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In JoVE (1)
Other Publications (4)
Articles by Angela R. Cantrell in JoVE
JoVE Neuroscience
Whole Cell Recording from an Organotypic Slice Preparation of Neocortex
Department of Anatomy and Neurobiology, University of Tennessee Health Science Center
This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.
Other articles by Angela R. Cantrell on PubMed
Molecular Mechanism of Convergent Regulation of Brain Na(+) Channels by Protein Kinase C and Protein Kinase A Anchored to AKAP-15
Activation of D1-like dopamine (DA) receptors reduces peak Na(+) current in hippocampal neurons voltage-dependent in a manner via phosphorylation of the alpha subunit. This modulation is dependent upon activation of cAMP-dependent protein kinase (PKA) and requires phosphorylation of serine 573 (S573) in the intracellular loop connecting homologous domains I and II (L(I-II)) by PKA anchored to A kinase anchoring protein-15 (AKAP-15). Activation of protein kinase C (PKC) also reduces peak Na(+) currents and enhances the strength of the PKA modulatory pathway. Here we probe the molecular mechanism responsible for the convergent effects of PKA and PKC on brain Na(v)1.2a channels. Analysis of the interaction of AKAP-15 with the intracellular loops of the Na(v)1.2a channel shows that it binds to L(I-II), thereby targeting PKA directly to its sites of phosphorylation on the Na(+) channel by specific protein-protein interactions. Mutagenesis and expression experiments indicate that reduction of peak Na(+) current by PKC requires S554 and S573 in L(I-II) in addition to S1506 in the inactivation gate. In addition, PKC-dependent phosphorylation of S576 in L(I-II) is necessary for enhancement of PKA modulation of brain Na(+) channels. When S576 is phosphorylated by PKC, the increase in modulation by PKA activation requires phosphorylation of S687 in L(I-II). Thus, the maximal modulation of these Na(+) channels by concurrent activation of PKA and PKC requires phosphorylation at four distinct sites in L(I-II): S554, S573, S576, and S687. This convergent regulation provides a novel mechanism by which information from multiple signaling pathways may be integrated at the cellular level in the hippocampus and throughout the central nervous system.
Transmitter Modulation of Slow, Activity-dependent Alterations in Sodium Channel Availability Endows Neurons with a Novel Form of Cellular Plasticity
Voltage-gated Na+ channels are major targets of G protein-coupled receptor (GPCR)-initiated signaling cascades. These cascades act principally through protein kinase-mediated phosphorylation of the channel alpha subunit. Phosphorylation reduces Na+ channel availability in most instances without producing major alterations of fast channel gating. The nature of this change in availability is poorly understood. The results described here show that both GPCR- and protein kinase-dependent reductions in Na+ channel availability are mediated by a slow, voltage-dependent process with striking similarity to slow inactivation, an intrinsic gating mechanism of Na+ channels. This process is strictly associated with neuronal activity and develops over seconds, endowing neurons with a novel form of cellular plasticity shaping synaptic integration, dendritic electrogenesis, and repetitive discharge.
Specific Modulation of Na+ Channels in Hippocampal Neurons by Protein Kinase C Epsilon
Acetylcholine binding to muscarinic acetylcholine receptors activates G-proteins, phospholipase C, and protein kinase C (PKC), which phosphorylates brain Na+ channels and reduces peak Na+ current in hippocampal neurons. Because multiple PKC isozymes with different regulatory properties are expressed in hippocampal neurons, we investigated which ones are responsible for mediating this effect. The diacylglycerol analog oleoylacetylglycerol (OAG) reduced the amplitude of Na+ current in dissociated mouse hippocampal neurons by 28.5 +/- 5.3% (p < 0.01). The reduction of peak Na+ current was similar with Ca2+-free internal solution and in 92 nm internal Ca2+, suggesting that calcium-dependent, conventional PKC isozymes were unlikely to mediate this response. Gö6976, which inhibits conventional PKC isozymes, reduced the effect of PKC activators only slightly, whereas rottlerin, which inhibits PKCdelta preferentially at 5 microm, had no effect. Ro-31-8425 (20 nm), which inhibits conventional PKC isozymes, did not reduce the response to OAG. However, higher concentrations of Ro-31-8425 (100 nm or 1 microm) that inhibit novel PKC isozymes effectively blocked OAG inhibition of Na+ current. Inclusion of a selective PKCepsilon-anchoring inhibitor peptide (PKCepsilon-I) in the recording pipette prevented the reduction of peak Na+ current by OAG, whereas an anchoring inhibitor peptide specific for PKCbeta and an inactive scrambled PKCepsilon-I peptide had no effect. In addition, OAG had no effect on Na+ current in hippocampal neurons from PKCepsilon null mice. Overall, our data from four experimental approaches indicate that anchored PKCepsilon is the isozyme responsible for PKC-mediated reduction of peak Na+ currents in mouse hippocampal neurons.
Presenilin 1 Deficiency Alters the Activity of Voltage-gated Ca2+ Channels in Cultured Cortical Neurons
Presenilins 1 and 2 (PS1 and PS2, respectively) play a critical role in mediating gamma-secretase cleavage of the amyloid precursor protein (APP). Numerous mutations in the presenilins are known to cause early-onset familial Alzheimer's disease (FAD). In addition, it is well established that PS1 deficiency leads to altered intracellular Ca(2+) homeostasis involving endoplasmic reticulum Ca(2+) stores. However, there has been little evidence suggesting Ca(2+) signals from extracellular sources are influenced by PS1. Here we report that the Ca(2+) currents carried by voltage-dependent Ca(2+) channels are increased in PS1-deficient cortical neurons. This increase is mediated by a significant increase in the contributions of L- and P-type Ca(2+) channels to the total voltage-mediated Ca(2+) conductance in PS1 (-/-) neurons. In addition, chelating intracellular Ca(2+) with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) produced an increase in Ca(2+) current amplitude that was comparable to the increase caused by PS1 deficiency. In contrast to this, BAPTA had no effect on voltage-dependent Ca(2+) conductances in PS1-deficient neurons. These data suggest that PS1 deficiency may influence voltage-gated Ca(2+) channel function by means that involve intracellular Ca(2+) signaling. These findings reveal that PS1 functions at multiple levels to regulate and stabilize intracellular Ca(2+) levels that ultimately control neuronal firing behavior and influence synaptic transmission.
