The amino terminus of high-voltage-activated calcium channels: CaM you or can't you?

Voltage-gated calcium channels (VGCCs), calmodulin (CaM), and calmodulin kinase II (CaMKII) are essential for various nervous system functions. CaM and CaMKII differentially regulate calcium dependent facilitation (CDF) and calcium dependent inactivation (CDI) of the Cav1 and Cav2 families of VGCCs. It is generally accepted that conserved structures in the C-terminus of these channels regulate CDF and CDI, and yet recent evidence indicates that other intracellular regions may be involved. We recently discovered that N-terminal sequences in Cav1.2 bind CaM and CaMKII, and function to regulate CDI as well as surface expression and open probability, respectively. Cav1 and Cav2 share significant portions of N-terminal sequence and therefore we explored whether homologous binding sites might exist in Cav2.1. Here, we show that like the proximal N-terminus of Cav1.2, the homologous region of Cav2.1 contains sequences which interact either directly or indirectly with CaM.     

Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

L-type calcium channels mediate acetylcholine receptor aggregation on cultured muscle

Agrin activation of muscle specific kinase (MuSK) initiates postsynaptic development on skeletal muscle that includes the aggregation of acetylcholine receptors (AChRs; Glass et al. [1996]: Cell 85: 513-523; Gautam et al. [1996]: Cell 85: 525-535). Although the agrin/MuSK signaling pathway remains largely unknown, changes in intracellular calcium levels are required for agrin-induced AChR aggregation (Megeath and Fallon [1998]: J Neurosci 18: 672-678). Here, we show that L-type calcium channels (L-CaChs) are required for full agrin-induced aggregation of AChRs and sufficient to induce agrin-independent AChR aggregation. Blockade of L-CaChs in muscle cultures inhibited agrin-induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Activation of L-CaChs in the absence of agrin induced AChR aggregation but not tyrosine phosphorylation of MuSK or AChR beta subunits. Agrin responsiveness was significantly reduced in primary muscle cultures from the muscular dysgenesis mouse, a natural mutant, which does not express the L-CaCh. Our results establish a novel role for L-CaChs as important sources of the intracellular calcium necessary for the aggregation of AChRs.     

Inactivation of calcium channels in vascular smooth muscle myocytes

Many of the structural domains involved in Ca²⁺ channel (CACN) inactivation are also involved in determining their sensitivity to antagonist inhibition. We hypothesize that differences in inactivation properties and their structural determinants may suggest candidate domains as targets for the development of novel, selective antagonists. The characteristics of Ca²⁺ current (I_Ca) inactivation, steady-state inactivation (SSIN), and recovery from inactivation were studied in freshly dispersed smooth muscle cells from rabbit portal vein (RPV) using whole-cell, voltage-clamp methods. The time course of inactivation could be represented by two time constants. Increasing I_Ca by increasing [Ca²⁺]_o or with more negative holding potentials decreased both time constants. With Sr²⁺, Ba²⁺, or Na⁺ as the charge carrier, I_Ca inactivation was also represented by two time constants, both of which were larger than those found with Ca²⁺. With Ca²⁺, Sr²⁺, or Ba²⁺ as the charge carrier, both time constants had minimum values near the voltage associated with maximum current. When Na⁺ (140 mM) was the charge carrier, voltages for I_max (−20 mV) or τ_min (o mV) did not correspond. SSIN of I_Ca had a half-maximum voltage of −32±4 mV for Ca²⁺, −43 mV±5 mV for Sr²⁺, −41±5 mV for Ba²⁺, and −68±6 mV for Na⁺. The slope factor for SSIN per e-fold voltage change was 6.5±0.2 mV for Ca²⁺, 6.8±0.3 for Sr²⁺, and 6.6±0.2 for Ba²⁺, representing four equivalent charges. When Na⁺ or Li⁺ was the charge carrier, the slope factor was 13.5±0.7 mV, representing two equivalent charges. For I_Ca in rat left ventricular (rLV) myocytes, there was no difference in the slope factor of SSIN for Ca²⁺ and Na⁺. The rate of recovery of I_Ca from inactivation varied inversely with recovery voltage and was independent of the charge carrier. These results suggest that inactivation of I_Ca in PV myocytes possess an intrinsic voltage dependence that is modified by Ca²⁺. For RPV but not rLV I_Ca, the charge of the permeating ion confers the voltage-dependency of SSIN.     

