Department of Biochemistry
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Item The NaV1.5 auxiliary subunit FGF13 modulates channels by regulating membrane cholesterol independent of channel bindingGade, A.R.; Malvezzi, M.; Das, L.T.; Matsui, M.; Ma, C.-I.J.; Mazdisnian, K.; Marx, S.O.; Maxfield, F.R.; Pitt, G.S. (American Society for Clinical Investigation, 2025-08-12)Fibroblast growth factor homologous factors (FHFs) bind to the cytoplasmic carboxy terminus of voltage-gated sodium channels (VGSCs) and modulate channel function. Variants in FHFs or VGSCs perturbing that bimolecular interaction are associated with arrhythmias. Like some channel auxiliary subunits, FHFs exert additional cellular regulatory roles, but whether these alternative roles affect VGSC regulation is unknown. Using a separation-of-function strategy, we show that a structurally guided, binding incompetent mutant FGF13 (the major FHF in mouse heart) confers complete regulation of VGSC steady-state inactivation (SSI), the canonical effect of FHFs. In cardiomyocytes isolated from Fgf13 knockout mice, expression of the mutant FGF13 completely restores wild-type regulation of SSI. FGF13 regulation of SSI derives from effects on local accessible membrane cholesterol, which is unexpectedly polarized and concentrated in cardiomyocytes at the intercalated disc (ID) where most VGSCs localize. Fgf13 knockout eliminates the polarized cholesterol distribution and causes loss of VGSCs from the ID. Moreover, we show that the previously described FGF13-dependent stabilization of VGSC currents at elevated temperatures depends on the cholesterol mechanism. These results provide new insights into how FHFs affect VGSCs and alter the canonical model by which channel auxiliary subunits exert influence.Item Real-time pH imaging of macrophage lysosomes using the pH-sensitive probe ApHID.Sole-Domenech, S.; Singh, P.K.; Funes, L.; Ma, C.-I.J.; Warren, J.D.; Maxfield, F.R. (Cell Press, 2025-10-14)Active endolysosomal pH regulation is essential for optimal enzymatic activity. To measure acidification, pH sensors can be delivered to acidic compartments using labeled dextran polymers or proteins. However, commercial probes have limited sensitivity in the acidic endolysosomal range or their fluorescence undergoes degradation. Herein, we introduce the new pH-sensitive probe ApHID, a green-emitting sensor with optimal dynamic range matching the acidity of endosomes and lysosomes. Acid pH indicator dye (ApHID) has a pKa near 5, increasing brightness with acidity, and withstands oxidation and photobleaching. We used ApHID dextrans to measure endolysosomal pH in macrophages and compared it to other commercially available sensors. ApHID reported pH accurately and stably over time in cell culture and was sensitive to subtle variations in organelle acidification in real time. Overall, ApHID circumvents limitations of currently available commercial probes and can provide utility in demanding applications such as intravital imaging of tissues.
Item A pyrophosphatase that regulates lipid precursors of N-glycosylation.Menon, A.K. (Rockefeller University Press, 2025-10-10)The oligosaccharide used for protein N-glycosylation in the ER is built as a glycolipid. A recent study by Li, Suzuki, and colleagues (https://doi.org/10.1083/jcb.202501239) identifies a long-sought enzyme that hydrolyzes this lipid as part of a possible homeostatic/quality control mechanism.Item Triglycerides are an important fuel reserve for synapse function in the brain.Kumar, M.; Wu, Y.; Knapp, J.; Pontius, C.L.; Park, D.; Witte, R.E.; McAllister, R.; Gupta, K.; Rajagopalan, K.N.; De Camilli, P.; Ryan, T.A. (Nature Research, 2025-07-01)Proper fuelling of the brain is critical to sustain cognitive function, but the role of fatty acid (FA) combustion in this process has been elusive. Here we show that acute block of a neuron-specific triglyceride lipase, DDHD2 (a genetic driver of complex hereditary spastic paraplegia), or of the mitochondrial lipid transporter CPT1 leads to rapid onset of torpor in adult male mice. These data indicate that in vivo neurons are probably constantly fluxing FAs derived from lipid droplets (LDs) through β-oxidation to support neuronal bioenergetics. We show that in dissociated neurons, electrical silencing or blocking of DDHD2 leads to accumulation of neuronal LDs, including at nerve terminals, and that FAs derived from axonal LDs enter mitochondria in an activity-dependent fashion to drive local mitochondrial ATP production. These data demonstrate that nerve terminals can make use of LDs during electrical activity to provide metabolic support and probably have a critical role in supporting neuron function in vivo.Item Itraconazole and posaconazole, inhibitors of NPC1 sterol transport, act as pharmacological chaperones after washoutWang, W.A.; Ma, C.-I.J.; Steinfeld, N.; Maxfield, F.R. (Elsevier, 2025-06-16)Niemann-Pick type C (NPC) disease is a rare lysosomal storage disorder primarily caused by mutations in the NPC Cholesterol Transporter 1 (NPC1) gene, resulting in cholesterol and lipid accumulation in late endosomes and lysosomes. While several therapeutic drugs show promise in reducing cholesterol accumulation, none of the current treatments are highly effective. Itraconazole and posaconazole, widely used antifungal drugs, have been shown to stabilize misfolded NPC1 proteins, enabling their escape from endoplasmic reticulum-associated degradation. This chaperone-like property makes them attractive candidates for testing chaperones as possible treatments for NPC disease, but both drugs also inhibit NPC1 function. In this study, we employed a washout approach to reverse the inhibitory effects of these drugs, leveraging the fact that wildtype NPC1 proteins have a half-life of about 42 hours. Treating NPC1I1061T/I1061T human fibroblasts with itraconazole or posaconazole for 72 hours followed by 24-48 hours of washout, we observed a significant reduction in lysosomal cholesterol accumulation. A modest rebound was observed 72 hours after drug removal, likely due to protein turnover. We also tested a repeated pulsed exposure treatment, in which short drug treatments were followed by extended washout periods. This strategy preserved the functional benefit of NPC1 stabilization while minimizing inhibitory effects. These findings indicate that a washout strategy can enhance the functional benefits of pharmacological chaperones, offering a potential future therapeutic approach for NPC disease.