Ca2+/calmodulin binds to and modulates P/Q-type calcium channels.

Neurotransmitter release at many central synapses is initiated by an influx of calcium ions through P/Q-type calcium channels, which are densely localized in nerve terminals. Because neurotransmitter release is proportional to the fourth power of calcium concentration, regulation of its entry can profoundly influence neurotransmission. N- and P/Q-type calcium channels are inhibited by G proteins, and recent evidence indicates feedback regulation of P/Q-type channels by calcium. Although calcium-dependent inactivation of L-type channels is well documented, little is known about how calcium modulates P/Q-type channels. Here we report a calcium-dependent interaction between calmodulin and a novel site in the carboxy-terminal domain of the alpha1A subunit of P/Q-type channels. In the presence of low concentrations of intracellular calcium chelators, calcium influx through P/Q-type channels enhances channel inactivation, increases recovery from inactivation and produces a long-lasting facilitation of the calcium current. These effects are prevented by overexpression of a calmodulin-binding inhibitor peptide and by deletion of the calmodulin-binding domain. Our results reveal an unexpected association of Ca2+/calmodulin with P/Q-type calcium channels that may contribute to calcium-dependent synaptic plasticity.     

Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2

Voltage-gated calcium channels (VGCCs) conduct calcium into cells after membrane depolarization and are vital for diverse biological events. They are regulated by various signalling pathways, which has profound functional consequences. The activity of VGCCs decreases with time in whole-cell and inside-out patch-clamp recordings. This rundown reflects persistent intrinsic modulation of VGCCs in intact cells. Although several mechanisms have been reported to contribute to rundown of L-type channels, the mechanism of rundown of other types of VGCC is poorly understood. Here we show that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), an essential regulator of ion channels and transporters, is crucial for maintaining the activity of P/Q- and N-type channels. Activation of membrane receptors that stimulate hydrolysis of PtdIns(4,5)P2 causes channel inhibition in oocytes and neurons. PtdIns(4,5)P2 also inhibits P/Q-type channels by altering the voltage dependence of channel activation and making the channels more difficult to open. This inhibition is alleviated by phosphorylation by protein kinase A. The dual actions of PtdIns(4,5)P2 and the crosstalk between PtdIns(4,5)P2 and protein kinase A set up a dynamic mechanism through which the activity of VGCCs can be finely tuned by various neurotransmitters, hormones and trophic factors.     

Inactivation of the sarcoplasmic reticulum calcium channel by protein kinase.

The ryanodine receptor protein of skeletal muscle sarcoplasmic reticulum (SR) membranes is a calcium ion channel which allows movement of calcium from the SR lumen into the cytoplasm during muscle activation. Gating of this channel is modulated by a number of physiologically important substances including calcium. Interestingly, calcium has both activating and inactivating effects which are concentration- and tissue-specific. In skeletal muscle, calcium-dependent inactivation of calcium release occurs at concentrations reached physiologically, suggesting that calcium may modulate the release process by a negative feedback mechanism. To determine the cellular mechanism responsible for calcium-dependent inactivation, we have investigated the ability of protein phosphorylation to affect single channel gating behaviour using the patch clamp technique. Here we demonstrate that the ryanodine receptor protein/calcium release channel of skeletal muscle SR is inactivated under conditions permissive for protein phosphorylation. This inactivation is reversed by the application of phosphatase and prevented by a peptide inhibitor specific for calcium/calmodulin-dependent protein kinase II. The results provide evidence for an endogenous protein kinase which is closely associated with the ryanodine receptor protein and regulates channel gating.     

Calcium-dependent enhancement of calcium current in smooth muscle by calmodulin-dependent protein kinase II.

