Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

A novel transgenic mouse model of lysosomal storage disorder.

Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat CaVß2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas; decreased adipose tissue; thickened, orange intestines; and enlarged liver, spleen, and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria, while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic acid-Schiff-positive, diastase-resistant, iron-negative, Oil Red O-positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. Electron microscopic analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build up of cholesterol, cholesterol esters, and triglycerides, along with changes in liver metabolic enzyme levels, were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site, an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95 Loss of gene function can account for the altered lipid processing, along with hypertrophy of the immune system, which define this phenotype, and serendipitously provides a novel mouse model of lysosomal storage disorder.

Role of cilia in normal pancreas function and in diseased states.

Primary cilia play an essential role in modulating signaling cascades that shape cellular responses to environmental cues to maintain proper tissue development. Mutations in primary cilium proteins have been linked to several rare developmental disorders, collectively known as ciliopathies. Together with other disorders associated with dysfunctional cilia/centrosomes, affected individuals have increased risk of developing metabolic syndrome, neurologic disorders, and diabetes. In pancreatic tissues, cilia are found exclusively in islet and ductal cells where they play an essential role in pancreatic tissue organization. Their absence or disorganization leads to pancreatic duct abnormalities, acinar cell loss, polarity defects, and dysregulated insulin secretion. Cilia in pancreatic tissues are hubs for cellular signaling. Many signaling components, such as Hh, Notch, and Wnt, localize to pancreatic primary cilia and are necessary for proper development of pancreatic epithelium and ß-cell morphogenesis. Receptors for neuroendocrine hormones, such as Somatostatin Receptor 3, also localize to the cilium and may play a more direct role in controlling insulin secretion due to somatostatin's inhibitory function. Finally, unique calcium signaling, which is at the heart of ß-cell function, also occurs in primary cilia. Whereas voltage-gated calcium channels trigger insulin secretion and serve a variety of homeostatic functions in ß-cells, transient receptor potential channels regulate calcium levels within the cilium that may serve as a feedback mechanism, regulating insulin secretion. This review article summarizes our current understanding of the role of primary cilia in normal pancreas function and in the diseased state.

The acid-sensitive, anesthetic-activated potassium leak channel, KCNK3, is regulated by 14-3-3ß-dependent, protein kinase C (PKC)-mediated endocytic trafficking.

The acid-sensitive neuronal potassium leak channel, KCNK3, is vital for setting the resting membrane potential and is the primary target for volatile anesthetics. Recent reports demonstrate that KCNK3 activity is down-regulated by PKC; however, the mechanisms responsible for PKC-induced KCNK3 down-regulation are undefined. Here, we report that endocytic trafficking dynamically regulates KCNK3 activity. Phorbol esters and Group I metabotropic glutamate receptor (mGluR) activation acutely decreased both native and recombinant KCNK3 currents with concomitant KCNK3 surface losses in cerebellar granule neurons and cell lines. PKC-mediated KCNK3 internalization required the presence of both 14-3-3ß and a novel potassium channel endocytic motif, because depleting either 14-3-3ß protein levels or ablating the endocytic motif completely abrogated PKC-regulated KCNK3 trafficking. These results demonstrate that neuronal potassium leak channels are not static membrane residents but are subject to 14-3-3ß-dependent regulated trafficking, providing a straightforward mechanism to modulate neuronal excitability and synaptic plasticity by Group I mGluRs.

Mutations in DISC1 alter IP3R and voltage-gated Ca2+ channel functioning, implications for major mental illness.

Disrupted in Schizophrenia 1 (DISC1) participates in a wide variety of developmental processes of central neurons. It also serves critical roles that underlie cognitive functioning in adult central neurons. Here we summarize DISC1's general properties and discuss its use as a model system for understanding major mental illnesses (MMIs). We then discuss the cellular actions of DISC1 that involve or regulate Ca2+ signaling in adult central neurons. In particular, we focus on the tethering role DISC1 plays in transporting RNA particles containing Ca2+ channel subunit RNAs, including IP3R1, CACNA1C and CACNA2D1, and in transporting mitochondria into dendritic and axonal processes. We also review DISC1's role in modulating IP3R1 activity within mitochondria-associated ER membrane (MAM). Finally, we discuss DISC1-glycogen synthase kinase 3ß (GSK3ß) signaling that regulates functional expression of voltage-gated Ca2+ channels (VGCCs) at central synapses. In each case, DISC1 regulates the movement of molecules that impact Ca2+ signaling in neurons.

L- and N-current but not M-current inhibition by M1 muscarinic receptors requires DAG lipase activity.

