M\u3csub\u3e1\u3c/sub\u3e Muscarinic Modulation of N-Type Calcium Channels: A Dissertation

The influx of calcium through N-type calcium channels (N-current) affects a myriad of neuronal functions. These include the triggering of synaptic release of neurotransmitter, adjustment of membrane potential and changes in gene transcription. N-channels are highly modulated proteins, so that N-current is attenuated or potentiated in response to environmental changes. In turn, the modulation of N-current has a direct effect on the downstream events, making the N-channel a focal point in neural signaling, and its modulation a mechanism for short term plasticity. The modulation of N-current by M1 muscarinic receptors (M1Rs) is of particular interest for several reasons. The M1R is instrumental in both cognition and memory formation as indicated by studies using either pharmacological agents aimed at M1Rs or knockout animals lacking M1Rs. Clinically, the M1R is an important target in the treatment of Alzheimer’s disease. Thus, like the N-channel, the M1R is an important element of neural signaling. Moreover, the stimulation of M1Rs affects N-current by through signaling pathways which despite being studied for decades, are not completely understood. For my dissertation I have investigated of M1R signaling on N-current using electrophysiological recordings of N-current from freshly dissociated neurons and from HEK cells expressing N-channels and M1Rs. Asking how one receptor affects one type of calcium channel would seem to be a simple question. However, the answer has many facets. Since M1Rs have multiple downstream effects and N-channels are highly modulated proteins, stimulation of M1Rs initiates several different pathways which modulate N-current. This thesis aims to unravel some of the complexities of the interactions of two vital components of neuronal signaling. Here I present the results of studies elucidating three different actions of M1signaling of N-current modulation. The first study I present here examines the effect of N-channel subunit composition on modulation of N-current. The stimulation of M1Rs in superior cervical ganglion (SCG) neurons elicits a distinct pattern of modulation; inhibiting N-current elicited by strong depolarizations and enhancing current elicited by lesser depolarizations. Thus M1Rs cause two simultaneous modulatory effects on N-current; increasing voltage sensitivity and decreasing overall conductance. I found the expression of the N-channel’s β subunit (CaVβ) determines the observed effect. Specifically when the isoform CaVβ2a is expressed M1 stimulation elicits enhancement without inhibition. Conversely, when CaVβ1b, CaVβ3, or CaVβ4 are expressed M1 stimulation elicits inhibition with out enhancement. These results fit a model in which both the enhancing and inhibiting effects of M1stimulation occur in all channels, but typically inhibition dominates. CaVβ2a blocks inhibition unmasking latent enhancement. Moreover, using mutants and chimeras I found palmitoylation of CaVβ2a at the N-terminus plays a key role in blocking inhibition. My findings predict the expression and localization of different CaVβ isoforms would dramatically alter modulation of N-current and thus may represent a previously unrecognized form of plasticity. The inhibition of N-current by M1Rs is controversial. It has been proposed recently that inhibition is directly attributable to the depletion of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during M1 stimulation. However, in our lab, we have found arachidonic acid (AA) release, which occurs subsequent to PtdIns(4,5)P2 hydrolysis, is both necessary and sufficient to elicit inhibition. Therefore, in a second study, I tested the effect of CaVβ expression on N-current during exogenous AA application and found a pattern of modulation identical to M1R stimulation. Furthermore, I took part in a collaborative project identifying the AA producing enzyme, diacylglycerol lipase (DAGL), to be a necessary component of the inhibitory pathway elicited by M1Rs. These findings provide increased evidence for AA release being a key factor in the M1R stimulated pathway of inhibition. Moreover, these discoveries identify the expression of CaVβ2a and use of specific DAGL inhibitors as a molecular and pharmacological strategy to block inhibition of N-current, respectively. These tools allow the dissection of downstream effects of M1R stimulation, so that other modulatory effects may be observed. The phosphorylation of N-channels by protein kinase C (PKC) blocks inhibition of current brought on by G-protein β and γ subunits (Gβγ) binding directly to the channel. Relief of Gβγ inhibition by other means has been identified as a mechanism of short term plasticity. M1Rs are known to simulate PKC, but a connection between M1Rs and PKC phosphorylation of Nchannels had not been demonstrated. I hypothesized that PKC stimulation may be occluded by other downstream effects of M1Rs. Therefore in a third study, I used a pharmacological approach on SCG neurons to dissect the PKC activating pathway from the other downstream effects of M1 stimulation. I observed modulation of N-current indicating a loss of Gβγ&#; inhibition, thus consistent with PKC phosphorylation of channels. This conclusion reveals another aspect of M1 modulation, which can function as a means of short term plasticity

Clinical decision making in a high-risk primary care environment: a qualitative study in the UK

OBJECTIVE: Examine clinical reasoning and decision making in an out of hours (OOH) primary care setting to gain insights into how general practitioners (GPs) make clinical decisions and manage risk in this environment. DESIGN: Semi-structured interviews using open-ended questions. SETTING: A 2-month qualitative interview study conducted in Oxfordshire, UK. PARTICIPANTS: 21 GPs working in OOH primary care. RESULTS: The most powerful themes to emerge related to dealing with urgent potentially high-risk cases, keeping patients safe and responding to their needs, while trying to keep patients out of hospital and the concept of 'fire fighting'. There were a number of well-defined characteristics that GPs reported making presentations easy or difficult to deal with. Severely ill patients were straightforward, while the older people, with complex multisystem diseases, were often difficult. GPs stopped collecting clinical information and came to clinical decisions when high-risk disease and severe illness requiring hospital attention has been excluded; they had responded directly to the patient's needs and there was a reliable safety net in place. Learning points that GPs identified as important for trainees in the OOH setting included the importance of developing rapport in spite of time pressures, learning to deal with uncertainty and learning about common presentations with a focus on critical cues to exclude severe illness. CONCLUSIONS: The findings support suggestions that improvements in primary care OOH could be achieved by including automated and regular timely feedback system for GPs and individual peer and expert clinician support for GPs with regular meetings to discuss recent cases. In addition, trainee support and mentoring to focus on clinical skills, knowledge and risk management issues specific to OOH is currently required. Investigating the stopping rules used for diagnostic closure may provide new insights into the root causes of clinical error in such a high-risk setting

Additional preliminary soil and foundation investigation -- proposed 6, 7, and 9-story apartment structures and 2-story parking structures, Hui Iwa Street, Kaneohe, Hawaii

W.O. 414-00Includes field investigation, site conditions, groundwater, recommendations, site plan, allowable load in kips, boring logs, and laboratory test data.Dan Ostrow Construction Compan

Addendum soils investigation -- Kuikahi Gardens, Kalihi Street, Kalihi-Uka, Honolulu, Hawaii

W.O. 344-10Sections: field exploration, soils, history of sliding, recommendations, grading plan, cross sections, boring logs, laboratory testing, and laboratory data.Mid-Pac Development, Ltd

Feasibility level, soils and geology investigation -- Lilipuna Hillside Residence development, Kaneohe, Hawaii

tax map key: 4-6-01W.O. 435-00Includes proposed development, field exploration, subsurface conditions, recommendations, site plan, test pit logs, and grading specifications.Brian Gray & Associate

Preliminary soils and geology investigation -- Puu Alii Residential PUD, Heeia, Oahu, Hawaii

tax map key: 4-6-02W.O. 436-00Includes proposed development, field exploration, subsurface conditions, recommendations, site plan, boring logs, and test pit logs.Brian Gray & Associate

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\u27s 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