Biology subjects

Brumback, A. C.

Publications and source records attributed to Brumback, A. C..

6 recordsLinked to original sources

bioRxiv2026

Single-molecule nanophotonic resolution of binding dynamics from apo to fully liganded for a cyclic nucleotide-gated ion channel in cell-derived vesicles

Many biological macromolecules are activated upon ligand binding at multiple specific binding domains. However, how these domains interact and the transient intermediate conformations that connect binding events to protein activity are typically unknown. Ensemble-averaged measures over stochastic binding events are challenged to resolve the underlying asynchronous dynamics. Single-molecule resolution of these dynamics offers an attractive approach to investigate the ligand-activation process. Optical methods using fluorescently labeled ligands enable observation of individual binding events that report on the energetics of early ligand-bound conformational changes. However, diffraction-limited microscopy limits these methods to low ligand concentrations, often below what is required for physiologically relevant activation. Here, we overcome this limitation using nanophotonic zero-mode waveguides to observe the sequential binding of a fluorescent cyclic nucleotide to each of four subunits in TAX-4 cyclic nucleotide-gated ion channels in cell-derived vesicles. Our observations suggest that binding at one domain positively promotes binding at other domains, and that binding induces an isomerization of the binding domain in individual subunits which we attribute to a sequence of pre-activated intermediate states. This approach provides a broadly applicable tool to dissect the energetic landscape of ligand-binding at macromolecules in native cell membranes.

biophysics↗

bioRxiv2026

The human GABAA receptor α4 subunit variant Ile114Asn linked to epilepsy impairs membrane expression with β3 and δ subunits

GABAA receptors mediate fast inhibitory neurotransmission in the brain, and variants in their subunits are linked to neurological disorders like epilepsy. For many identified variants, it remains to be determined whether the variant is pathogenic. Here, we functionally characterized the 4 subunit variant p.I114N (p.I79N in the mature protein), found in an individual with epilepsy, using patch-clamp electrophysiology in combination with {beta}3 and {delta} subunits in HEK293T cells. Whereas the variant has little to no impact on basal activity or GABA-sensitivity, lower maximal GABA-elicited currents as compared to wild-type channels suggest that it impairs assembly or trafficking to reduce expression of channels in the plasma membrane.

biophysics↗

bioRxiv2025

HCN channel currents underlie distinct neurophysiology of mediodorsal thalamus subnuclei

The mediodorsal thalamus (MD) is a hub coordinating cortical and subcortical brain regions to support executive and social/emotional functioning. MD can be subdivided into medial (M), central (C), and lateral (L) based on synaptic coupling, molecular identity, and physiology. Recently, we identified differential intrinsic properties between thalamocortical M and L neurons projecting to the medial prefrontal cortex (mPFC). L neurons projecting to mPFC showed increased hyperpolarization activated cyclic nucleotide gated (HCN) channel activity compared with M neurons, which caused L neurons to have lower cellular resistance and shorter time windows for integration of inputs. In addition to their role in synaptic integration, HCN channels are critical for thalamic rhythm generation. In this study, we used a combination of patch clamp electrophysiology and in situ hybridization to investigate how differences in HCN impact intrinsic oscillatory dynamics in M, C, and L neurons. We found that HCN current differed across MD subnuclei with C > L >> M. Clustering neurons based on HCN properties was sufficient to classify subnuclei with >95% accuracy, highlighting the differences in HCN function between subnuclei. Greater HCN activity in MD neurons was associated with decreased input resistance, decreased action potential firing, and higher resonant frequency. These findings provide an ionic basis for differences in cellular resonance across MD subnuclei, with implications for thalamic rhythm generation and information processing.