Stimulation of postsynaptic M(1) muscarinic receptors (M(1)Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M(1)R stimulation modulates currents through various voltage-gated ion channels, including KCNQ K+ channels (M-current) and both L- and N-type Ca2+ channels (L- and N-current) by a pertussis toxin-insensitive, slow signaling pathway. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) during M(1)R stimulation suffices to inhibit M-current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L- and N-current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC-80267, for its capacity to antagonize M(1)R-mediated modulation of whole-cell Ca2+ currents. RHC-80267 significantly reduced L- and N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) but did not affect their inhibition by exogenous AA. Moreover, voltage-dependent inhibition of N-current by Oxo-M remained in the presence of RHC-80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N-current. In contrast, RHC-80267 had no effect on native M-current inhibition. These data are consistent with a role for DAGL in mediating L- and N-current inhibition. These results extend our previous findings that the signaling pathway mediating L- and N-current inhibition diverges from the pathway initiating M-current inhibition.

Modulation of CaV1.3b L-type calcium channels by M1 muscarinic receptors varies with CaVß subunit expression.

OBJECTIVES: We examined whether two G protein-coupled receptors (GPCRs), muscarinic M1 receptors (M1Rs) and dopaminergic D2 receptors (D2Rs), utilize endogenously released fatty acid to inhibit L-type Ca2+ channels, CaV1.3. HEK-293 cells, stably transfected with M1Rs, were used to transiently transfect D2Rs and CaV1.3b with different CaVß-subunits, allowing for whole-cell current measurement from a pure channel population. RESULTS: M1R activation with Oxotremorine-M inhibited currents from CaV1.3b coexpressed with α2δ-1 and a ß1b, ß2a, ß3, or ß4-subunit. Surprisingly, the magnitude of inhibition was less with ß2a than with other CaVß-subunits. Normalizing currents revealed kinetic changes after modulation with ß1b, ß3, or ß4, but not ß2a-containing channels. We then examined if D2Rs modulate CaV1.3b when expressed with different CaVß-subunits. Stimulation with quinpirole produced little inhibition or kinetic changes for CaV1.3b coexpressed with ß2a or ß3. However, quinpirole inhibited N-type Ca2+ currents in a concentration-dependent manner, indicating functional expression of D2Rs. N-current inhibition by quinpirole was voltage-dependent and independent of phospholipase A2 (PLA2), whereas a PLA2 antagonist abolished M1R-mediated N-current inhibition. These findings highlight the specific regulation of Ca2+ channels by different GPCRs. Moreover, tissue-specific and/or cellular localization of CaV1.3b with different CaVß-subunits could fine tune the response of Ca2+ influx following GPCR activation.

cPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α's importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α's functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

M1 muscarinic receptors inhibit L-type Ca2+ current and M-current by divergent signal transduction cascades.

Ion channels reside in a sea of phospholipids. During normal fluctuations in membrane potential and periods of modulation, lipids that directly associate with channel proteins influence gating by incompletely understood mechanisms. In one model, M(1)-muscarinic receptors (M(1)Rs) may inhibit both Ca(2+) (L- and N-) and K(+) (M-) currents by losing a putative interaction between channels and phosphatidylinositol-4,5-bisphosphate (PIP(2)). However, we found previously that M(1)R inhibition of N-current in superior cervical ganglion (SCG) neurons requires loss of PIP(2) and generation of a free fatty acid, probably arachidonic acid (AA) by phospholipase A(2) (PLA(2)). It is not known whether PLA(2) activity and AA also participate in L- and M-current modulation in SCG neurons. To test whether PLA(2) plays a similar role in M(1)R inhibition of L- and M-currents, we used several experimental approaches and found unanticipated divergent signaling. First, blocking resynthesis of PIP(2) minimized M-current recovery from inhibition, whereas L-current recovered normally. Second, L-current inhibition required group IVa PLA(2) [cytoplasmic PLA(2) (cPLA(2))], whereas M-current did not. Western blot and imaging studies confirmed acute activation of cPLA(2) by muscarinic stimulation. Third, in type IIa PLA(2) [secreted (sPLA(2))](-/-)/cPLA(2)(-/-) double-knock-out SCG neurons, muscarinic inhibition of L-current decreased. In contrast, M-current inhibition remained unaffected but recovery was impaired. Our results indicate that L-current is inhibited by a pathway previously shown to control M-current over-recovery after washout of muscarinic agonist. Our findings support a model of M(1)R-meditated channel modulation that broadens rather than restricts the roles of phospholipids and fatty acids in regulating ion channel activity.

