Regulation Of Voltage-dependent Calcium Channels Research Articles

Vascular Dysfunction in Diabetes Mellitus

Vascular smooth muscle membrane potential is a key determinant of vessel tone primarily through regulation of voltage-dependent calcium channels (VDCCs). The opening probability of VDCCs is increased by membrane depolarization, facilitating influx of calcium and producing contraction of vascular smooth muscle cells (VSMCs). Control of membrane potential and VDCC activity is orchestrated by a complex series of signals that occur just below the sarcolemmal membrane. Article, see p 607 Large-conductance calcium-activated potassium channels (also known as maxi-K or BK channels) play a pivotal role in modulating vasomotor tone in both health and disease. BK channels, expressed on VSMCs, act as breaks for the increase in vascular tone that occurs after membrane depolarization and elevation of cytosolic calcium. An intracellular rise in cytosolic calcium through VDCC leads to activation of ryanodine receptors that release quanta of calcium from the sarcoplasmic reticulum (calcium sparks). It is these intracellularly generated sparks that elevate local submembrane concentrations of calcium and activate BK channels which increase potassium conductance and lower membrane potential.1 This reduces opening of VDCCs, thus modulating contraction (Figure2). Figure. Pathways for activation and modulation of vascular large-conductance calcium-activated potassium (BK) channels . Diabetes mellitus reduces BK channel activity by ≥3 mechanisms. First, as demonstrated by Nystoriak et al,2 through activation of calcineurin, dephosphorylated nuclear factor of activated T cells, c3 isoform (NFATc3) enters the nucleus where it inhibits transcription of the β1 subunit of BK, thereby reducing BK sensitivity to calcium. Second, diabetes mellitus–induced elevations in reactive oxygen species (ROS), particularly hydrogen peroxide, directly oxidize cysteine residues in the bowl region of the α subunit of BK to reduce opening. Finally, reduced insulin activation of phosphoinositol-3-kinase (PI3) kinase in diabetes mellitus reduces phosphorylation of forkhead box O family transcription factor (FOXO)-3a, allowing nuclear localization and transcription of f-box only protein …

Structure of the GDP-bound G domain of the RGK protein Rem2.

RGK proteins are atypical small GTP-binding proteins that are involved in the regulation of voltage-dependent calcium channels and actin cytoskeleton remodelling. The structure of the Rem2 G domain bound to GDP is reported here in a monoclinic crystal form at 2.66 Å resolution. It is very similar to the structure determined previously from an orthorhombic crystal form. However, differences in the crystal-packing environment revealed that the switch I and switch II regions are flexible and not ordered as previously reported. Comparison of the available RGK protein structures along with those of other small GTP-binding proteins highlights two structural features characteristic of this atypical family and suggests that the conserved tryptophan residue in the DXWEX motif may be a structural determinant of the nucleotide-binding affinity.

Diabetes-Induced Inhibition of Voltage-Dependent Calcium Channels in the Retinal Microvasculature: Role of Spermine

Although decentralized control of blood flow is particularly important in the retina, knowledge of the functional organization of the retinal microvasculature is limited. Here, the authors characterized the distribution and regulation of L-type voltage-dependent calcium channels (VDCCs) within the most decentralized operational complex of the retinal vasculature--the feeder vessel/capillary unit--which consists of a capillary network plus the vessel linking it with a myocyte-encircled arteriole. Perforated-patch recordings, calcium-imaging, and time-lapse photography were used to assess VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter, in microvascular complexes freshly isolated from the rat retina. Topographical heterogeneity was found in the distribution of functional VDCCs; VDCC activity was markedly greater in feeder vessels than in capillaries. Experiments showed that this topographical distribution occurs, in large part, because of the inhibition of capillary VDCCs by a mechanism dependent on the endogenous polyamine spermine. An operational consequence of functional VDCCs predominantly located in the feeder vessels is that voltage-driven vasomotor responses are generated chiefly in this portion of the feeder vessel/capillary unit. However, early in the course of diabetes, this ability to generate voltage-driven vasomotor responses becomes profoundly impaired because of the inhibition of feeder vessel VDCCs by a spermine-dependent mechanism. The regulation of VDCCs by endogenous spermine not only plays a critical role in establishing the physiological organization of the feeder vessel/capillary unit, but also may contribute to dysfunction of this decentralized operational unit in the diabetic retina.