Decalmodulation of Cav1 channels by CaBPs

Ca²⁺-dependent inactivation (CDI) is a negative feedback regulation of voltage-gated Ca_v1 and Ca_v2 channels that is mediated by the Ca²⁺ sensing protein, calmodulin (CaM), binding to the pore-forming Ca_v α₁ subunit. David Yue and his colleagues made seminal contributions to our understanding of this process, as well as factors that regulate CDI. Important in this regard are members of a family of Ca²⁺ binding proteins (CaBPs) that are related to calmodulin. CaBPs are expressed mainly in neural tissues and can antagonize CaM-dependent CDI for Ca_v1 L-type channels. This review will focus on the roles of CaBPs as Ca_v1-interacting proteins, and the significance of these interactions for vision, hearing, and neuronal Ca²⁺ signaling events.     

A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca²⁺ signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.     

Modulation of distinct isoforms of L-type calcium channels by Gq-coupled receptors in Xenopus oocytes

L-type voltage dependent Ca²⁺ channels (L-VDCCs; Ca_v1.2) are crucial in cardiovascular physiology. In heart and smooth muscle, hormones and transmitters operating via G_q enhance L-VDCC currents via essential protein kinase C (PKC) involvement. Heterologous reconstitution studies in Xenopus oocytes suggested that PKC and G_q-coupled receptors increased L-VDCC currents only in cardiac long N-terminus (NT) isoforms of α_1C, whereas known smooth muscle short-NT isoforms were inhibited by PKC and G_q activators. We report a novel regulation of the long-NT α_1C isoform by Gβγ. Gβγ inhibited whereas a Gβγ scavenger protein augmented the G_q- but not phorbol ester-mediated enhancement of channel activity, suggesting that Gβγ acts upstream from PKC. In vitro binding experiments reveal binding of both Gβγ and PKC to α_1C-NT. However, PKC modulation was not altered by mutations of multiple potential phosphorylation sites in the NT, and was attenuated by a mutation of C-terminally located serine S1928. The insertion of exon 9a in intracellular loop 1 rendered the short-NT α_1C sensitive to PKC stimulation and to Gβγ scavenging. Our results suggest a complex antagonistic interplay between G_q-activated PKC and Gβγ in regulation of L-VDCC, in which multiple cytosolic segments of α_1C are involved.     

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.     

Dihydropyridine sensitive calcium channels in a smooth muscle cell line

The pharmacological properties of voltage sensitive calcium channels (VSCC) were examined in a rat aortic smooth muscle cell line (A10). The inorganic VSCC blockers Co2+ and Cd2+ blocked 45Ca2+ uptake into these cells in both 5 mM K+ and 50 mM K+ (depolarizing) conditions. The organic VSCC antagonists nitrendipine, nimodipine, D-600 and diltiazem also blocked 45Ca2+ uptake at low concentrations. The relative potencies of blockade were similar to those found in intact vascular smooth muscle. The VSCC "agonist" BAY K8644 enhanced 45Ca2+ uptake and this effect could be reversed by nitrendipine. These results indicate that A10 cells possess VSCC and that these VSCC behave similarly to those in authentic smooth muscle.     

Calcineurin activation by slow calcium release from intracellular stores suppresses protein kinase C regulation of L-type calcium channels in L6 cells

