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Research

Current Projects in the Rittenhouse Laboratory

The Rittenhouse lab examines protein-protein, protein-lipid, protein-small molecule, and/or protein-nucleic acid interactions of voltage-gated calcium channels (VGCCs). Disruptions of these interactions lead to certain diseases. This research focus highlights functional aspects of molecular interactions across a spectrum of preparations from established immortalized cell lines to primary neurons, human pancreatic b-cells and human iPSCs.  Many of our studies involve measuring activity of molecules in real time using state-of-the art imaging methodologies, secretion assays, and electrophysiological methodologies. Our goals are to reveal how specific molecules interact and function under normal and pathological conditions.

Diseases result from physiological changes in our bodies that adversely affect our health. Ultimately, these disease precipitating changes occur at the molecular level. Every molecule has binding partners that form a functional cassette often used in many cell types. Using this information, we build upon these molecular interactions to predict function at the systems level and identify therapeutic targets for treating brain diseases, such as Alzheimer’s Disease, Stroke, and Schizophrenia.

The Rittenhouse lab is asking three questions that interrogate the regulation of VGCCs in health and disease:

  1. How do neurotransmitters and second messengers alter VGCC activity?
  2. Do vicinal palmitoyl groups from palmitoylated proteins bind to VGCCs to change their responsiveness to modulatory mechanisms initiated by neurotransmitters?
  3. Does the protein Disrupted in Schizophrenia 1 (DISC1) control VGCC levels at synapses?

Project 1: Modulation of voltage-gated Ca2+ channels by neurotransmitters and signaling molecules.

The Rittenhouse lab has a long-standing interest in N-type VGCCs because of their special position in the nervous system. They coordinate electrical activity occurring at the cell membrane with underlying biochemical and transcriptional events. N-VGCCs are found only in nerve cells and neuronally-derived tissues, are associated with the regulation of transmitter synthesis, and release from most presynaptic nerve endings. They are the most extensively modulated VGCCs in the brain in that more pathways exist for their modulation than for any other type.

While plasticity of the brain at the level of complex human behavior is quite obvious, it is also apparent at the cellular and molecular level. One initial site for plasticity occurs with the influx of Ca2+ through VGCCs. Ca2+ influx serves a unique function of interfacing electrical signals with cellular biochemical and transcriptional changes. Variability in VGCC expression levels, in the location of cell-surface expression and in channel activity has profound effects on how much and where Ca2+ enters a nerve cell. This in turn influences the strength of synaptic contacts and on cellular and transcriptional processes. We are currently interrogating how phospholipids and free fatty acid metabolites modulate the N-VGCC using a variety of electro­physio­logical, imaging, biochemical, and molecular strategies. Superior cervical ganglion neurons (SCG) are used in this study because N-VGCCs are the dominant Ca2+ channel present.

Lab efforts on this project currently focus on two questions: 

  1. How does native N-VGCC activity change when modulated by neurotransmitters, which utilize small lipid signaling molecules such as fatty acids?
  2. What role does cPLA2a play in neurotransmitter-mediated channel modulation by small lipid signaling molecules?

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Figure 1: PIP2-AA Model of Phospholipid Metabolism by GqCRs. VGCCs are formed by a protein complex of three subunits: a pore-forming a1-sub­­­unit (CaVa1), a trans­membrane a2-d sub­unit, and a cytoplasmic b-subunit (CaVb) that binds to the AID segment of CaVa1’s I-II cytoplasmic linker.

Our findings support the following model: a phospholipid, possibly phosphatidylinositol 4,5-bisphosphate (PIP2) is the source of endogenous poly­un­satur­ated free fatty acidarachidonic acid (AA), is. AA is normally found as one of the fatty acid tails of PIP2 (Fig. 1). PIP2 binds to N-VGCCs; its presence couples the voltage sensor movement to opening the channel. Muscarinic M1 receptors (M1Rs) inhibit N-VGCC activity by a Gq signaling pathway involving metabolism of the bound phospholipid and thus has served as a model system to examine lipid effects on channels.

The lab currently is interrogating the following hypotheses:

  • M1R coupling to Gq activates phos­pho­lipase C (PLCb) to remove the inositol head group from the PIP2 associated with each channel. This first step is insuffi­cient to confer inhibition.
  • Both diacyl­glycerol lipase (DAGL) and group IVa phospholipase A2 (cPLA2a) must cleave the two fatty acid tails (normally stearic acid in the sn-1 position and AA in the sn-2 position) from the glycerol backbone to observe VGCC inhi­bition.
  • The two fatty acid tails are predicted to remain bound to the channel. By occupying sites where PIP2’s fatty acid tails normally reside, the free fatty acids block a PIP2 molecule from re-binding to the PIP2 binding site and thus uncouples voltage sensing from channel opening.

This simple model resolves previous con­cep­tual disagreements over the mechan­ism of N-VGCC inhi­bi­tion by Gq signaling and provides a frame­work for pursuing additional questions surrounding Ca2+ channel regulation by lipid molecules.

Additionally, we have interrogated the functional importance of cPLA2a in regulating action potential firing properties. We found that SCG neurons from cPLA2a’s knockout mice exhibit neuronal hyper­excita­bility, indicating the enzyme provides tonic regula­tion of phospholipid interaction with multiple types of ion channels.

Significance: Currently, antago­nizing cPLA2a is a therapeutic strategy for decreasing excito­toxi­city during stroke. The main target of SCG neurons is cerebral blood vessels. These surprising findings raise questions about this therapeutic strategy over the long term since inhibiting cPLA2a could cause severe cerebral vasospasm. Thus, cPLA2a may be a useful target for debilitating transient ischemic attacks.