G010 Regulation of voltage-dependent calcium channels by Nedd4-1

Calcium entry into excitable cells can be regulated by controlling both the activity of calcium channels and the amount of channels available at the plasma membrane. Little is known about their internalisation and degradation. One post-translational modification shown to be involved in membrane protein internalisation and subsequent degradation is the attachment of ubiquitin moieties by ubiquitin-ligases. Previously, it has been shown that cardiac ion channels such as Nav1.5 and KCNQ1 are down-regulated by ubiquitin-ligases of the Nedd4 family. These regulations involve the interaction between the PY motif of the target channels and the WW motif of the ubiquitin-ligases. Despite the absence of such PY motif in the cardiac voltage-gated calcium channel Cav1.2, we investigated whether this channel could by regulated in the same manner than other cardiac channels as previously described. We co-expressed, in HEK293 cells, L-type calcium channels and its two regulatory subunits Cavbeta and Cavalpha2delta1 together with ubiquitin-ligases, and examined by voltage-clamp whole-cell recordings the calcium current. We next determined by western blot and surface biotinylation assays the availability of the different subunits of calcium channels in total HEK293 lysates, and at the cell surface. Levels of ubiquitylation of the different subunits were assessed by pull-down GST-S5A and immunoprecipitation of Cav1.2 and its subunits. We found that co-expressing the ubiquitin-ligase Nedd4-1 significantly reduced Cav currents, and decreased Cavalpha and its subunit protein levels. This effect was Nedd4-1 specific since none of the other members of the Nedd4 family we tested produced a similar effect. We also found that the effect of Nedd4-1 was dependent on the co-expression of the Cavbeta subunit. No Nedd4-1-dependent increase in ubiquitylation of the Cavalpha protein was found; and unexpectedly, the two regulating subunits Cavbeta and Cavalpha2delta1 were detected to be deubiquitylated upon Nedd4-1 co-expression. Our data suggest that Nedd4-1 regulates the expression of Cav channels and its subunits by Nedd4-1 via an indirect mechanism constituting a new regulatory pathway to be determined. Further experiments will focus on the role of adrenergic receptors known to be ubiquitylated by Nedd4 and to bind to Cav1.2.

β-Adrenergic Receptor Activation Induces Internalization of Cardiac Cav1.2 Channel Complexes through a β-Arrestin 1-mediated Pathway

Voltage-dependent calcium channels (VDCCs) play a pivotal role in normal excitation-contraction coupling in cardiac myocytes. These channels can be modulated through activation of beta-adrenergic receptors (beta-ARs), which leads to an increase in calcium current (I(Ca-L)) density through cardiac Ca(v)1 channels as a result of phosphorylation by cAMP-dependent protein kinase A. Changes in I(Ca-L) density and kinetics in heart failure often occur in the absence of changes in Ca(v)1 channel expression, arguing for the importance of post-translational modification of these channels in heart disease. The precise molecular mechanisms that govern the regulation of VDCCs and their cell surface localization remain unknown. Our data show that sustained beta-AR activation induces internalization of a cardiac macromolecular complex involving VDCC and beta-arrestin 1 (beta-Arr1) into clathrin-coated vesicles. Pretreatment of myocytes with pertussis toxin prevents the internalization of VDCCs, suggesting that G(i/o) mediates this response. A peptide that selectively disrupts the interaction between Ca(V)1.2 and beta-Arr1 and tyrosine kinase inhibitors readily prevent agonist-induced VDCC internalization. These observations suggest that VDCC trafficking is mediated by G protein switching to G(i) of the beta-AR, which plays a prominent role in various cardiac pathologies associated with a hyperadrenergic state, such as hypertrophy and heart failure.

Introducing an alternative biophysical method to analyze direct G protein regulation of voltage-dependent calcium channels

Direct G protein inhibition of voltage-dependent calcium channels is currently indirectly assessed by the gain of current produced by depolarizing prepulse potentials (PP). Indeed, PPs produce a channel opening- and time-dependent dissociation of G proteins from the channel that is responsible for the increase in Ca 2+ permeation. Parameters of G protein dissociation are essential to describe the characteristic landmark modifications in channel activities that underlie G protein regulation. From the kinetics and opening-dependence of this dissociation, crucial biophysical parameters are extracted such as the extent and the rate of G protein unbinding from the channel. Unfortunately, the method used so far assumes that G protein regulated channels undergo the same inactivation kinetics than control channels. Herein, we demonstrate for the first time that G protein-bound channels undergo a much slower inactivation than control channels. We thus introduce a novel simple-to-use method that avoids the use of PPs and that is not affected by potential changes in channel inactivation kinetics conferred by G protein binding. This method extracts G protein unbinding parameters from ionic currents induced by regular depolarizing pulses by separating the ionic currents due to non-regulated channels from the ionic currents that result from a progressive departure of G proteins from regulated channels.