Calcium entry through voltage-activated Ca2+ channels is important in regulating many cellular functions. Activation of these channels in many cell types results in feedback regulation of channel activity. Mechanisms linking Ca2+ channel activity with its downregulation have been described, but little is known of the events responsible for the enhancement of Ca2+ current that in many cells follows Ca2+ channel activation and an increase in cytoplasmic Ca2+ concentration. Here we investigate how this positive feedback is achieved in single smooth muscle cells. We find that in these cells voltage-activated calcium current is persistently but reversibly enhanced after periods of activation. This persistent enhancement of the Ca2+ current is mediated by activation of calmodulin-dependent protein kinase II because it is blocked when either the rise in cytoplasmic Ca2+ is inhibited or activation of calmodulin-dependent protein kinase II is prevented by specific peptide inhibitors of calcium-calmodulin or calmodulin-dependent protein kinase II itself. This mechanism may be important in different forms of Ca2+ current potentiation, such as those that depend on prior Ca2+ channel activation or are a result of agonist-induced release of Ca2+ from internal stores.     

Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cyclic AMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB co-activator CRTC2 (ref. 1). Glucagon promotes CRTC2 dephosphorylation in part through the protein kinase A (PKA)-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases seem to be capable of dephosphorylating CRTC2 (refs 2, 3), but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show in mice that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin-dependent Ser/Thr-phosphatase calcineurin (also known as PP3CA). Glucagon increased cytosolic calcium concentration through the PKA-mediated phosphorylation of inositol-1,4,5-trisphosphate receptors (InsP(3)Rs), which associate with CRTC2. After their activation, InsP(3)Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signalling reduced CRTC2 activity via the AKT-mediated inactivation of InsP(3)Rs. InsP(3)R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic downregulation of InsP(3)Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how interactions between cAMP and calcium pathways at the level of the InsP(3)R modulate hepatic glucose production under fasting conditions and in diabetes.     

Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels

Acute modulation of P/Q-type (alpha1A) calcium channels by neuronal activity-dependent changes in intracellular Ca2+ concentration may contribute to short-term synaptic plasticity, potentially enriching the neurocomputational capabilities of the brain. An unconventional mechanism for such channel modulation has been proposed in which calmodulin (CaM) may exert two opposing effects on individual channels, initially promoting ('facilitation') and then inhibiting ('inactivation') channel opening. Here we report that such dual regulation arises from surprising Ca2+-transduction capabilities of CaM. First, although facilitation and inactivation are two competing processes, both require Ca2+-CaM binding to a single 'IQ-like' domain on the carboxy tail of alpha1A; a previously identified 'CBD' CaM-binding site has no detectable role. Second, expression of a CaM mutant with impairment of all four of its Ca2+-binding sites (CaM1234) eliminates both forms of modulation. This result confirms that CaM is the Ca2+ sensor for channel regulation, and indicates that CaM may associate with the channel even before local Ca2+ concentration rises. Finally, the bifunctional capability of CaM arises from bifurcation of Ca2+ signalling by the lobes of CaM: Ca2+ binding to the amino-terminal lobe selectively initiates channel inactivation, whereas Ca2+ sensing by the carboxy-terminal lobe induces facilitation. Such lobe-specific detection provides a compact means to decode local Ca2+ signals in two ways, and to separately initiate distinct actions on a single molecular complex.     

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.     

Sphingosine-1-phosphate induces Ca2+ mobilization via TRPC6 channels in SH-SY5Y cells and hippocampal neurons

Sphingosine-1-phosphate (S1P) is a widely expressed biologically active sphingolipid that plays an important role in cell differentiation, migration, proliferation, metabolism and apoptosis. S1P activates various signaling pathways, some of which evoke Ca2+ signals in the cytosol. Few studies have focused on the mechanism by which S1P evokes Ca2+ signals in neurons. Here, we show that S1P evokes global Ca2+ signals in SH-SY5Y cells and hippocampal neurons. Removal of extracellular calcium largely abolished the S1P-induced increase in intracellular Ca2+, suggesting that the influx of extracellular Ca2+ is the major contributor to this process. Moreover, we found that S1P-induced Ca2+ mobilization is independent of G protein-coupled S1P receptors. The TRPC6 inhibitor SAR7334 suppressed S1P-induced calcium signals, indicating that the TRPC6 channel acts as the downstream effector of S1P. Using patch-clamp recording, we showed that S1P activates TRPC6 currents. Two Src tyrosine kinase inhibitors, Src-I1 and PP2, dramatically inhibited the activation of TRPC6 by S1P. Taken together, our data suggest that S1P activates TRPC6 channels in a Src-dependent way to induce Ca2+ mobilization in SH-SY5Y cells and hippocampal neurons.     

