Membrane Stretch Gates NMDA Receptors.

NMDARs are ionotropic glutamate receptors widely expressed in the CNS, where they mediate phenomena as diverse as neurotransmission, information processing, synaptogenesis, and cellular toxicity. They function as glutamate-gated Ca2+-permeable channels, which require glycine as coagonist, and can be modulated by many diffusible ligands and cellular cues, including mechanical stimuli. Previously, we found that, in cultured astrocytes, shear stress initiates NMDAR-mediated Ca2+ entry in the absence of added agonists, suggesting that more than being mechanosensitive, NMDARs may be mechanically activated. Here, we used controlled expression of rat recombinant receptors and noninvasive on-cell single-channel current recordings to show that mild membrane stretch can substitute for the neurotransmitter glutamate in gating NMDAR currents. Notably, stretch-activated currents maintained the hallmark features of the glutamate-gated currents, including glycine-requirement, large unitary conductance, high Ca2+ permeability, and voltage-dependent Mg2+ blockade. Further, we found that the stretch-gated current required the receptor's intracellular domain. Our results are consistent with the hypothesis that mechanical forces can gate endogenous NMDAR currents even in the absence of synaptic glutamate release, which has important implications for understanding mechanotransduction and the physiological and pathologic effects of mechanical forces on cells of the CNS.SIGNIFICANCE STATEMENT We show that, in addition to enhancing currents elicited with low agonist concentrations, membrane stretch can gate NMDARs in the absence of the neurotransmitter glutamate. Stretch-gated currents have the principal hallmarks of the glutamate-gated currents, including requirement for glycine, large Na+ conductance, high Ca2+ permeability, and voltage-dependent Mg2+ block. Therefore, results suggest that mechanical forces can initiate cellular processes presently attributed to glutamatergic neurotransmission, such as synaptic plasticity and cytotoxicity. Given the ubiquitous presence of mechanical forces in the CNS, this discovery identifies NMDARs as possibly important mechanotransducers during development and across the lifespan, and during pathologic processes, such as those associated with traumatic brain injuries, shaken infant syndrome, and chronic traumatic encephalopathy.

Separate intramolecular targets for protein kinase A control N-methyl-D-aspartate receptor gating and Ca2+ permeability.

Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca(2+) flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca(2+) permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca(2+) conductance, because neither Na(+) conductance nor Ca(2+)-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca(2+) permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca(2+) permeability.

Sustaining "meaningful use" of health information technology in low-resource practices.

PURPOSE: The implementation of electronic health records (EHRs) has been extensively studied, but their maintenance once implemented has not. The Regional Extension Center (REC) program provides implementation assistance to priority practices-those with limited financial, technical, and organizational resources-but the assistance is time limited. Our objective was to identify potential barriers to maintenance of meaningful use of EHRs in priority primary care practices using a qualitative observational study for federally qualified health centers (FQHCs) and priority practices in Michigan. METHODS: We conducted cognitive task analysis (CTA) interviews and direct observations of health information technology implementation in FQHCs. In addition, we conducted semistructured interviews with implementation specialists serving priority practices to detect emergent themes relevant to maintenance. RESULTS: Maintaining EHR technology will require ongoing expert technical support indefinitely beyond implementation to address upgrades and security needs. Maintaining meaningful use for quality improvement will require ongoing support for leadership and change management. Priority practices not associated with larger systems lack access to the necessary technical expertise, financial resources, and leverage with vendors to continue alone. Rural priority practices are particularly challenged, because expertise is often not available locally. CONCLUSIONS: Priority practices, especially in rural areas, are at high risk for falling on the wrong side of a "digital divide" as payers and regulators enact increasing expectations for EHR use and information management. For those without affiliation to maintain the necessary expert staff, ongoing support will be needed for those practices to remain viable.

C-terminal domains of N-methyl-D-aspartic acid receptor modulate unitary channel conductance and gating.

NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor.

An Approach to Evaluating Multisector Partnerships to Support Evidence-Based Quality Improvement in Primary Care.

BACKGROUND: Quality improvement (QI) interventions in primary care are increasingly designed and implemented by multisector partnerships, yet little guidance exists on how to best monitor or evaluate these partnerships. The goal of this project was to describe an approach for evaluating the development and effectiveness of a multisector partnership using data from the first year of the Healthy Hearts for Michigan (HH4M) Cooperative, a multisector partnership of nine organizations tasked with designing and implementing evidence-based QI strategies for hypertension management and tobacco cessation in 50 rural primary care practices. METHODS: The researchers developed a 49-item online survey focused on factors that facilitate or hinder multisector partnerships, drawing on implementation science and partnership, engagement, and collaboration research. The team surveyed all 44 members of the HH4M Cooperative (79.5% response rate) and conducted interviews with 14 members. The interviews focused on implementation phase-specific goals, accomplishments, and challenges. Descriptive analysis was used for the survey results, and thematic analysis for the interview data. RESULTS: Respondents reported strong overall performance by the Cooperative during its first year, which facilitated the successful completion of several intervention design tasks. Strengths included having a clear purpose and trust and respect among members. Areas for improvement included a need for common terminology, clarification of roles and functions, and improvement in communication across workgroups. Lack of engagement from physician practices due to capacity constraints, exacerbated by the COVID-19 pandemic, was the Cooperative's biggest challenge. CONCLUSION: This multimethod approach to evaluating the development and effectiveness of a multisector partnership yielded practical, actionable feedback to program leaders.