L-type Ca²⁺ channel activity was assayed in L6 cells as the rate of nifedipine-sensitive Ba²⁺ influx in a depolarizing medium. In the absence of extracellular Ca²⁺, activation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or thymeleatoxin (TMX) inhibited Ba²⁺ influx by 38%. Thapsigargin (Tg), a selective inhibitor of the Ca²⁺-ATPase in the sarcoplasmic reticulum, evoked a rise in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in a Ca²⁺-free medium from 30 to 80 nM. This [Ca²⁺]_i increase declined slowly, giving rise to a modest elevation of [Ca²⁺]_i that persisted for >5 min. The inhibitory effects of PMA and TMX on channel activity were abolished when tested in Tg-treated cells in a Ca²⁺-free medium. However, when the Ca²⁺ ionophore, ionomycin, was applied with Tg, PMA and TMX retained their inhibitory effect on L-type Ca²⁺ channel activity, suggesting that a lower amplitude and prolonged release of Ca²⁺ stores is necessary for abrogating PKC-mediated inhibition of LCC. Cyclosporin A (5 μM) and ascomycin (5 μM), inhibitors of the Ca²⁺/calmodulin-dependent protein phosphatase, calcineurin, fully restored the inhibitory effect of PMA and TMX on channel activity. Addition of 1 mM CaCl₂ to the Tg-treated cells increased [Ca²⁺]_i to 165 nM and also restored the inhibitory effects of PMA and TMX. These results indicate that a small, relatively prolonged [Ca²⁺]_i increase elicited by passive depletion of internal Ca²⁺ stores led to activation of calcineurin, giving rise to an increase in protein phosphatase activity that counteracted the inhibitory effects of PKC on channel activity. A larger increase in [Ca²⁺]_i via store-dependent Ca²⁺ entry enhanced the activity of PKC sufficiently to overcome the protein phosphatase activity of calcineurin. This study is the first to demonstrate that the regulation of L-type Ca²⁺ channels in a myocyte model involves a balance between the differential Ca²⁺ sensitivities and opposing actions of PKC and calcineurin.     

Impact of phosphomimetic and non‐phosphorylatable mutations of phospholemman on L‐type calcium channels gating in HEK 293T cells

Background : Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L‐type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods : The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild‐type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results : WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage‐dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM‐induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions : Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects.     

Spectrum of Cav1.4 dysfunction in congenital stationary night blindness type 2

Defective retinal synaptic transmission in patients affected with congenital stationary night blindness type 2 (CSNB2) can result from different dysfunction phenotypes in Cav1.4 L-type calcium channels. Here we investigated two prototypical Cav1.4 variants from either end of the functional spectrum. Using whole-cell and single-channel patch-clamp techniques, we provide analysis of the biophysical characteristics of the point mutation L860P and the C-terminal truncating mutation R1827X. L860P showed a typical loss-of-function phenotype attributed to a reduced number of functional channels expressed at the plasma membrane as implied by gating current and non-stationary noise analyses. This phenotype can be rationalized, because the inserted proline is predicted to break an amphipatic helix close to the transmembrane segment IIIS1 and thus to reduce channel stability and promote misfolding. In fact, L860P was subject to an increased turnover. In contrast, R1827X displayed an apparent gain-of-function phenotype, i.e., due to a hyperpolarizing shift of the IV-curve and increased single-channel activity. However, truncation also resulted in the loss of functional C-terminal modulation and thus unmasked calcium-dependent inactivation. Thus R1827X failed to support continuous calcium influx. Current inactivation curtails the dynamic range of photoreceptors (e.g., when adapting to variation in illumination). Taken together, the analysis of two representative mutations that occur in CSNB2 patients revealed fundamental differences in the underlying defect. These may explain subtle variations in the clinical manifestation and must be taken into account, if channel function is to be restored by pharmacochaperones or related approaches.     

Gain-of-function nature of Cav1.4 L-type calcium channels alters firing properties of mouse retinal ganglion cells

Proper function of Cav1.4 L-type calcium channels is crucial for neurotransmitter release in the retina. Our understanding about how different levels of Cav1.4 channel activity affect retinal function is still limited. In the gain-of-function mouse model Cav1.4-IT we expected a reduction in the photoreceptor dynamic range but still transmission toward retinal ganglion cells. A fraction of Cav1.4-IT ganglion cells responded to light stimulation in multielectrode array recordings from whole-mounted retinas, but showed a significantly delayed response onset. Another significant number of cells showed higher activity in darkness. In addition to structural remodeling observed at the first retinal synapse of Cav1.4-IT mice the functional data suggested a loss of contrast enhancement, a fundamental feature of our visual system. In fact, Cav1.4-IT mouse retinas showed a decline in spatial response and changes in their contrast sensitivity profile. Photoreceptor degeneration was obvious from the nodular structure of cone axons and enlarged pedicles which partly moved toward the outer nuclear layer. Loss of photoreceptors was also expressed as reduced expression of proteins involved in chemical and electrical transmission, as such metabotropic glutamate receptor mGluR6 and the gap junction protein Connexin 36. Such gross changes in retinal structure and function could also explain the diminished visual performance of CSNB2 patients. The expression pattern of the plasma-membrane calcium ATPase 1 which participates in the maintenance of the intracellular calcium homeostasis in photoreceptors was changed in Cav1.4-IT mice. This might be part of a protection mechanism against increased calcium influx, as this is suggested for Cav1.4-IT channels.     

