site.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.     

Determinants for calmodulin binding on voltage-dependent Ca2+ channels

Calmodulin, bound to the alpha(1) subunit of the cardiac L-type calcium channel, is required for calcium-dependent inactivation of this channel. Several laboratories have suggested that the site of interaction of calmodulin with the channel is an IQ-like motif in the carboxyl-terminal region of the alpha(1) subunit. Mutations in this IQ motif are linked to L-type Ca(2+) current (I(Ca)) facilitation and inactivation. IQ peptides from L, P/Q, N, and R channels all bind Ca(2+)calmodulin but not Ca(2+)-free calmodulin. Another peptide representing a carboxyl-terminal sequence found only in L-type channels (designated the CB domain) binds Ca(2+)calmodulin and enhances Ca(2+)-dependent I(Ca) facilitation in cardiac myocytes, suggesting the CB domain is functionally important. Calmodulin blocks the binding of an antibody specific for the CB sequence to the skeletal muscle L-type Ca(2+) channel, suggesting that this is a calmodulin binding site on the intact protein. The binding of the IQ and CB peptides to calmodulin appears to be competitive, signifying that the two sequences represent either independent or alternative binding sites for calmodulin rather than both sequences contributing to a single binding site.     

Energetics of divalent selectivity in a calcium channel: the ryanodine receptor case study

A model of the ryanodine receptor (RyR) calcium channel is used to study the energetics of binding selectivity of Ca²⁺ versus monovalent cations. RyR is a calcium-selective channel with a DDDD locus in the selectivity filter, similar to the EEEE locus of the L-type calcium channel. While the affinity of RyR for Ca²⁺ is in the millimolar range (as opposed to the micromolar range of the L-type channel), the ease of single-channel measurements compared to L-type and its similar selectivity filter make RyR an excellent candidate for studying calcium selectivity. A Poisson-Nernst-Planck/density functional theory model of RyR is used to calculate the energetics of selectivity. Ca²⁺ versus monovalent selectivity is driven by the charge/space competition mechanism in which selectivity arises from a balance of electrostatics and the excluded volume of ions in the crowded selectivity filter. While electrostatic terms dominate the selectivity, the much smaller excluded-volume term also plays a substantial role. In the D4899N and D4938N mutations of RyR that are analyzed, substantial changes in specific components of the chemical potential profiles are found far from the mutation site. These changes result in the significant reduction of Ca²⁺ selectivity found in both theory and experiments.     

How do calcium channels transport calcium ions?

Calcium channel activity is crucial for many fundamental physiological processes ranging from the heart beat to synaptic transmission. The channel-forming protein, of about 2000 amino acids, comprises four domains internally homologous to each other. Voltage-dependent Ca2+ channels are the most selective ion channels known. Under physiological conditions, they prefer Ca2+ over Na+ by a ratio of about 1000:1. To explain at the same time the exquisite ion selectivity and the large Ca2+ ion turnover rate of Ca2+ channels (approximately 3 x 10(6) ions/s), two kind models have been proposed. In one, the conduction pathway possesses two high-affinity binding sites. When two Ca2+ ions are bound to each site, the mutual repulsion between them speeds the exit rate for the ions, causing greater ion permeation through the pore. The second model hypothesizes the existence of a single site having a charged structure able to attract multiple, interacting ions, simultaneously. Recent studies that combine mutagenesis and electrophysiology show that the high-affinity binding site is formed by a ring of glutamate residues located in the pore forming region of the Ca2+ channel. As proposed in the second class of models, the results suggest that four glutamate residues, one glutamate donated by each repeat, combine to form a single high-affinity site. In this review the different conduction models for Ca2+ channels are discussed and confronted with structural data.     

Probing ion binding in the selectivity filter of the Cav1.1 channel with molecular dynamics

Ca_v1.1 is the voltage-gated calcium channel essential for the contraction of skeletal muscles upon membrane potential changes. Structural determination of the Ca_v1.1 channel opens the avenue toward understanding of the structure-function relationship of voltage-gated calcium channels. Here, we show that there exist two Ca²⁺-binding sites, termed S1 and S2, within the selectivity filter of Ca_v1.1 through extensive molecular dynamics simulations on various initial ion arrangement configurations. The formation of both binding sites is associated with the four Glu residues (Glu292/614/1014/1323) that constitute the so-called EEEE locus. At the S1 site near the extracellular side, the Ca²⁺ ion is coordinated with the negatively charged carboxylic groups of these Glu residues and of the Asp615 residue either in a direct way or via an intermediate water molecule. At the S2 site, Ca²⁺ binding shows two distinct states: an upper state involving two out of the four Glu residues in the EEEE locus and a lower state involving only one Glu residue. In addition, there exist two recruitment sites for Ca²⁺ above the entrance of the filter. These findings promote the understanding of mechanism for ion permeation and selectivity in calcium channels.     

