Amy Lee

Essential role of Ca2+-binding protein 4, a Cav1.4 channel regulator, in photoreceptor synaptic function

CaBP1–8 are neuronal Ca2+-binding proteins with similarity to calmodulin (CaM). Here we show that CaBP4 is specifically expressed in photoreceptors, where it is localized to synaptic terminals. The outer plexiform layer, which contains the photoreceptor synapses with secondary neurons, was thinner in the Cabp4−/− mice than in control mice. Cabp4−/− retinas also had ectopic synapses originating from rod bipolar and horizontal cells tha HJt extended into the outer nuclear layer. Responses of Cabp4−/− rod bipolars were reduced in sensitivity about 100-fold. Electroretinograms (ERGs) indicated a reduction in cone and rod synaptic function. The phenotype of Cabp4−/− mice shares similarities with that of incomplete congenital stationary night blindness (CSNB2) patients. CaBP4 directly associated with the C-terminal domain of the Cav1.4 α1-subunit and shifted the activation of Cav1.4 to hyperpolarized voltages in transfected cells. These observations indicate that CaBP4 is important for normal synaptic function, probably through regulation of Ca2+ influx and neurotransmitter release in photoreceptor synaptic terminals.

Molecular determinants of Ca2+/calmodulin-dependent regulation of Cav2.1 channels

Ca2+-dependent facilitation and inactivation (CDF and CDI) of Cav2.1 channels modulate presynaptic P/Q-type Ca2+ currents and contribute to activity-dependent synaptic plasticity. This dual feedback regulation by Ca2+ involves calmodulin (CaM) binding to the α1 subunit (α12.1). The molecular determinants for Ca2+-dependent modulation of Cav2.1 channels reside in CaM and in two CaM-binding sites in the C-terminal domain of α12.1, the CaM-binding domain (CBD) and the IQ-like domain. In transfected tsA-201 cells, CDF and CDI were both reduced by deletion of CBD. In contrast, alanine substitution of the first two residues of the IQ-like domain (IM-AA) completely prevented CDF but had little effect on CDI, and glutamate substitutions (IM-EE) greatly accelerated voltage-dependent inactivation but did not prevent CDI. Mutational analyses of the Ca2+ binding sites of CaM showed that both the N- and C-terminal lobes of CaM were required for full development of facilitation, but only the N-terminal lobe was essential for CDI. In biochemical assays, CaM12 and CaM34 were unable to bind CBD, whereas CaM34 but not CaM12 retained Ca2+-dependent binding to the IQ-like domain. These findings support a model in which Ca2+ binding to the C-terminal EF-hands of preassociated CaM initiates CDF via interaction with the IQ-like domain. Further Ca2+ binding to the N-terminal EF-hands promotes secondary CaM interactions with CBD, which enhance facilitation and cause a conformational change that initiates CDI. This multifaceted mechanism allows positive regulation of Cav2.1 in response to local Ca2+ increases (CDF) and negative regulation during more global Ca2+ increases (CDI).

Drug excretion into breast milk--overview.

Breastfeeding is the optimal form of infant feeding for the first months of an infants life, and the majority of healthy women initiate breastfeeding after the birth of their infant. However, women on medication may default to formula feeding or not taking their drug therapy for fear of exposing their infant to the medication through the breast milk. Although the majority of medications are considered to be compatible with breastfeeding, cases of significant infant toxicity exist, suggesting a case by case risk assessment to be made before the mother initiates breastfeeding or drug therapy. Unfortunately, current clinical risk assessment is often compromised by the paucity of data, as studies in breastfeeding women and their infants are ethically difficult to conduct. Circumventing the ethical constraints, approaches have been proposed to estimate drug excretion into milk from physicochemical characteristics of the drug, which diffuses through the mammary gland epithelia. However, as our understanding on drug transfer mechanisms increases, it has become abundantly clear that carrier-mediated processes are involved with excretion of a number of drugs into milk. This article provides an overview of the benefits of breastfeeding, the effect of medication use during breastfeeding on maternal decisions and infant health, and factors determining infant exposure to medication through the breast milk.

Cav1 L-type Ca2+ channel signaling complexes in neurons

Cav1 L‐type Ca2+ channels play crucial and diverse roles in the nervous system. The pre‐ and post‐synaptic functions of Cav1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G‐protein coupled receptors, scaffolding proteins, and Ca2+‐binding proteins. The cytoplasmic domains of the main pore forming α1 subunit of Cav1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca2+ entry. Through effects on Cav1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Cav1 Ca2+ signals that control neuronal excitability, synaptic plasticity, and gene expression.

Ca2+-binding proteins tune Ca2+-feedback to Cav1.3 channels in mouse auditory hair cells

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Cav1.3 voltage‐gated Ca2+ channels. Central to their role in this regard, Cav1.3 channels in inner hair cells show little Ca2+‐dependent inactivation, a fast negative feedback regulation by incoming Ca2+ ions, which depends on calmodulin association with the Ca2+ channel α1 subunit. Ca2+‐dependent inactivation characterizes nearly all voltage‐gated Ca2+ channels including Cav1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Cav1.3 in inner hair cells remains a mystery. Previously, we established calmodulin‐like Ca2+‐binding proteins in the brain and retina (CaBPs) as essential modulators of voltage‐gated Ca2+ channels. Here, we demonstrate that CaBPs differentially modify Ca2+ feedback to Cav1.3 channels in transfected cells and explore their significance for Cav1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca2+‐dependent inactivation of Cav1.3. CaBP1 and CaBP4 both interact with calmodulin‐binding sequences in Cav1.3, but CaBP4 more weakly inhibits Ca2+‐dependent inactivation than CaBP1. Ca2+‐dependent inactivation is marginally greater in inner hair cells from CaBP4−/− than from wild‐type mice, yet CaBP4−/− mice are not hearing‐impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild‐type and CaBP4−/− mice and therefore is positioned to modulate native Cav1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca2+ channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Cav1.3 Ca2+ currents required for auditory transmission.

