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.     

Critical determinants of Ca(2+)-dependent inactivation within an EF-hand motif of L-type Ca(2+) channels

L-type (alpha(1C)) calcium channels inactivate rapidly in response to localized elevation of intracellular Ca(2+), providing negative Ca(2+) feedback in a diverse array of biological contexts. The dominant Ca(2+) sensor for such Ca(2+)-dependent inactivation has recently been identified as calmodulin, which appears to be constitutively tethered to the channel complex. This Ca(2+) sensor induces channel inactivation by Ca(2+)-dependent CaM binding to an IQ-like motif situated on the carboxyl tail of alpha(1C). Apart from the IQ region, another crucial site for Ca(2+) inactivation appears to be a consensus Ca(2+)-binding, EF-hand motif, located approximately 100 amino acids upstream on the carboxyl terminus. However, the importance of this EF-hand motif for channel inactivation has become controversial since the original report from our lab implicating a critical role for this domain. Here, we demonstrate not only that the consensus EF hand is essential for Ca(2+) inactivation, but that a four-amino acid cluster (VVTL) within the F helix of the EF-hand motif is itself essential for Ca(2+) inactivation. Mutating these amino acids to their counterparts in non-inactivating alpha(1E) calcium channels (MYEM) almost completely ablates Ca(2+) inactivation. In fact, only a single amino acid change of the second valine within this cluster to tyrosine (V1548Y) supports much of the functional knockout. However, mutations of presumed Ca(2+)-coordinating residues in the consensus EF hand reduce Ca(2+) inactivation by only approximately 2-fold, fitting poorly with the EF hand serving as a contributory inactivation Ca(2+) sensor, in which Ca(2+) binds according to a classic mechanism. We therefore suggest that while CaM serves as Ca(2+) sensor for inactivation, the EF-hand motif of alpha(1C) may support the transduction of Ca(2+)-CaM binding into channel inactivation. The proposed transduction role for the consensus EF hand is compatible with the detailed Ca(2+)-inactivation properties of wild-type and mutant V1548Y channels, as gauged by a novel inactivation model incorporating multivalent Ca(2+) binding of CaM.     

Analysis of the modal hypothesis of Ca2+-dependent inactivation of L-type Ca2+ channels

A kinetic model of Ca2+-dependent inactivation (CDI) of L-type Ca2+ channels was developed. The model is based on the hypothesis that postulates the existence of four short-lived modes with lifetimes of a few hundreds of milliseconds. Our findings suggest that the transitions between the modes is primarily determined by the binding of Ca2+ to two intracellular allosteric sites located in different motifs of the CI region, which have greatly differing binding rates for Ca2+ (different k(on)). The slow-binding site is controlled by local Ca2+ near a single open channel that is consistent with the "domain" CDI model, and Ca2+ binding to the fast-binding site(s) depends on Ca2+ arising from distant sources that is consistent with the "shell" CDI model. The model helps to explain numerous experimental findings that are poorly understood so far.     

Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.

Ca(2+)-dependent inactivation (CDI) of L-type Ca(2+) channels plays a critical role in controlling Ca(2+) entry and downstream signal transduction in excitable cells. Ca(2+)-insensitive forms of calmodulin (CaM) act as dominant negatives to prevent CDI, suggesting that CaM acts as a resident Ca(2+) sensor. However, it is not known how the Ca(2+) sensor is constitutively tethered. We have found that the tethering of Ca(2+)-insensitive CaM was localized to the C-terminal tail of alpha(1C), close to the CDI effector motif, and that it depended on nanomolar Ca(2+) concentrations, likely attained in quiescent cells. Two stretches of amino acids were found to support the tethering and to contain putative CaM-binding sequences close to or overlapping residues previously shown to affect CDI and Ca(2+)-independent inactivation. Synthetic peptides containing these sequences displayed differences in CaM-binding properties, both in affinity and Ca(2+) dependence, leading us to propose a novel mechanism for CDI. In contrast to a traditional disinhibitory scenario, we suggest that apoCaM is tethered at two sites and signals actively to slow inactivation. When the C-terminal lobe of CaM binds to the nearby CaM effector sequence (IQ motif), the braking effect is relieved, and CDI is accelerated.     