Calcium channels: critical targets of toxicants and diseases.

The structure and regulation of voltage-dependent calcium channels and their role ininherited and toxicant-induced neurologicdisorders was the topic of a conference spon-sored and hosted by the National Institute ofEnvironmental Health Sciences, the U.S.Environmental Protection Agency, SibiaNeurosciences, and the Muscular DystrophyAssociation on 6–8 December 1999. Theconference, “Calcium Channels: CriticalTargets of Toxicants and Diseases,” broughttogether experts in a variety of disciplines todiscuss our current knowledge of calciumchannel structure and function, channel reg-ulation by intracellular signaling molecules,and the effects of mutations, aging, and toxi-cants on channel function.Calcium channels play important anddiverse roles in neurologic function. It hasbeen known for many years that calciumchannels are likely targets of a variety oforganic and inorganic toxicants. More recent-ly, calcium channel mutations have beenimplicated in several inherited neurologic dis-orders, including migraine headaches, variousmovement disorders, and some types ofmalignant hyperthermia. The last decade hasseen great strides in determining the subunitstructure of calcium channels, how the genet-ic diversity of subunits generates a variety offunctionally distinct channels, and how intra-cellular signaling pathways regulate channelfunction. The meeting was convened to guidefuture research by applying this knowledge ofcalcium channel biology to further our under-standing of how toxicants and mutations alterchannel functions. The presentations discussed four majoraspects of calcium channels that are relevantfor understanding their roles in genetic andenvironmental neurologic disorders: thegenetics and molecular biology of channelstructure; interactions between calcium chan-nels and intracellular signaling pathways; theeffects of toxicants on calcium channel func-tion, both through direct interactions withchannel subunits and by altering intracellularsignaling; and the genetics, molecular biolo-gy, and physiology of inherited disorderscaused by mutations in the genes encodingcalcium channel subunits or from autoim-mune disorders caused by the generation ofantibodies against subunits of those proteins.

P21-ras is involved in regulation of voltage-dependent calcium channels in cultured rat dorsal root ganglion cells.

P21-ras in combination with ras-GAP has been found to block activation of atrial K+ channels by M2 muscarinic receptors (mAChRs), an effect which was suggested to involve interference with receptor-G-protein coupling [l]. Since both M2 and M4 mAChRs are known to inhibit Ca2' channels directly via the pertussis toxin (PTX)-sensitive G-protein, Go, p21-ras may also be involved in regulation of neuronal Ca2+ channels [2, 31. Here we report the results from a study in which the whole cell patch clamp technique has been used to examine the involvement of endogenous p21-ras in regulation of Ca channels in primary cultured rat dorsal root ganglion cells (DRGs). In preliminary ex eriments, the presence of the muscarinic agonist, carbachol(10' M) in the external medium caused a 32.0 * 4.0 YO (mean i s.e.m.) inhibition of calcium channel current, IBa. Use of the M 1 M4-selective muscarinic antagonist, pirenzepine ( M), the M4-selective antagonist, himbacine ( M), and PTX (230 ng/ml for 15 h) suggested the involvement of a PTXsensitive M4 receptor in rat DRGs. In DRGs microinjected with the p21-ras neutralising antibody (Y13-259, 0.1 mg/ml, 50 pl/cell) 2 h prior to experiments, a 43.7 f 4.0 % inhibition of IBa was observed in the presence of carbachol. This result was not significantly different from control cells injected with 1 mg/ml of mouse IgG (36.2 5 4.0 %) or from noninjected cells (42.9 * 4.0 %). The voltage-dependence of both activation and inactivation of IB, shifted between 5-10 mV in a depolarising direction in all three treatments following application of carbachol, although these shifts were not significant either within, or, between treatments. Thus, microinjection of Y 13-259 had no effect on the ability of the muscarinic agonist to inhibit I B ~ Importantly, however, there was a significant reduction in both peak amplitude of maximum IBa (36.0 * 3.0 %, p < 0.05) and the maximum tail current amplitude (31.0 f 3.0 %, p < 0.05) in antibody-injected cells versus controls. No significant differences in the voltage-dependence of either activation or inactivation of maximum peak amplitude of IB, were observed between treatments. Thus, acute blockade of a pathway involving endogenous p21 -ras reduces calcium currents. Intracellular application of phosphopeptides (10-1 5 amino acids) with the sequences of tyrosine autophosphorylation sites on protein tyrosine kinases involved in growth factor activation of p21-ras (NGFR, TRK490; EGFR1068; and CRK adaptor protein, CRK221; at 0.1 mg/ml) also reduced both the peak amplitude of IBa, and maximum tail current amplitude. Effects of these phosphopeptides are thought to be due to the blockade of productive SH2-SH3 interactions which are initiated during the activation of p21-ras by growth factors. Inclusion of TRK490 in the patch pipette for up to 10 min caused a significant reduction of both peak IB, (1 5.8 f 2 %, p < 0.05) and tail current (34.0 f 6.4 %, p < 0.05). Application of EGFR1068 for 10 min resulted in a 19.8 f 4.0 % reduction of IB, (significant at p < 0.05) , and reduced the tail current by 12.8 f 3.0 %, which was not significant. CRK221 caused a significant reduction of peak I B ~ by 18.3 f 2.0 % (p < 0.05), and maximum tail current by 29.2 f 2.0 % (p < 0.01), with time. In control cells, neither the peak amplitude of Is,, nor the tail current amplitude changed significantly with time (up to 10 min). Similarly, internal application of a control (scrambled) phosphopeptide, also had no ! . significant effects on either the maximum peak amplitude of IB,, or the maximum tail current amplitude. No significant changes in voltage-dependence of either current activation or inactivation were observed either within, or, between any of the above treatments. Thus, blocking a pathway possibly involving growth factor activation of p21-ras also reduced calcium current amplitude in these cells. In contrast, microinjection of oncogenic p21-K-ras (2 mg/ml, 24 h prior to experiments) caused a significant increase in the peak amplitude of maximum IBa (61 f 3.0 %, p < 0.01) when compared with control cells. Similarly, the maximum tail current amplitude also increased significantly by 59.7 * 3.0 % (p < 0.01) The voltagedependence of both activation and inactivation of the currents exhibited no significant differences in p21-K-ras-injected, versus control cells. Thus, we have provided evidence that endogenous p2 1 -ras, possibly activated via a protein tyrosine kinase pathway, is involved in the regulation of Ca2+ channels in rat DRGs, although unlike in cardiac myocytes, it is not involved in muscarinic inhibition of these Ca currents. These results tie in with other studies in which the injection of oncogenic p21-ras has been found to increase Ca2+ current in neuronal cells [4,5,6].

