Browsing by Subject "Patch-Clamp Techniques"

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Open Access
Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization.
(Journal of the American College of Cardiology, 2013-11) Beavers, DL; Wang, W; Ather, S; Voigt, N; Garbino, A; Dixit, SS; Landstrom, AP; Li, N; Wang, Q; Olivotto, I; Dobrev, D; Ackerman, MJ; Wehrens, XHT
This study sought to study the role of junctophilin-2 (JPH2) in atrial fibrillation (AF).JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca(2+) handling and modulation of ryanodine receptor Ca(2+) channels (RyR2). Whereas defective RyR2-mediated Ca(2+) release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias.Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudoknock-in (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation.PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice expressing a hypertrophic cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca(2+) release events. These changes were attributed to reduced binding of E169K-JPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca(2+) spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients and an increased frequency of spontaneous Ca(2+) release events.Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2/RyR2 ratios can promote SR Ca(2+) leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.
Open Access
Novel hybrid action of GABA mediates inhibitory feedback in the mammalian retina.
(PLoS biology, 2019-04) Grove, James CR; Hirano, Arlene A; de Los Santos, Janira; McHugh, Cyrus F; Purohit, Shashvat; Field, Greg D; Brecha, Nicholas C; Barnes, Steven
The stream of visual information sent from photoreceptors to second-order bipolar cells is intercepted by laterally interacting horizontal cells that generate feedback to optimize and improve the efficiency of signal transmission. The mechanisms underlying the regulation of graded photoreceptor synaptic output in this nonspiking network have remained elusive. Here, we analyze with patch clamp recording the novel mechanisms by which horizontal cells control pH in the synaptic cleft to modulate photoreceptor neurotransmitter release. First, we show that mammalian horizontal cells respond to their own GABA release and that the results of this autaptic action affect cone voltage-gated Ca2+ channel (CaV channel) gating through changes in pH. As a proof-of-principle, we demonstrate that chemogenetic manipulation of horizontal cells with exogenous anion channel expression mimics GABA-mediated cone CaV channel inhibition. Activation of these GABA receptor anion channels can depolarize horizontal cells and increase cleft acidity via Na+/H+ exchanger (NHE) proton extrusion, which results in inhibition of cone CaV channels. This action is effectively counteracted when horizontal cells are sufficiently hyperpolarized by increased GABA receptor (GABAR)-mediated HCO3- efflux, alkalinizing the cleft and disinhibiting cone CaV channels. This demonstrates how hybrid actions of GABA operate in parallel to effect voltage-dependent pH changes, a novel mechanism for regulating synaptic output.
Open Access
Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.
(Neuron, 2007-09) Lu, Jiuyi; Helton, Thomas D; Blanpied, Thomas A; Rácz, Bence; Newpher, Thomas M; Weinberg, Richard J; Ehlers, Michael D
Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.
Open Access
"Silent" NMDA Synapses Enhance Motion Sensitivity in a Mature Retinal Circuit.
(Neuron, 2017-12) Sethuramanujam, Santhosh; Yao, Xiaoyang; deRosenroll, Geoff; Briggman, Kevin L; Field, Greg D; Awatramani, Gautam B
Retinal direction-selective ganglion cells (DSGCs) have the remarkable ability to encode motion over a wide range of contrasts, relying on well-coordinated excitation and inhibition (E/I). E/I is orchestrated by a diverse set of glutamatergic bipolar cells that drive DSGCs directly, as well as indirectly through feedforward GABAergic/cholinergic signals mediated by starburst amacrine cells. Determining how direction-selective responses are generated across varied stimulus conditions requires understanding how glutamate, acetylcholine, and GABA signals are precisely coordinated. Here, we use a combination of paired patch-clamp recordings, serial EM, and large-scale multi-electrode array recordings to show that a single high-sensitivity source of glutamate is processed differentially by starbursts via AMPA receptors and DSGCs via NMDA receptors. We further demonstrate how this novel synaptic arrangement enables DSGCs to encode direction robustly near threshold contrasts. Together, these results reveal a space-efficient synaptic circuit model for direction computations, in which "silent" NMDA receptors play critical roles.
Open Access
Simultaneous transcranial magnetic stimulation and single-neuron recording in alert non-human primates.
(Nat Neurosci, 2014-08) Mueller, Jerel K; Grigsby, Erinn M; Prevosto, Vincent; Petraglia, Frank W; Rao, Hrishikesh; Deng, Zhi-De; Peterchev, Angel V; Sommer, Marc A; Egner, Tobias; Platt, Michael L; Grill, Warren M
Transcranial magnetic stimulation (TMS) is a widely used, noninvasive method for stimulating nervous tissue, yet its mechanisms of effect are poorly understood. Here we report new methods for studying the influence of TMS on single neurons in the brain of alert non-human primates. We designed a TMS coil that focuses its effect near the tip of a recording electrode and recording electronics that enable direct acquisition of neuronal signals at the site of peak stimulus strength minimally perturbed by stimulation artifact in awake monkeys (Macaca mulatta). We recorded action potentials within ∼1 ms after 0.4-ms TMS pulses and observed changes in activity that differed significantly for active stimulation as compared with sham stimulation. This methodology is compatible with standard equipment in primate laboratories, allowing easy implementation. Application of these tools will facilitate the refinement of next generation TMS devices, experiments and treatment protocols.
Open Access
STIM1-Ca(2+) signaling is required for the hypertrophic growth of skeletal muscle in mice.
(Molecular and cellular biology, 2012-08) Li, Tianyu; Finch, Elizabeth A; Graham, Victoria; Zhang, Zhu-Shan; Ding, Jin-Dong; Burch, Jarrett; Oh-hora, Masatsugu; Rosenberg, Paul
Immediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.