Volume regulatory decrease in UMR-106.01 cells is mediated by specific α1 subunits of L-type calcium channels

An early cellular response of osteoblasts to swelling is plasma membrane depolarization, accompanied by a transient increase in intracellular calcium ([Ca2+]i), which initiates regulatory volume decrease (RVD). The authors have previously demonstrated a hypotonically induced depolarization of the osteoblast plasma membrane, sufficient to open L-type Ca channels and mediate Ca2+ influx. Herein is described the initiation of RVD in UMR-106.01 cells, mediated by hypotonically induced [Ca2+]i transients resulting from the activation of specific isoforms of L-type Ca channels. The authors further demonstrate that substrate interaction determines which specific alpha1 Ca channel subunit isoform predominates and mediates Ca2+ entry and RVD. Swelling-induced [Ca2+]i transients, and RVD in cells grown on a type I collagen matrix, are inhibited by removal of Ca from extracellular solutions, dihydropyridines, and antisense oligodeoxynucleotides directed exclusively to the alpha1C isoform of the L-type Ca channel. Ca2+ transients and RVD in cells grown on untreated glass cover slips were inhibited by similar maneuvers, but only by antisense oligodeoxynucleotides directed to the alpha1S isoform of the L-type Ca channel. This represents the first molecular identification of the Ca channels that transduce the initiation signal for RVD by osteoblastic cells.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Nonglutamate pore residues in ion selection and conduction in voltage-gated Ca(2+) channels

High-affinity, intrapore binding of Ca(2+) over competing ions is the essential feature in the ion selectivity mechanism of voltage-gated Ca(2+) channels. At the same time, several million Ca(2+) ions can travel each second through the pore of a single open Ca(2+) channel. How such high Ca(2+) flux is achieved in the face of tight Ca(2+) binding is a current area of inquiry, particularly from a structural point of view. The ion selectivity locus comprises four glutamate residues within the channel's pore. These glutamates make unequal contributions to Ca(2+) binding, underscoring a role for neighboring residues in pore function. By comparing two Ca(2+) channels (the L-type alpha(1C), and the non-L-type alpha(1A)) that differ in their pore properties but only differ at a single amino acid position near the selectivity locus, we have identified the amino-terminal neighbor of the glutamate residue in motif III as a determinant of pore function. This position is more important in the function of alpha(1C) channels than in alpha(1A) channels. For a systematic series of mutations at this pore position in alpha(1C), both unitary Ba(2+) conductance and Cd(2+) block of Ba(2+) current varied with residue volume. Pore mutations designed to make alpha(1C) more like alpha(1A) and vice versa revealed that relative selectivity for Ba(2+) over K(+) depended almost solely on pore sequence and not channel type. Analysis of thermodynamic mutant cycles indicates that the motif III neighbor normally interacts in a cooperative fashion with the locus, molding the functional behavior of the pore.     

Ion interactions in the high-affinity binding locus of a voltage-gated Ca(2+) channel

The selectivity filter of voltage-gated Ca(2+) channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca(2+) channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca(2+) channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd(2+), which binds very tightly within the EEEE locus, and Ba(2+) or Li(+), which bind less tightly and hence exhibit high flux rates: Cd(2+) binds to the EEEE locus approximately 10(4)x more tightly than does Ba(2+), and approximately 10(8)x more tightly than does Li(+). For wild-type channels, Cd(2+) entry into the EEEE locus was 400x faster when Li(+) rather than Ba(2+) was the current carrier, reflecting the large difference between Ba(2+) and Li(+) in affinity for the EEEE locus. For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba(2+) vs. Li(+)). The dependence of Cd(2+) block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.     

Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L-type calcium channels

Elevated intracellular Ca2+ triggers inactivation of L-type calcium channels, providing negative Ca2+ feedback in many cells. Ca2+ binding to the main alpha1c channel subunit has been widely proposed to initiate such Ca2+ -dependent inactivation. Here, we find that overexpression of mutant, Ca2+ -insensitive calmodulin (CaM) ablates Ca2+ -dependent inactivation in a "dominant-negative" manner. This result demonstrates that CaM is the actual Ca2+ sensor for inactivation and suggests that CaM is constitutively tethered to the channel complex. Inactivation is likely to occur via Ca2+ -dependent interaction of tethered CaM with an IQ-like motif on the carboxyl tail of alpha1c. CaM also binds to analogous IQ regions of N-, P/Q-, and R-type calcium channels, suggesting that CaM-mediated effects may be widespread in the calcium channel family.     