Mu- and kappa-opioid receptors selectively reduce the same transient components of high-threshold calcium current in rat dorsal root ganglion sensory neurons

Whole-cell patch-clamp recordings were used to examine the regulation of voltage-dependent calcium channels by mu- and kappa-opioid receptors in acutely isolated rat dorsal root ganglion (DRG) sensory neurons. Agonists selective for either mu- (Tyr-Pro-NMePhe-D-Pro-NH2, PLO17) or kappa-opioid receptors (dynorphin A, U69,593) inhibited high-threshold calcium currents in a reversible and naloxone-sensitive manner, whereas administration of D-Pen2,5-enkephalin, a delta-selective agonist, was without effect. However, none of the opioids reduced low-threshold T-type currents. The inhibitory effects of PLO17 were blocked by the irreversible mu-opioid antagonist beta-funaltrexamine but not the kappa-opioid antagonist nor-binaltorphimine, while responses to kappa-opioid agonists showed the opposite pattern of antagonist sensitivity. In addition, many cells responded to both PLO17 and dynorphin A (or U69,593), and in these neurons the inhibitory response to one agonist was occluded when tested in the presence of the other. These data suggest that mu- and kappa-opioid receptors are coexpressed on at least some DRG neurons and appear to be functionally coupled to a common pool of calcium channels. Both rapidly inactivating (transient) and sustained components of high-threshold current, arising from pharmacologically distinct types of calcium channels, were identified in our neurons. Activation of mu-opioid receptors selectively reduced the transient component of currents evoked at +10 mV from Vh = -80 mV, while sparing the sustained component. The transient component was irreversibly blocked by the N-type channel antagonist omega-conotoxin GVIA (omega-CgTx), and in one-half of the neurons there was a concomitant loss of the response to PLO17. In the remaining neurons, PLO17 continued to reduce a small fraction of omega-CgTx-insensitive current and subsequent administration of the L-type channel blocker nifedipine in saturating concentrations failed to reduce the opioid-induced inhibitory effect. These data demonstrate that mu-opioid receptors are negatively coupled to several pharmacologically distinct types of calcium channels in DRG sensory neurons, one that was blocked by omega-CgTx and thus likely to be N-type, and a second that was resistant to blockade by N- and L-type channel blockers.