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.     

Sensory transmitters regulate intracellular calcium in dorsal horn neurons

Primary afferent terminals in the dorsal horn of the spinal cord release excitatory amino acid and peptide transmitters that initiate the central processing of nociceptive information. The postsynaptic actions of amino acid transmitters on spinal neurons have been well characterized, but the cellular basis of peptide actions remains unclear. Substance P is the best characterized of the peptides present in sensory neurons and has been shown to depolarize dorsal horn neurons and to facilitate nociceptive reflexes. To determine the mechanisms by which substance P contributes to afferent synaptic transmission, we have monitored the levels of intracellular calcium in single isolated rat dorsal horn neurons and report that substance P can produce a prolonged elevation in calcium concentration by mobilizing its release from intracellular stores. This elevation may contribute to the long-term changes in the excitable properties of dorsal horn neurons that occur following afferent fibre stimulation. We have also found that L-glutamate elevates intracellular calcium in substance P-sensitive dorsal horn neurons by increasing calcium influx. These results provide a direct demonstration of intracellular calcium changes in response to neuropeptides in mammalian central neurons. They also indicate that there is convergent regulation of intracellular calcium in dorsal horn neurons by two different classes of sensory transmitters that are co-released from the same afferent terminals.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells

The neurotrophin receptor TrkB is essential for normal function of the mammalian brain. It is expressed in three splice variants. Full-length receptors (TrkB(FL)) possess an intracellular tyrosine kinase domain and are considered as those TrkB receptors that mediate the crucial effects of brain-derived neurotrophic factor (BDNF) or neurotrophin 4/5 (NT-4/5). By contrast, truncated receptors (TrkB-T1 and TrkB-T2) lack tyrosine kinase activity and have not been reported to elicit rapid intracellular signalling. Here we show that astrocytes predominately express TrkB-T1 and respond to brief application of BDNF by releasing calcium from intracellular stores. The calcium transients are insensitive to the tyrosine kinase blocker K-252a and persist in mutant mice lacking TrkB(FL). By contrast, neurons produce rapid BDNF-evoked signals through TrkB(FL) and the Na(v)1.9 channel. Expression of antisense TrkB messenger RNA strongly reduces BDNF-evoked calcium signals in glia. Thus, our results show that, unexpectedly, TrkB-T1 has a direct signalling role in mediating inositol-1,4,5-trisphosphate-dependent calcium release; in addition, they identify a previously unknown mechanism of neurotrophin action in the brain.     

Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400

Cloning and expression of functional P400 protein from cerebellar Purkinje neurons shows that this protein is a receptor for inositol 1,4,5-trisphosphate, a second messenger that mediates the release of intracellular calcium.     

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.     

Alpha 1-adrenoceptor subtypes linked to different mechanisms for increasing intracellular Ca2+ in smooth muscle