Rationale and design for Healthy Hearts for Michigan (HH4M): A pragmatic single-arm hybrid effectiveness-implementation study.

Background: The burden of cardiovascular disease (CVD) is particularly high in several US states, which include the state of Michigan. Hypertension and smoking are two major risk factors for mortality due to CVD. Rural Michigan is disproportionally affected by CVD and by primary care shortages. The Healthy Hearts for Michigan (HH4M) study aims to promote hypertension management and smoking cessation through practice facilitation and quality improvement efforts and is part of the multi-state EvidenceNOW: Building State Capacity initiative to provide external support to primary care practices to improve care delivery. Methods: Primary care practices in rural and underserved areas of Michigan were recruited to join HH4M, a pragmatic, single-arm hybrid Type 2 effectiveness-implementation study during which practice facilitation was delivered at the practice level for 12 months, followed by a 3-month maintenance period. Results: Fifty-four practices were enrolled over a 12-month recruitment period. At baseline, the mean proportion (standard deviation) of patients at the practice level meeting the clinical quality measures were: blood pressure, 0.72 (0.12); tobacco screening, 0.80 (0.30); tobacco cessation intervention, 0.57 (0.28); tobacco screening and cessation intervention: 0.78 (0.26). Conclusion: This three-year research program will evaluate the ability of rural and medically underserved primary care practices to implement the quality improvement model by identifying drivers of and barriers to sustainable implementation, and test whether the model improves (a) blood pressure control and (b) tobacco use screening and cessation.

Mechanical stress activates NMDA receptors in the absence of agonists.

While studying the physiological response of primary rat astrocytes to fluid shear stress in a model of traumatic brain injury (TBI), we found that shear stress induced Ca2+ entry. The influx was inhibited by MK-801, a specific pore blocker of N-Methyl-D-aspartic acid receptor (NMDAR) channels, and this occurred in the absence of agonists. Other NMDA open channel blockers ketamine and memantine showed a similar effect. The competitive glutamate antagonists AP5 and GluN2B-selective inhibitor ifenprodil reduced NMDA-activated currents, but had no effect on the mechanically induced Ca2+ influx. Extracellular Mg2+ at 2 mM did not significantly affect the shear induced Ca2+ influx, but at 10 mM it produced significant inhibition. Patch clamp experiments showed mechanical activation of NMDAR and inhibition by MK-801. The mechanical sensitivity of NMDARs may play a role in the normal physiology of fluid flow in the glymphatic system and it has obvious relevance to TBI.

Extracellular Ca(2+) ions reduce NMDA receptor conductance and gating.

Brief intracellular Ca(2+) transients initiate signaling routines that direct cellular activities. Consequently, activation of Ca(2+)-permeable neurotransmitter-gated channels can both depolarize and initiate remodeling of the postsynaptic cell. In particular, the Ca(2+) transient produced by NMDA receptors is essential to normal synaptic physiology, drives the development and plasticity of excitatory central synapses, and also mediates glutamate excitotoxicity. The amplitude and time course of the Ca(2+) signal depends on the receptor's conductance and gating kinetics; these properties are themselves influenced both directly and indirectly by fluctuations in the extracellular Ca(2+) concentration. Here, we used electrophysiology and kinetic modeling to delineate the direct effects of extracellular Ca(2+) on recombinant GluN1/GluN2A receptor conductance and gating. We report that, in addition to decreasing unitary conductance, Ca(2+) also decreased channel open probability primarily by lengthening closed-channel periods. Using one-channel current recordings, we derive a kinetic model for GluN1/GluN2A receptors in physiological Ca(2+) concentrations that accurately describes macroscopic channel behaviors. This model represents a practical instrument to probe the mechanisms that control the Ca(2+) transients produced by NMDA receptors during both normal and aberrant synaptic signaling.

One-channel cell-attached patch-clamp recording.

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel's conductance properties and the temporal sequence of open-closed conformations that make up the channel's activation mechanism, thus helping to understand their functions in health and disease.

Two serine residues on GluN2A C-terminal tails control NMDA receptor current decay times.

NMDA receptors are glutamate-activated, Ca ( 2+) -permeable ion channels with critical roles in synaptic transmission and plasticity. The shape and size of their current is modulated by several kinase/phosphatase systems, and numerous residues located on the receptors' intracellular C-termini are phosphorylated in vivo. To investigate the mechanisms by which phosphorylation may control channel gating, we examined the single-channel behaviors of receptors carrying the S900A or S929A substitution in their GluN2A subunits and thus were rendered resistant to phosphorylation at those sites. We found that the mutations reduced channel open probability primarily by increasing the frequency of desensitized events. The kinetic models we developed revealed complex but similar changes in mechanism for the two mutants, leading to the view that dephosphorylation at either site may cause receptors to activate slower, deactivate faster and desensitize more frequently. This modulatory mechanism is consistent with the proposed roles for these residues in Ca ( 2+) -dependent desensitization and calcineurin-mediated reduction of current during brain development.