Inositol phosphate regulation of voltage-dependent calcium channels in cerebellar granule neurons

The effects of intracellularly applied inositol phosphates on voltage-dependent calcium channel currents were assessed in rat cerebellar neurons using the whole-cell recording configuration of the patch-clamp technique. Intraneuronal perfusion of 10 μM inositol 1,4,5-trisphosphate (IP3) increased the amplitude of currents elicited by depolarization from a holding potential of -40 mV. IP 3 did not modify current activation, but shifted the steady-state inactivation curve toward more positive values. The dose-response curve indicated an ECso of 0.5 μM for IP3. Inositol 1,3,4,5, tetrakisphosphate (IP 4), but not inositol 4,5, bisphosphate, mimicked the effect of IP 3. The effect of IP 3 persisted in the presence of 100 μg/ ml heparin and did not depend on intracellular calcium mobilization, as similar responses were not produced by 10 mM caffeine or by intrapipette calcium buffering at pCa 6 instead of pCa 7.7. Preincubation with ω-conotoxin led to a 5596 inhibition of barium current; however, inhibition was reversed by I P3, which reestablished the control current amplitude. These results imply that IP 3 and IP 4 can elicit calcium entry by modifying both the gating characteristics and the pharmacological properties of voltage-dependent calcium channels.

The effects of strychnine on the regulation of voltage-dependent calcium channels by dihydropyridines in brain and heart

The effects of strychnine (STR) were investigated on K +-stimulated 45Ca 2+-uptake into mouse brain neurons, the contractile activity of spontaneously beating rat atria and on [ 3H]nitrendipine and [ 3H]BAY K 8644 binding to dihydropyridine calcium channel antagonist and agonist binding sites on brain and cardiac membranes. STR (10 −6−10 −4 M) had no effect on neuronal 45Ca 2+-uptake. When combined at equimolar concentrations (10 −5 M), STR and nifedipine produced a potent (nM) inhibition (40%) of neuronal 45Ca 2+-uptake. In the spontaneously beating rat atria, STR produced a dose-dependent (10 −7−3 × 10 −4 M) decrease in chronotropy but did not affect inotropy. STR (10 −4 M) completely inhibited the positive chronotropic, but did not affect the positive inotropic effects of (−)-S-BAY K 8644. [ 3H]Nitrendipine and [ 3H]BAY K 8644 binding to brain and cardiac membranes was enhanced by STR in a concentration-dependent manner (EC 50 8 × 10 −6 M). Scatchard analysis revealed that STR increased the affinity (decreased the K d) of [ 3H]BAY K 8644 to a greater degree than that of [ 3H]nitrendipine for dihydropyridine binding sites. STR decreased the K d of [ 3H]nitrendipine binding by increasing and decreasing the microassociation and microdissociation constants respectively. STR enhanced [ 3H]nitrendipine binding to the same extent in the cerebral cortex, striatum, hippocampus, cerebellum, brainstem and spinal cord. The enhancement of [ 3H]nitrendipine binding in brain was completely inhibited by Ca 2+ and partially inhibited by Na + in a concentration-dependent manner. Glycine (10 −2 M) did not affect the STR enhancement of [ 3H]nitrendipine binding. Extensive rinsing of brain membranes increased (∼two-fold) the affinity of STR for enhancement of [ 3H]nitrendipine binding. The effects of STR on [ 3H]nitrendipine binding both resembled and were unique to those of phencyclidine. These findings suggest that STR interacts specifically with calcium channels in brain and cardiac tissues at a site similar, but not identical to that of phencyclidine and which is distinct from the dihydropyridine calcium channel regulatory site.

Homologous and heterologous regulation of voltage-dependent calcium channels

Homologous and heterologous regulation of voltage-dependent calcium channels

Drug- and disease-induced regulation of voltage-dependent calcium channels.

Drug- and disease-induced regulation of voltage-dependent calcium channels.

Homologous regulation of voltage-dependent calcium channels by 1,4-dihydropyridines

Chronic treatment of PC 12 cells with the 1,4-dihydropyridine Ca2+ channel antagonist nifedipine [ 5 × 10-8M/5 days ] and the activator S Bay K 8644 [ 5 × 10-7M/5 days ] resulted in up- and down-regulation of 1,4-dihydropyridine binding site density by 29 and 24%, respectively, without change in affinity. These changes in binding site density represent functional changes as indicated by the corresponding changes in K+ depolarization-induced 45Ca2+ uptake and in whole cell currents carried by Ba2+ ions. This homologous regulation of voltage-dependent Ca2+ channels [ VDCC ] by potent and specific ligands parallels that observed for other classes of membrane receptors.