Project 2: Do vicinal palmitoyl groups from proteins interact with VGCCs to change their responsiveness to modulatory mechanisms initiated by neurotransmitters?

Expression of CaVb2a vs other CaVb subunits appears to block inhibition of N- and L-VGCC activity by M1 muscarinic receptor signaling in sympathetic neurons. Genetic, electrophysiological, biochemical and cell biological methodology are used to:

  1. Test whether palmitoylation of CaVb2a’s two N-terminus cysteines (C3,C4) are responsible for this block and/or whether additional residues of CaVb2a are involved.
  2. Test whether the CaVb2a’s palmitoyl groups compete with PIP2 for binding to the channel.
  3. Identify the lipid interaction domain where the palmitoyl groups interact with VGCCs.

 If proven correct, the findings would document a new role for palmitoylation of cytoplasmic proteins – that of serving as a phospholipid mimic that regulates transmembrane proteins.

Figure 2. M1R agonist or free AA inhibits N-VGCC activity from recombinant channels made up of a b1, b3, or b4 subunit coexpressed with CaV2.2e, the N-VGCC pore-forming subunit, and a2d1, an accessory subunit. Comparison of N-VGCCs protected with pal­mi­toy­lated β2a (Protected) to N-VGCCs with β3 that are susceptible (Available) to inhibition by GqPCR signaling (Inhibited). The exaggerated size of PIP2 serves to illustrate the 3 different lipids predicted to interact with N-VGCCs.

In contrast, co­expression with a CaVb2 splice variant, CaVb2a, blocks N-VGCC inhibition revealing a latent enhancement of current. Enhancement occurs at a separate phospholipid -binding site from inhibition. Coexpression of CaV2.2e, and a2d with depalmitoylated CaVb2a restores inhi­bi­tion, indicating that palmitoylation is necessary for block.

A similar vari­ability in current modulation occurs with a different class of channels, the L-VGCCs CaV1.3, when co-expressed with different CaVb sub­units, indicating that CaVb2a interacts with other pore-forming subunits to block VGCC inhibition by M1Rs.

Significance: These studies reveal a new role for protein palmitoylation; that of regulating VGCC modula­tion. More­over, our findings raise the possibility that the palmitoyl residues of CaVb2a directly interact with N-VGCCs at the phospholipid binding site. If the palmitoyl groups substitute for PIP2’s fatty acid tails, no phospholipid metabolism would occur upon M1R signaling because no phospholipid is bound: the channels are protected from inhibition. Palmitoy­la­tion-de­pal­mi­toy­la­tion events allow M1R responses to toggle between current inhibi­tion and en­hance­ment from moment to moment depending on the activity of phos­pho­li­pases, acyl hydro­lases, and palmitoyl acyl trans­ferases. Moreover, sequentially palmitoyaled proteins serving as a phospholipid mimic provides a unique mechanism for regulating transmembrane proteins.

Project-3: Do vicinal palmitoyl groups of other proteins bind to VGCCs?

Respiratory syncytial virus (RSV) is a significant threat to elderly populations, a threat that rivals that of influenza virus. Currently it is estimated that RSV infections in the elderly result in 11,000 to 17,000 deaths per year in the US alone and ten times that number of RSV associated hospitalizations. The world-wide population over age 60 is predicted to reach 2.1 billion, more than 20% of the total population, by 2050. In some developed countries, this percentage is already at 20-25%. Such an expansion of this population over the next few decades will pose a greatly increased public health burden making development of RSV vaccines for the elderly an important priority. However, attempts to develop effective RSV vaccines, which have proceeded since the late 1960s, have failed. This problem is compounded by the requirement that an RSV vaccine for the elderly must overcome immune senescence that accompanies aging. This project is developing novel virus-like particles (VLPs) as an RSV vaccine for elderly populations. These VLPs contain the RSV pre-fusion F protein and the RSV G protein. They have been shown to be an effective vaccine in mice and cotton rats (CRs). In recent studies, we have defined effects of prior RSV infection on immune responses to pre-fusion F VLP immunization and reported results consistent with our novel conclusion that RSV infection can induce healthy RSV memory responses, however, a second RSV infection cannot activate this memory, a result consistent with the inability of RSV infections of humans to protect from subsequent RSV infections. Strikingly, however, a single immunization with a pre-fusion F containing VLP can robustly activate memory established by prior RSV infections resulting in high titer neutralizing antibodies with high avidity and robust levels of F protein specific splenic memory B cells and bone marrow associated plasma cells (LLPC) while a second RSV infection cannot. These observations suggest that the challenge for a vaccine for the elderly, who have experienced RSV infections multiple times, is to efficiently recall this memory. This proposal is based on the hypothesis that pre-fusion F containing VLPs can accomplish this recall. This project is exploring different ways to optimize activation of memory responses to RSV.

Project 4: Immune responses to different forms of SARS-CoV-2 S protein.

The goal of this project is to identify the optimal conformational form of the SARS-CoV2 S protein for induction of protective responses. We are employing the approaches we have developed for generation of vaccine candidates for respiratory syncytial virus (RSV). The attachment protein for SARS-CoV-2 is the S protein, which binds to the ACE-2 cell receptor and is the primary target for vaccine development. Our focus is on immune responses to VLPs assembled with different versions of the SARS-CoV-2 S protein. This project is proceeding by assembling and validating of VLPs with different conformations of the S protein or a subset of S protein sequences, immunization of mice with the VLPs in order to characterize of the antibody responses and durability of protective responses to different S protein sequences, and assessment of the safety of the immunization.