Characterization of ST14A Cells for Studying Modulation of Voltage-Gated Calcium Channels.

In medium spiny neurons (MSNs) of the striatum, dopamine D2 receptors (D2Rs) specifically inhibit the Ca(v)1.3 subtype of L-type Ca(2+) channels (LTCs). MSNs are heterogeneous in their expression of dopamine receptors making the study of D2R pathways difficult in primary neurons. Here, we employed the ST14A cell line, derived from embryonic striatum and characterized to have properties of MSNs, to study Ca(v)1.3 current and its modulation by neurotransmitters. Round, undifferentiated ST14A cells exhibited little to no endogenous Ca(2+) current while differentiated ST14A cells expressed endogenous Ca(2+) current. Transfection with LTC subunits produced functional Ca(v)1.3 current from round cells, providing a homogeneous model system compared to native MSNs for studying D(2)R pathways. However, neither endogenous nor recombinant Ca(v)1.3 current was modulated by the D(2)R agonist quinpirole. We confirmed D(2)R expression in ST14A cells and also detected D(1)Rs, D(4)Rs, D(5)Rs, G(q), calcineurin and phospholipase A2 using RT-PCR and/or Western blot analysis. Phospholipase C ß-1 (PLCß-1) expression was not detected by Western blot analysis which may account for the lack of LTC modulation by D2Rs. These findings raise caution about the assumption that the presence of G-protein coupled receptors in cell lines indicates the presence of complete signaling cascades. However, exogenous arachidonic acid inhibited recombinant Ca(v)1.3 current indicating that channels expressed in ST14A cells are capable of modulation since they respond to a known signaling molecule downstream of D(2)Rs. Thus, ST14A cells provide a MSN-like cell line for studying channel modulation and signaling pathways that do not involve activation of PLCß-1.

A photosensitive surface capable of inducing electrophysiological changes in NG108-15 neurons.

Retinal prostheses promise to be a viable therapy for many forms of blindness. Direct stimulation of neurons using an organic light-sensitive, self-assembled monolayer surface offers a simple alternative to conventional semiconductor technology. For this purpose we have derivatized an indium tin oxide (ITO) substrate with the photosensitive dye, NK5962, using 3-(aminopropyl)trimethoxysilane (APTMS) as cross-linker. The surface was characterized through contact angle goniometry, electrochemical impedance spectroscopy, grazing angle infrared and ultraviolet-visible spectrophotometry. NG108-15 neurons were grown on the ITO-APTMS-NK5962 surface and neural responses from electrical stimulation vs. photostimulation through the ITO-APTMS-NK5962 surface were measured using patch clamp electrophysiology. Under these conditions, photostimulation of depolarized cells caused an approximate 2-fold increase in voltage-gated sodium (Na(+)) current amplitude at a membrane potential of -30mV. Our results demonstrate the feasibility of stimulating neurons, grown on light-sensitive surfaces, with light impulses, which ultimately may facilitate the fabrication of a simple, passive retinal prosthetic.

The calcium channel ligand FPL 64176 enhances L-type but inhibits N-type neuronal calcium currents.

One strategy for isolating neuronal L-type calcium (Ca(2+)) currents, which typically comprise a minority of the whole cell current in neurons, has been to use pharmacological agents that increase channel activity. This study examines the effects of the benzoyl pyrrole FPL 64176 (FPL) on L-type Ca(2+) currents and compares them to those of the dihydropyridine (+)-202-791. At micromolar concentrations, both agonists increased whole cell current amplitude in PC12 cells. However, FPL also significantly slowed the rate of activation and elicited a longer-lasting slow component of the tail current compared to (+)-202-791. In single channel cell-attached patch recordings, FPL increased open probability, first latency, mean closed time and mean open time more than (+)-202-791, with no difference in unitary conductance. These gating differences suggest that, compared to (+)-202-791, FPL decreases transition rates between open and closed conformations. Where examined, the actions of FPL and (+)-202-791 on whole cell L-type currents in sympathetic neurons appeared similar to those in PC12 cells. In contrast to its effects on L-type current, 10 microM FPL inhibited the majority of the whole cell current in HEK cells expressing a recombinant N-type Ca(2+) channel, raising caution concerning the use of FPL as a selective L-type Ca(2+) channel agonist in neurons.

Pharmacological discrimination between muscarinic receptor signal transduction cascades with bethanechol chloride.