Extremely slow inactivation of the ion channels formed by transfected α2 of L-type Ca2+ channelsof L-type Ca2+ channels

Barium currents through ion channels formed by α1-subunit of L-type Ca²⁺ channel (I _α1) were recorded from cultured chinese hamster ovary (CHO) cells. The cells were stably transfected with either a cardiac or a smooth muscle (SM) variant of α1-subunit. TheI _α1 in both cases exhibited similar fast voltage-dependent activation kinetics and slow apparent inactivation kinetics. With 10 mM Ba²⁺ in the bath solution,I _α1 was activated at potentials more positive than −40 mV, peaked between 0 and +10 mV, and reversed at about +50 mV. In addition to slow apparent inactivation of inward current, both subunits provided an extremely slow voltage-dependent inactivation at potentials more positive than −100 mV, with half-maximum inactivation at −43.4 mV for cardiac and −41.4 mV for SM α1-subunits. The onset of inactivation as well as recovery from this process were within a time range of minutes. The voltage dependence of steady-state inactivation could be fitted by the sum of two Boltzmann's equations with slope factors of about 12 mV and 5 mV. A less sloped component has its midpoints at −75.6 and −63.7 mV, and a steeper component has its midpoints at −42.8 and −37.7 mV for cardiac and SM α1-subunits, respectively. Relative contribution of the steeper component was higher in both subunits (0.86 and 0.66 for cardiac and SM subunits, respectively). For comparison, the inactivation curves for 5-sec-long conditioning prepulses could be fitted by single Boltzmann's distribution with a 20 mV more positive midpoint and a slope factor of about 13 mV. In contrast to the steady-state inactivation curves, they showed considerable overlap with the steady-state activation curve. Our results reflect functional consequences of known sequence differences between α1-subunits of the cardiac and SM L-type Ca²⁺ channels and could be used in structural modeling of Ca²⁺ channel gating. In addition, they show that depolarization-induced window current has a transient nature and decays with the development of extremely slow inactivation. This is the first demonstration that slow inactivation of the L-type Ca²⁺ channel is an intrinsic property of its α1-subunits.     

Modulation of the conductance of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions

The accompanying paper (Josephson, I. R., A. Guia, E. G. Lakatta, and M. D. Stern. 2002. Biophys. J. 83:2575-2586) examined the effects of conditioning prepulses on the kinetics of unitary L-type Ca(2+) channel currents using Ca(2+) and Ba(2+) ions to determine the ionic-dependence of gating mechanisms responsible for channel inactivation and facilitation. Here we demonstrate that in addition to alterations in gating kinetics, the conductance of single L-type Ca(2+) channels was also dependent on the prior conditioning voltage and permeant ions. All recordings were made in the absence of any Ca(2+) channel agonists. Strongly depolarizing prepulses produced an increased frequency of long-duration (mode 2) openings during the test voltage steps. Mode 2 openings also displayed >25% larger single channel current amplitude (at 0 mV) than briefer (but well-resolved) mode 1 openings. The conductance of mode 2 openings was 26 pS for 105 mM Ba(2+), 18 pS for 5 mM Ba(2+), and 6 pS for 5 mM Ca(2+) ions; these values were 70% greater than the conductance of Ca(2+) channel openings of all durations (mode 1 and mode 2). Thus, the prepulse-driven shift into mode 2 gating results in a longer-lived Ca(2+) channel conformation that, in addition, displays altered permeation properties. These results, and those in the accompanying paper, support the hypothesis that multiple aspects of single L-type Ca(2+) channel behavior (gating kinetics, modal transitions, and ion permeation) are interrelated and are modulated by the magnitude of the conditioning depolarization and the nature and concentration of the ions permeating the channel.     

Modulation of the gating of unitary cardiac L-type Ca(2+) channels by conditioning voltage and divalent ions

Although a considerable number of studies have characterized inactivation and facilitation of macroscopic L-type Ca(2+) channel currents, the single channel properties underlying these important regulatory processes have only rarely been examined using Ca(2+) ions. We have compared unitary L-type Ca(2+) channel currents recorded with a low concentration of Ca(2+) ions with those recorded with Ba(2+) ions to elucidate the ionic dependence of the mechanisms responsible for the prepulse-dependent modulation of Ca(2+) channel gating kinetics. Conditioning prepulses were applied across a wide range of voltages to examine their effects on the subsequent Ca(2+) channel activity, recorded at a constant test potential. All recordings were made in the absence of any Ca(2+) channel agonists. Moderate-depolarizing prepulses resulted in a decrease in the probability of opening of the Ca(2+) channels during subsequent test voltage steps (inactivation), the extent of which was more dramatic with Ca(2+) ions than Ba(2+) ions. Facilitation, or increase of the average probability of opening with strong predepolarization, was due to long-duration mode 2 openings with Ca(2+) ions and Ba(2+) ions, despite a decrease in Ca(2+) channel availability (inactivation) under these conditions. The degree of both prepulse-induced inactivation and facilitation decreased with increasing Ba(2+) ion concentration. The time constants (and their proportions) describing the distributions of Ca(2+) channel open times (which reflect mode switching) were also prepulse-, and ion-dependent. These results support the hypothesis that both prior depolarization and the nature and concentration of permeant ions modulate the gating properties of cardiac L-type Ca(2+) channels.     