Ca2+-Binding Protein-1 Facilitates and Forms a Postsynaptic Complex with Cav1.2 (L-Type) Ca2+ Channels

Ca2+-binding protein-1 (CaBP1) is a Ca2+-binding protein that is closely related to calmodulin (CaM) and localized in somatodendritic regions of principal neurons throughout the brain, but how CaBP1 participates in postsynaptic Ca2+ signaling is not known. Here, we describe a novel role for CaBP1 in the regulation of Ca2+ influx through Cav1.2 (L-type) Ca2+ channels. CaBP1 interacts directly with the α1 subunit of Cav1.2 at sites that also bind CaM. CaBP1 binding to one of these sites, the IQ domain, is Ca2+ dependent and competitive with CaM binding. The physiological significance of this interaction is supported by the association of Cav1.2 and CaBP1 in postsynaptic density fractions purified from rat brain. Moreover, in double-label immunofluorescence experiments, CaBP1 and Cav1.2 colocalize in numerous cell bodies and dendrites of neurons, particularly in pyramidal cells in the CA3 region of the hippocampus and in the dorsal cortex. In electrophysiological recordings of cells transfected with Cav1.2, CaBP1 greatly prolonged Ca2+ currents, prevented Ca2+-dependent inactivation, and caused Ca2+-dependent facilitation of currents evoked by step depolarizations and repetitive stimuli. These effects contrast with those of CaM, which promoted strong Ca2+-dependent inactivation of Cav1.2 with these same voltage protocols. Our findings reveal how Ca2+-binding proteins, such as CaM and CaBP1, differentially adjust Ca2+ influx through Cav1.2 channels, which may specify diverse modes of Ca2+ signaling in neurons.

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Molecular Mechanism for Divergent Regulation of Cav1.2 Ca2+ Channels by Calmodulin and Ca2+-binding Protein-1

Ca2+-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca2+-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca2+ channels. Whereas CaM enhances inactivation of Ca2+ currents through Cav1.2 (L-type) Ca2+ channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Cav1.2 inactivation is unknown. Here, we identified molecular determinants in the α1-subunit of Cav1.2 (α11.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of α11.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca2+-independent binding of CaBP1 to the N-terminal domain (NT) of α11.2, which was in contrast to Ca2+-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Cav1.2 Ca2+ currents, but spared Ca2+-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of α11.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Cav1.2.

Ultrastructural evidence for pre‐ and postsynaptic localization of Cav1.2 L‐type Ca2+ channels in the rat hippocampus

In the hippocampal formation, Cav1.2 (L‐type) voltage‐gated Ca2+ channels mediate Ca2+ signals that can trigger long‐term alterations in synaptic efficacy underlying learning and memory. Immunocytochemical studies indicate that Cav1.2 channels are localized mainly in the soma and proximal dendrites of hippocampal pyramidal neurons, but electrophysiological data suggest a broader distribution of these channels. To define the subcellular substrates underlying Cav1.2 Ca2+ signals, we analyzed the localization of Cav1.2 in the hippocampal formation by using antibodies against the pore‐forming α1‐subunit of Cav1.2 (α11.2). By light microscopy, α11.2‐like immunoreactivity (α11.2‐IR) was detected in pyramidal cell soma and dendritic fields of areas CA1–CA3 and in granule cell soma and fibers in the dentate gyrus. At the electron microscopic level, α11.2‐IR was localized in dendrites, but also in axons, axon terminals, and glial processes in all hippocampal subfields. Plasmalemmal immunogold particles representing α11.2‐IR were more significant for small‐ than large‐caliber dendrites and were largely associated with extrasynaptic regions in dendritic spines and axon terminals. These findings provide the first detailed ultrastructural analysis of Cav1.2 localization in the brain and support functionally diverse roles of these channels in the hippocampal formation. J. Comp. Neurol. 506:569–583, 2008.

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a.

Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (effectors) directly into host cells. The YopJ/HopZ superfamily of effectors found in animal and plant pathogens can modify host kinase proteins to suppress host immunity. In the model plant Arabidopsis, HopZ1a is recognized by the resistance protein ZAR1 to induce a robust immune response that blocks pathogen growth. Here, we show that the HopZ1a effector from the plant pathogen Pseudomonas syringae targets the Arabidopsis nonfunctional pseudokinase ZED1 and that ZED1 is required for recognition of HopZ1a by ZAR1. We hypothesize that HopZ1a targets kinases to promote pathogen virulence, and Arabidopsis ZED1 evolved as a decoy to trap HopZ1a in the ZAR1 complex for recognition by the plant immune system. Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1–resistance complex, resulting in ETI activation.