1. Muscarinic agonist specificity is limited, making it difficult to match receptor subtypes with signal transduction cascades that mediate ion channel modulation. We have characterized the inhibitory effects of two muscarinic agonists, oxotremorine-M (Oxo-M) and bethanechol chloride (BeCh), on Ca(2+) currents in neonatal rat superior cervical ganglion neurons. 2. Oxo-M-mediated (10 micro M) inhibition occurred via two signaling pathways. The first pathway inhibited whole cell peak currents, consisting primarily of N-type current, but not FPL 64176-induced, long-lasting tail currents, comprised entirely of L-type current. Inhibited currents displayed slowed activation kinetics and voltage dependence, characteristics of membrane-delimited inhibition. Current inhibition was blocked by the selective M(2) receptor antagonist, methoctramine (METH; 100 nM), or following pertussis toxin (PTX) pretreatment. 3. Activation of the second pathway inhibited both peak and long-lasting tail currents. This pathway was voltage-independent, PTX-insensitive, but sensitive to internal Ca(2+) chelator concentration. Muscarinic toxin 7 (MT-7, 100 nM), an irreversible M(1) receptor antagonist, eliminated this inhibition. Oxo-M (100 micro M) decreased L- and N-type channel activities in cell-attached patches, indicating that a diffusible second messenger is involved. 4. BeCh (100 micro M) also inhibited whole cell currents via the membrane-delimited pathway. Blocking M(4) receptors with 100 nM pirenzepine (in the presence of MT-7) had no effect, while antagonizing M(2) receptors with METH abolished inhibition. Concentrations of BeCh as high as 3 mM failed to inhibit either peak or long-lasting tail currents following PTX pretreatment. 5. These results indicate that BeCh may be an effective tool for selectively activating M(2) receptor stimulation of the membrane-delimited pathway.

Arachidonic acid inhibition of L-type calcium (CaV1.3b) channels varies with accessory CaVbeta subunits.

Arachidonic acid (AA) inhibits the activity of several different voltage-gated Ca(2+) channels by an unknown mechanism at an unknown site. The Ca(2+) channel pore-forming subunit (Ca(V)alpha(1)) is a candidate for the site of AA inhibition because T-type Ca(2+) channels, which do not require accessory subunits for expression, are inhibited by AA. Here, we report the unanticipated role of accessory Ca(V)beta subunits on the inhibition of Ca(V)1.3b L-type (L-) current by AA. Whole cell Ba(2+) currents were measured from recombinant channels expressed in human embryonic kidney 293 cells at a test potential of -10 mV from a holding potential of -90 mV. A one-minute exposure to 10 microM AA inhibited currents with beta(1b), beta(3), or beta(4) 58, 51, or 44%, respectively, but with beta(2a) only 31%. At a more depolarized holding potential of -60 mV, currents were inhibited to a lesser degree. These data are best explained by a simple model where AA stabilizes Ca(V)1.3b in a deep closed-channel conformation, resulting in current inhibition. Consistent with this hypothesis, inhibition by AA occurred in the absence of test pulses, indicating that channels do not need to open to become inhibited. AA had no effect on the voltage dependence of holding potential-dependent inactivation or on recovery from inactivation regardless of Ca(V)beta subunit. Unexpectedly, kinetic analysis revealed evidence for two populations of L-channels that exhibit willing and reluctant gating previously described for Ca(V)2 channels. AA preferentially inhibited reluctant gating channels, revealing the accelerated kinetics of willing channels. Additionally, we discovered that the palmitoyl groups of beta(2a) interfere with inhibition by AA. Our novel findings that the Ca(V)beta subunit alters kinetic changes and magnitude of inhibition by AA suggest that Ca(V)beta expression may regulate how AA modulates Ca(2+)-dependent processes that rely on L-channels, such as gene expression, enzyme activation, secretion, and membrane excitability.

Regulation of voltage-gated Ca2+ channels by lipids.

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca(2+) channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca(V)2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP(2)), a palmitoylated accessory subunit (beta(2a)) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M(1) muscarinic receptors may stimulate multiple lipases to break down the PIP(2) associated with VGCCs and leave PIP(2)'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.

Orientation of palmitoylated CaVbeta2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation.

The G(q)-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca(2+) channel (Ca(V)2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca(V)beta is coexpressed with Ca(V)2.2. When palmitoylated Ca(V)beta2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca(V)beta3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca(V)beta2a may occupy an inhibitory site on Ca(V)2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca(V)beta2a relative to Ca(V)2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca(V)beta2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the alpha interacting domain (AID) of Ca(V)2.2's I-II linker. Ca(V)betas bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca(V)beta's movements to Ca(V)2.2 gating. Although Bdel1/beta2a currents exhibited more variable enhancement by SP, Bdel2/beta2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca(V)2.2 coexpressed with Ca(V)beta3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/beta2a currents, suggesting that when palmitoylated Ca(V)beta2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca(V)beta2a's palmitoyl groups directly interact with an inhibitory site on Ca(V)2.2 to block N-current inhibition by SP.