电压门控钙通道钙依赖性易化和失活的分子机制

电压门控钙通道受钙依赖性易化和失活两种相互对立的反馈机制调节.不同浓度的钙离子,通过作为钙感受器的钙调蛋白的介导,主要与钙通道α1亚基羧基端的多个不连续片段发生复杂的相互作用,分别引发钙依赖性易化和失活.钙/钙调蛋白依赖性蛋白激酶Ⅱ及其它钙结合蛋白等也参与此调节过程.新近研究表明,钙通道的钙依赖性调节机制失衡与心律失常等的发病机制密切相关.     

Regulation of CRAC channels by Ca2+-dependent inactivation

CRAC channels are a major route for Ca²⁺ influx in eukaryotic cells. The channels show prominent Ca²⁺-dependent inactivation through two spatially and temporally distinct mechanisms: fast inactivation, which develops over milliseconds and is triggered by Ca²⁺ near the mouth of the channel and slow inactivation, which arises over tens of seconds and requires a rise in global cytosolic Ca²⁺. Slow inactivation is controlled physiologically by Ca²⁺ uptake into mitochondria through the MCU. Site-directed mutagenesis studies on STIM1 and Orai1 have led to new molecular insight into how fast inactivation occurs. This review describes properties and molecular mechanisms that contribute to these important Ca²⁺-dependent inhibitory pathways.     

Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents inHelix aspersa neurons

The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca²⁺ currents (L-type ICa²⁺) and their Ca²⁺-dependent inactivation, were studied in identifiedHelix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca²⁺]_i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa²⁺ (IC₅₀=28 μM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca²⁺-dependent inactivation of L-type ICa²⁺. Trifluoperazine sulfoxide (50 μM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa²⁺. TFP (20 μM) increased basal [Ca²⁺]_i from 147±37 nM to 650±40nM (N=7). The increase in [Ca²⁺]_i was prevented by removal of external Ca²⁺ and curtailed by depletion of caffeine-sensitive intracellular Ca²⁺ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-O-tetradecanoyl-phorbol-13-acetate) on L-type Ca²⁺ currents. This compound produced an increase in L-type ICa²⁺ without enhancing Ca²⁺-dependent inactivation. The results show that 1) TFP reduces L-type ICa²⁺ while enhancing the efficacy of Ca²⁺-dependent inactivation. 2) TFP produces an increase in basal [Ca²⁺]_i which may contribute to the enhancement of Ca²⁺-dependent inactivation. 3) PKC up-regulates L-type ICa²⁺ without altering the efficacy of Ca²⁺ dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.     

Species-specific difference in distribution of voltage-gated L-type Ca(2+) channels of cardiac myocytes

Ca²⁺influx via sarcolemmal voltage-dependent Ca²⁺ channels(L-type Ca²⁺ channels) is the fundamental step inexcitation-contraction (E-C) coupling in cardiac myocytes.Physiological and pharmacological studies reveal species-specificdifferences in E-C coupling resulting from a difference in thecontribution of Ca²⁺ influx and intracellularCa²⁺ release to activation of contraction. We investigatedthe distribution of L-type Ca²⁺ channels in isolatedcardiac myocytes from rabbit and rat ventricle by correlativeimmunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In ratmyocytes, labeling appeared more intense in T tubules than in thesurface sarcolemma. Immunogold electron microscopy extended thesefindings, showing that the number of gold particles in the surfaceplasma membrane was significantly higher in rabbit than rat myocytes.In rabbit myocyte plasma membrane, the gold particles were distributedas clusters in both regions that were associated with junctionalsarcoplasmic reticulum and those that were not. The findings areconsistent with the idea that influx of Ca²⁺ via surfacesarcolemmal Ca²⁺ channels contributes to intracellularCa²⁺ to a greater degree in rabbit than in rat myocytes.

     

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.     

L-type Ca2+ channels in Ca2+ channelopathies

Voltage-gated L-type Ca2+ channels (LTCCs) mediate depolarization-induced Ca2+ entry in electrically excitable cells, including muscle cells, neurons, and endocrine and sensory cells. In this review we summarize the role of LTCCs for human diseases caused by genetic Ca2+ channel defects (channelopathies). LTCC dysfunction can result from structural aberrations within pore-forming alpha1 subunits causing incomplete congenital stationary night blindness, malignant hyperthermia sensitivity or hypokalemic periodic paralysis. However, studies in mice revealed that LTCC dysfunction also contributes to neurological symptoms in Ca2+ channelopathies affecting non-LTCCs, such as Ca(v)2.1 alpha1 in tottering mice. Ca2+ channelopathies provide exciting molecular tools to elucidate the contribution of different LTCC isoforms to human diseases.     