Receptor-mediated increases in intracellular Ca2+ levels can be caused by release from intracellular organelles and/or influx from the extracellular fluid. Noradrenaline (NA) released from sympathetic nerves acts on alpha 1-adrenoceptors to increase cytosolic Ca2+ and promote smooth muscle contraction. In many cells activation of alpha 1-adrenoceptors causes formation of inositol 1,4,5-trisphosphate which promotes Ca2+ release from intracellular stores. The mechanism by which receptor activation opens cell surface Ca2+ channels is not known, although in some cases it may be secondary to formation of inositol phosphates or release of stored intracellular Ca2+ (ref. 3). However, alpha 1-adrenoceptors have recently been shown to have different pharmacological properties in different tissues, and it has been proposed that different alpha 1-adrenoceptor subtypes may control mobilization of intracellular Ca2+ and gating of extracellular Ca2+ influx. We here report evidence for two subtypes of alpha 1-adrenoceptors which cause contractile responses through different molecular mechanisms. One subtype stimulates inositol phosphate (InsP) formation and causes contractions which are independent of extracellular Ca2+, and the other does not stimulate inositol phosphate formation and causes contractions which require the influx of extracellular Ca2+ through dihydropyridine-sensitive channels. These results suggest that neurotransmitters and hormones may control Ca2+ release from intracellular stores and influx through voltage-gated membrane channels through distinct receptor subtypes.     

Stimulation of protein kinase C recruits covert calcium channels in Aplysia bag cell neurons

The modulation of voltage-activated calcium currents by protein kinases provides excitable cells with a mechanism for regulating their electrical behaviour. At the single channel level, modulation of calcium current has, to date, been characterized only in cardiac muscle, where beta-adrenergic agonists, acting through cyclic AMP-dependent protein kinase, enhance the calcium current by increasing channel availability and opening. We now report that enhancement of calcium current in the peptidergic bag cell neurons of Aplysia by protein kinase C occurs through a different mechanism, the recruitment of a previously covert class of calcium channel. Under control conditions, bag cell neurons contain only one class of voltage-activated calcium channel with a conductance of approximately 12 pS. After exposure to agents that activate protein kinase C, these neurons also express a second class of calcium channel with a different unitary conductance (approximately 24 pS) that is never seen in untreated cells.     

Membrane potential has no direct role in evoking neurotransmitter release

Neurons communicate by secreting a transmitter that excites or inhibits other neurons at synapses. The role of presynaptic membrane potential in triggering transmitter release is still controversial. In one view, presynaptic action potentials trigger the release by the entry of calcium ions into presynaptic terminals through voltage-dependent calcium channels. Calcium acts at high local concentrations at release sites near channel mouths to cause neurosecretion. An opposing view is that, in addition to elevating presynaptic calcium, presynaptic potential stimulates transmitter release by a distinct direct action. The relative importance of depolarization and calcium entry in neurosecretion cannot be determined because the two events are tightly linked. To delineate the roles of presynaptic potential and calcium entry in transmitter release, we have used nitr-5, a photolabile calcium chelator, and a voltage-clamp technique to control intracellular calcium and membrane potential independently at a synapse formed between cell bodies of cultured neurons of the fresh water snail Helisoma trivolvis. We found transmitter release occurred when presynaptic calcium levels were elevated to concentrations of a few micromolar, and that presynaptic voltage had no direct effect on neurosecretion.     

Noradrenaline contracts arteries by activating voltage-dependent calcium channels

Noradrenaline (NA) regulates arterial smooth muscle tone and hence blood vessel diameter and blood flow. NA apparently increases tone by causing a calcium influx through the cell membrane. Two calcium influx pathways have been proposed: voltage-activated calcium channels and NA-activated calcium-permeable channels that are voltage-insensitive. Although voltage-activated calcium channels have been identified in arterial smooth muscle, voltage-insensitive calcium channels activated by NA have not. We show here that NA contractions of rabbit mesenteric arteries increase with depolarization. The increase parallels the elevation of open-state probability (P0) of single, voltage-dependent calcium channels. The action of noradrenaline can be explained by NA-activating voltage-dependent calcium channels, rather than by opening a second type of channel. We show directly that NA increases the open-state probability of single calcium channels. Thus, in the presence of NA, calcium entry through voltage-dependent calcium channels can regulate smooth muscle tone at physiological membrane potentials. These results may have relevance to pathophysiological conditions such as hypertension.