The Ca2+ channel beta subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current.

In superior cervical ganglion (SCG) neurons, stimulation of M(1) receptors (M(1)Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M(1)Rs. In addition, techniques that diminish AA's concentration during M(1)R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M(1)R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the beta subunit (Ca(V)beta) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M(1)Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca(V)2.2 and coexpressed with Ca(V)beta1b, Ca(V)beta3, or Ca(V)beta4 but enhanced activity of N channels containing Ca(V)beta2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca(V)beta2a, Ca(V)beta3, and Ca(V)beta4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca(V)beta with Ca(V)2.2. Further experiments with mutated, chimeric Ca(V)beta subunits and free palmitic acid revealed that palmitoylation of Ca(V)beta2a is essential for loss of inhibition. The data presented here fit a model in which Ca(V)beta2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca(V)beta2a toggles M(1)R- or NK-1R-mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M(1)Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca(V)beta2a.

Ca2+ influx through both L- and N-type Ca2+ channels increases c-fos expression by electrical stimulation of sympathetic neurons.

During direct membrane depolarization, Ca2+ influx primarily through L-type Ca2+ (L-) channels initiates activity-dependent gene transcription. This is surprising given that in most neurons a minority of the total Ca2+ current arises from L-channel activity. However, many studies have stimulated Ca2+ influx with unphysiological stimuli such as chronic membrane depolarization using high K+ medium. Few studies have tested whether other Ca2+ channels stimulate gene transcription in adult neurons as a consequence of direct electrical stimulation. Therefore, we evaluated the role of L- and N-type Ca2+ (N-) channel activity in regulating mRNA levels of c-fos, an activity-dependent transcription factor, in adult rat superior cervical ganglion (SCG) neurons as the majority of Ca2+ channels are N-type, while only a minority are L-type. Changes in c-fos mRNA levels were measured using semi-quantitative and single-cell RT-PCR. Phosphorylation of CREB (pCREB) and changes in c-Fos levels were visualized in dissociated cells by immunocytochemistry. Increases in pCREB, c-fos mRNA and c-Fos protein with either K+ or electrical depolarization required Ca2+ influx. These results support previous findings that elevated c-fos levels result from pCREB stimulating c-fos transcription. Elevation of pCREB, c-fos and c-Fos with K+ depolarization depended on L-channel activity. By contrast, antagonizing either channel at 10-Hz stimulation minimized these increases despite unequal numbers of the two channel types. Transition to exclusive L-channel involvement occurred with increasing frequency of stimulation (from 10 to 20 to 50 Hz). Our results demonstrate that N- and L-channel participation in regulating c-fos expression is encoded in the pattern of electrical stimulation.

Arachidonic acid mediates muscarinic inhibition and enhancement of N-type Ca2+ current in sympathetic neurons.

N-type Ca(2+) channels participate in acute activity-dependent processes such as regulation of Ca(2+)-activated K(+) channels and in more prolonged events such as gene transcription and long-term depression. A slow postsynaptic M(1) muscarinic receptor-mediated modulation of N-type current in superior cervical ganglion neurons may be important in regulating these processes. This slow pathway inhibits N-type current by using a diffusible second messenger that has remained unidentified for more than a decade. Using whole-cell patch-clamp techniques, which isolate the slow pathway, we found that the muscarinic agonist oxotremorine methiodide not only inhibits currents at positive potentials but enhances N-type current at negative potentials. Enhancement was also observed in cell-attached patches. These findings provide evidence for N-type Ca(2+)-current enhancement by a classical neurotransmitter. Moreover, enhancement and inhibition of current by oxotremorine methiodide mimics modulation observed with direct application of a low concentration of arachidonic acid (AA). Although no transmitter has been reported to use AA as a second messenger to modulate any Ca(2+) current in either neuronal or nonneuronal cells, we nevertheless tested whether a fatty acid signaling cascade was involved. Blocking phospholipase C, phospholipase A(2), or AA but not AA metabolism minimized muscarinic modulation of N-type current, supporting the participation of these molecules in the slow pathway. A role for the G protein G(q) was also confirmed by blocking muscarinic modulation of Ca(2+) currents with anti-G(qalpha) antibody. Our finding that AA participates in the slow pathway strongly suggests that it may be the previously unknown diffusible second messenger.