neuroscience↗

bioRxiv2025

Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity

BackgroundThe neurodevelopmental disorder Fragile X syndrome (FXS) results from hypermethylation of the FMR1 gene which prevents FMRP production. FMRP modulates the expression and function of a wide variety of proteins, including voltage-gated ion channels such as Hyperpolarization-Activated Cyclic Nucleotide gated (HCN) channels, which are integral to rhythmic activity in thalamic structures. Thalamocortical pathology, particularly involving the mediodorsal thalamus (MD), has been implicated in neurodevelopmental disorders. MD connectivity with mPFC is integral to executive functions like working memory and social behaviors that are disrupted in FXS. MethodsWe used a combination of retrograde labeling and ex vivo brain slice whole cell electrophysiology in 40 wild type and 42 Fmr1 KO male mice to investigate how a lack of Fmr1 affects intrinsic cellular properties in lateral (MD-L) and medial (MD-M) MD neurons that project to the medial prefrontal cortex (MD[->]mPFC neurons). ResultsIn MD-L neurons, Fmr1 knockout caused a decrease in HCN-mediated membrane properties such as voltage sag and membrane afterhyperpolarization. These changes in subthreshold properties were accompanied by changes in suprathreshold neuron properties such as the variability of action potential burst timing. ConclusionsIn Fmr1 knockout mice, reduced HCN channel activity in MD[->]mPFC neurons impairs both the timing and magnitude of HCN-mediated membrane potential regulation. Changes in response timing may adversely affect rhythm propagation in Fmr1 KO thalamocortical circuitry. MD thalamic neurons are critical for maintaining rhythmic activity involved in cognitive and affective functions. Understanding specific mechanisms of thalamocortical circuit activity may lead to therapeutic interventions for individuals with FXS.

neuroscience↗

bioRxiv2024

A novel mouse model for developmental and epileptic encephalopathy by Purkinje cell-specific deletion of Scn1b

Loss of function variants of SCN1B are associated with a range of developmental and epileptic encephalopathies (DEEs), including Dravet syndrome. These DEEs feature a wide range of severe neurological disabilities, including changes to social, motor, mood, sleep, and cognitive function which are notoriously difficult to treat, and high rates of early mortality. While the symptomology of SCN1B-associated DEEs indicates broad changes in neural function, most research has focused on epilepsy-related brain structures and function. Mechanistic studies of SCN1B/Scn1b have delineated diverse roles in development and adult maintenance of neural function, via cell adhesion, ion channel regulation, and other intra- and extra-cellular actions. However, use of mouse models is limited as knockout of Scn1b, globally and even in some cell-specific models (e.g., Parvalbumin+ interneuron-specific knockout) in adult mice, leads to severe and progressive epilepsy, health deterioration, and 100% mortality within weeks. Here, we report findings of a novel transgenic mouse line in which Scn1b was specifically deleted in cerebellar Purkinje cells. Unlike most existing models, these mice did not show failure to thrive or early mortality. However, we quantified marked decrements to Purkinje cell physiology as well as motor, social, and cognitive dysfunction. Our data indicate that cerebellar Purkinje cells are an important node for dysfunction and neural disabilities in SCN1B-related DEEs, and combined with previous work identify this as a potentially vital site for understanding mechanisms of DEEs and developing therapies that can treat these disorders holistically.

neuroscience↗

bioRxiv2021

Two contrasting mediodorsal thalamic circuits target the medial prefrontal cortex

At the heart of the prefrontal executive and limbic networks is the mediodorsal thalamus (MD). Despite the importance of MD in a broad range of behaviors and neuropsychiatric disorders, virtually nothing is known about the physiology of neurons in MD. Here, we injected the retrograde tracer cholera toxin subunit B (CTB) into the medial prefrontal cortex (mPFC) of adult (8 - 12 week old) male and female wildtype mice. We prepared acute brain slices and used current clamp electrophysiology to measure and compare the intrinsic properties of the neurons in MD that project to mPFC (MD[->]mPFC neurons). MD[->]mPFC neurons are located predominantly in the medial (MD-M) and lateral (MD-L) subnuclei of MD. We found that that MD-M[->]mPFC neurons have longer membrane time constants, higher membrane resistance, less Hyperpolarization and Cyclic Nucleotide gated (HCN) channel activity, and more readily generate action potentials compared to MD-L[->]mPFC neurons. Additionally, MD-M[->]mPFC neurons have larger and more complex dendritic arbors compared to MD-L[->]mPFC neurons. These data demonstrating that the two populations of MD[->]mPFC neurons have distinct physiologies and morphologies suggests a differential role in thalamocortical information processing and potentially behavior.

neuroscience↗