3H]PN200-110 binding in a fibroblast cell line transformed with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel

We examined the binding of the 1,4-dihydropyridine (DHP) [3H]PN200-110 to membranes from a fibroblast cell line transfected with the alpha 1 subunit (DHP receptor) of the L-type Ca2+ channel from rabbit skeletal muscle. Binding site affinity (KD) and density (Bmax) were 1.16 +/- 0.31 nM and 142 +/- 17 fmoles/mg protein, respectively. This affinity corresponded closely with that observed in native skeletal muscle. The Ca2+ channel antagonists diltiazem and MDL 12,330A stimulated [3H]PN200-110 binding in a dose-dependent manner while flunarizine, quinacrine and trifluoperazine inhibited binding. Surprisingly, D600 also stimulated [3H]PN200-110 binding in a dose-dependent and stereoselective manner. It is concluded that the fibroblast cells used in this study provide a unique system for interactions of the Ca2+ channel ligands with the alpha 1 subunit of the skeletal muscle L-type Ca2+ channel.     

Serine residue in the IIIS5-S6 linker of the L-type Ca2+ channel alpha 1C subunit is the critical determinant of the action of dihydropyridine Ca2+ channel agonists

The dihydropyridine (DHP)-binding site has been identified within L-type Ca(2+) channel alpha(1C) subunit. However, the molecular mechanism underlying modulation of Ca(2+) channel gating by DHPs has not been clarified. To search for novel determinants of high affinity DHP binding, we introduced point mutations in the rat brain Ca(2+) channel alpha(1C) subunit (rbCII or Ca(v)1.2c) based on the comparison of amino acid sequences between rbCII and the ascidian L-type Ca(2+) channel alpha(1) subunit, which is insensitive to DHPs. The alpha(1C) mutants (S1115A, S1146A, and A1420S) and rbCII were transiently expressed in BHK6 cells with beta(1a) and alpha(2)/delta subunits. The mutation did not affect the electrophysiological properties of the Ca(2+) channel, or the voltage- and concentration-dependent block of Ca(2+) channel currents produced by diltiazem and verapamil. However, the S1115A channel was significantly less sensitive to DHP antagonists. Interestingly, in the S1115A channel, DHP agonists failed to enhance whole-cell Ca(2+) channel currents and the prolongation of mean open time, as well as the increment of NP(o). Responsiveness to the non-DHP agonist FPL-64176 was also markedly reduced in the S1115A channel. When S1115 was replaced by other amino acids (S1115D, S1115T, or S1115V), only S1115T was slightly sensitive to S-(-)-Bay K 8644. These results indicate that the hydroxyl group of Ser(1115) in IIIS5-S6 linker of the L-type Ca(2+) channel alpha(1C) subunit plays a critical role in DHP binding and in the action of DHP Ca(2+) channel agonists.     

Binding and selectivity in L-type calcium channels: a mean spherical approximation

L-type calcium channels are Ca(2+) binding proteins of great biological importance. They generate an essential intracellular signal of living cells by allowing Ca(2+) ions to move across the lipid membrane into the cell, thereby selecting an ion that is in low extracellular abundance. Their mechanism of selection involves four carboxylate groups, containing eight oxygen ions, that belong to the side chains of the "EEEE" locus of the channel protein, a setting similar to that found in many Ca(2+)-chelating molecules. This study examines the hypothesis that selectivity in this locus is determined by mutual electrostatic screening and volume exclusion between ions and carboxylate oxygens of finite diameters. In this model, the eight half-charged oxygens of the tethered carboxylate groups of the protein are confined to a subvolume of the pore (the "filter"), but interact spontaneously with their mobile counterions as ions interact in concentrated bulk solutions. The mean spherical approximation (MSA) is used to predict ion-specific excess chemical potentials in the filter and baths. The theory is calibrated using a single experimental observation, concerning the apparent dissociation constant of Ca(2+) in the presence of a physiological concentration of NaCl. When ions are assigned their independently known crystal diameters and the carboxylate oxygens are constrained, e.g., to a volume of 0.375 nm(3) in an environment with an effective dielectric coefficient of 63.5, the hypothesized selectivity filter produces the shape of the calcium binding curves observed in experiment, and it predicts Ba(2+)/Ca(2+) and Na(+)/Li(+) competition, and Cl(-) exclusion as observed. The selectivities for Na(+), Ca(2+), Ba(2+), other alkali metal ions, and Cl(-) thus can be predicted by volume exclusion and electrostatic screening alone. Spontaneous coordination of ions and carboxylates can produce a wide range of Ca(2+) selectivities, depending on the volume density of carboxylate groups and the permittivity in the locus. A specific three-dimensional structure of atoms at the binding site is not needed to explain Ca(2+) selectivity.     

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.     

Ca2+- and voltage-dependent inactivation of the expressed L-type Ca(v)1.2 calcium channel

Ca2+-dependent regulation of the ion current through the alpha1Cbeta2aalpha2delta-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca2+ or Na+ ions were used as a charge carrier. Intracellular Ca2+ was either buffered by 10 mM EGTA or 200 microM Ca2+ was added into non-buffered intracellular solution. Free intracellular Ca2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, tau(fast) and tau(slow). Free intracellular calcium accelerated both time constants. Effect on the tau(slow) was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na+ ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.     

Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel''s open probability.

The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.