Gain-of-function nature of Cav1.4 L-type calcium channels alters firing properties of mouse retinal ganglion cells

Proper function of Cav1.4 L-type calcium channels is crucial for neurotransmitter release in the retina. Our understanding about how different levels of Cav1.4 channel activity affect retinal function is still limited. In the gain-of-function mouse model Cav1.4-IT we expected a reduction in the photoreceptor dynamic range but still transmission toward retinal ganglion cells. A fraction of Cav1.4-IT ganglion cells responded to light stimulation in multielectrode array recordings from whole-mounted retinas, but showed a significantly delayed response onset. Another significant number of cells showed higher activity in darkness. In addition to structural remodeling observed at the first retinal synapse of Cav1.4-IT mice the functional data suggested a loss of contrast enhancement, a fundamental feature of our visual system. In fact, Cav1.4-IT mouse retinas showed a decline in spatial response and changes in their contrast sensitivity profile. Photoreceptor degeneration was obvious from the nodular structure of cone axons and enlarged pedicles which partly moved toward the outer nuclear layer. Loss of photoreceptors was also expressed as reduced expression of proteins involved in chemical and electrical transmission, as such metabotropic glutamate receptor mGluR6 and the gap junction protein Connexin 36. Such gross changes in retinal structure and function could also explain the diminished visual performance of CSNB2 patients. The expression pattern of the plasma-membrane calcium ATPase 1 which participates in the maintenance of the intracellular calcium homeostasis in photoreceptors was changed in Cav1.4-IT mice. This might be part of a protection mechanism against increased calcium influx, as this is suggested for Cav1.4-IT channels.     

Influx of calcium through L‐type calcium channels in early postnatal regulation of chloride transporters in the rat hippocampus

During the early postnatal period, GABA_B receptor activation facilitates L‐type calcium current in rat hippocampus. One developmental process that L‐type current may regulate is the change in expression of the K⁺Cl^? co‐transporter (KCC2) and N⁺K⁺2Cl^? co‐transporter (NKCC1), which are involved in the maturation of the GABAergic system. The present study investigated the connection between L‐type current, GABA_B receptors, and expression of chloride transporters during development. The facilitation of L‐type current by GABA_B receptors is more prominent in the second week of development, with the highest percentage of cells exhibiting facilitation in cultures isolated from 7 day old rats (37.5%). The protein levels of KCC2 and NKCC1 were investigated to determine the developmental timecourse of expression as well as expression following treatment with an L‐type channel antagonist and a GABA_B receptor agonist. The time course of both chloride transporters in culture mimics that seen in hippocampal tissue isolated from various ages. KCC2 levels increased drastically in the first two postnatal weeks while NKCC1 remained relatively stable, suggesting that the ratio of the chloride transporters is important in mediating the developmental change in chloride reversal potential. Treatment of cultures with the L‐type antagonist nimodipine did not affect protein levels of NKCC1, but significantly decreased the upregulation of KCC2 during the first postnatal week. In addition, calcium current facilitation occurs slightly before the large increase in KCC2 expression. These results suggest that the expression of KCC2 is regulated by calcium influx through L‐type channels in the early postnatal period in hippocampal neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Selective fluorophore-assisted light inactivation of voltage-gated calcium channels

Fluorophore-assisted light inactivation (FALI) is an investigative tool to inactivate fluorescently labeled proteins by a mechanism of in situ photodestruction. We found that Cav 1.2 (L-type) and Cav 3.1 (T-type) calcium channels, labeled by genetic fusion with GFP derivatives, show differential sensitivity to FALI. Specifically, FALI silences Cav 1.2 calcium channels containing EYFP-labeled α 1C subunits but does not affect the EYFP-α 1G Cav 3.1 calcium channels or Cav 1.2 channels containing EYFP-labeled β subunits. Our findings limit the applicability of acceptor photobleaching for the measurements of FRET but open an opportunity to combine the fluorescent imaging of the live cell expressing labeled calcium channels with selective functional inactivation of their specific subsets.