Endogenous and exogenous Ca2+ buffers differentially modulate Ca2+-dependent inactivation of Ca(v)2.1 Ca2+ channels

Voltage-gated Ca2+ channels undergo a negative feedback regulation by Ca2+ ions, Ca2+-dependent inactivation, which is important for restricting Ca2+ signals in nerve and muscle. Although the molecular details underlying Ca2+-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca2+-dependent inactivation of Ca(v)2.1 (P/Q-type) Ca2+ channels is suppressed by strong cytoplasmic Ca2+ buffering, we investigated how factors that regulate cellular Ca2+ levels affect inactivation of Ca(v)2.1 Ca2+ currents in transfected 293T cells. We found that inactivation of Ca(v)2.1 Ca2+ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca2+ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca2+ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca2+-dependent inactivation of Ca(v)2.1. Cotransfection of Ca(v)2.1 with the EF-hand Ca2+-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca2+ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca2+ buffers. We conclude that Ca2+-dependent inactivation of Ca(v)2.1 depends on a subplasmalemmal Ca2+ microdomain that is affected by the amplitude of the Ca2+ current and differentially modulated by distinct Ca2+ buffers.     

Trypsin increases availability and open probability of cardiac L-type Ca2+ channels without affecting inactivation induced by Ca2+.

The patch-clamp technique was employed to investigate the response of single L-type Ca2+ channels to the protease trypsin applied to the intracellular face of excised membrane patches from guinea pig ventricular myocytes. Calpastatin and ATP were used to prevent run-down of Ca2+ channel activity monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)-BAYK 8644. Upon application of trypsin (100 micrograms/ml) channel activity was enhanced fourfold and remained elevated upon removal of trypsin, as expected of a proteolytic, irreversible modification. The trypsin effect was not mediated by a proteolytic activation of protein kinases, as evidenced by the insensitivity of this effect to protein kinase inhibitors. Trypsin-modified Ca2+ channels exhibited the usual run-down phanomenon upon removal of calpastatin and ATP. In ensemble average currents trypsin-induced changes of channel function are apparent as a threefold increase in peak current and a reduction in current inactivation. At the single channel level these effects were based on about a twofold increase in both Ca2+ channels' availability and open probability. Neither the actual number of channels in the patch nor their unitary conductance as well as reversal potential was changed by trypsin. The Ca(2+)-induced inactivation was not impaired, as judged by a comparable sensitivity of trypsin-modified Ca2+ channels to intracellular Ca2+. Similarly, trypsin treatment did not affect the sensitivity of Ca2+ channels to phenylalkylmine inhibition. The observed alterations in channel function are discussed in terms of possible structural correlates.     

Modification of annexin II expression in PC12 cell lines does not affect Ca(2+)-dependent exocytosis.

The Ca2+/phospholipid/cytoskeletal-binding protein annexin II has been proposed to play an important role in Ca(2+)-dependent exocytosis; however, the evidence for this role is inconclusive. More direct evidence obtained by manipulating annexin II levels in cells is still required. We have attempted to do this by generating stably transfected PC12 cell lines expressing proteins which elevate or lower functional annexin II levels and using these cell lines to investigate Ca(2+)-dependent exocytosis. Three cell lines were generated: one expressing an annexin II mutant which aggregates annexin II in at least a proportion of the cells, thereby removing functional protein from the cell; a mixed clonal cell line constitutively overexpressing human annexin II; and a clonal cell line capable of over-expressing annexin II in the presence of sodium butyrate. After digitonin permeabilization, Ca(2+)-dependent dopamine release from these cell lines was compared with that from control nontransfected cells, and, in addition, release was compared in induced to uninduced cells. There were no significant differences in Ca(2+)-dependent exocytosis between any of the transfected cell lines before or after induction and the control cells. In addition, nontransfected PC12 cells treated with nerve growth factor, which elevates annexin II levels severalfold, failed to increase Ca(2+)-dependent exocytosis after digitonin permeabilization, compared with control cells. We conclude that annexin II is not an important regulator of Ca(2+)-dependent exocytosis in PC12 cells.     

Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca(2+) channel currents

Weinvestigated the inactivation process of macroscopic cardiac L-typeCa²⁺ channel currents using the whole cell patch-clamptechnique with Na⁺ as the current carrier. The inactivationprocess of the inward currents carried by Na⁺ through thechannel consisted of two components >0 mV. The time constant of thefaster inactivating component (30.6 ± 2.2 ms at 0 mV) decreasedwith depolarization, but the time constant of the slower inactivatingcomponent (489 ± 21 ms at 0 mV) was not significantly influencedby the membrane potential. The inactivation process in the presence ofisoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currentsand attenuated the effects of isoproterenol. In the presence of cAMP(500 µM), the inactivation process consisted of a single slowcomponent. We propose that the faster inactivating component representsa kinetic of the dephosphorylated or partially phosphorylated channel,and phosphorylation converts the kinetics into one with a differentvoltage dependency.