Constitutive Plasma Membrane Turnover in T-REx293 cells via Ordered Membrane Domain Endocytosis under Mitochondrial Control.

Clathrin/dynamin-independent endocytosis of ordered plasma membrane domains (ordered membrane domain endocytosis, OMDE) can become massive in response to cytoplasmic Ca elevations, G protein activation by non-hydrolyzable GTP analogs, and enhanced oxidative metabolism. In patch-clamped murine bone marrow macrophages (BMMs), cytoplasmic succinate and pyruvate, but not ß-hydroxybutyrate, induce OMDE of 75% of the plasma membrane within 2 min. The responses require palmitoylation of membrane proteins, being decreased by 70% in BMMs lacking the acyltransferase, DHHC5, by treatment with carnitine to shift long-chain acyl groups from cytoplasmic to mitochondrial acyl-CoAs, by bromopalmitate/albumin complexes to block DHHCs, and by the mitochondria-specific cyclosporin, NIM811, to block permeability transition pores that may release mitochondrial coenzyme A into the cytoplasm. Using T-REx293 cells, OMDE amounts to 40% with succinate, pyruvate, or GTPγS, and it is inhibited by actin cytoskeleton disruption. Pyruvate-induced OMDE is blocked by the hydrophobic antioxidant, edaravone, which prevents permeability transition pore openings. Using fluorescent 3kD dextrans to monitor endocytosis, OMDE appears to be constitutively active in T-REx293 cells but not in BMMs. After 1 h without substrates or bicarbonate, pyruvate and hydroxybutyrate inhibit constitutive OMDE, as expected for a shift of CoA from long-chain acyl-CoAs to other CoA metabolites. In the presence of bicarbonate, pyruvate strongly enhances OMDE, which is then blocked by ß-hydroxybutyrate, bromopalmitate/albumin complexes, cyclosporines, or edaravone. After pyruvate responses, T-REx293 cells grow normally with no evidence for apoptosis. Fatty acid-free albumin (15 µM) inhibits basal OMDE in T-REx293 cells, as do cyclosporines, carnitine, and RhoA blockade. Surprisingly, OMDE in the absence of substrates and bicarbonate is not inhibited by siRNA knockdown of the acyltransferases, DHHC5 or DHHC2, which are required for activated OMDE in patch clamp experiments. We verify biochemically that small CoA metabolites decrease long-chain acyl-CoAs. We verify also that palmitoylations of many PM-associated proteins decrease and increase when OMDE is inhibited and stimulated, respectively, by different metabolites. STED microscopy reveals that vesicles formed during constitutive OMDE in T-REX293 cells have 90 to 130 nm diameters. In summary, OMDE is likely a major G-protein-dependent endocytic mechanism that can be constitutively active in some cell types, albeit not BMMs. OMDE depends on different DHHC acyltransferases in different circumstances and can be limited by local supplies of fatty acids, CoA, and long-chain acyl-CoAs.

Longitudinal diffusion barriers imposed by myofilaments and mitochondria in murine cardiac myocytes.

Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 µm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by ß-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 µM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.

Thiazides Attenuate Insulin Secretion Through Inhibition of Mitochondrial Carbonic Anhydrase 5b in ß -Islet Cells in Mice.

SIGNIFICANCE STATEMENT: Thiazide diuretics (thiazides) are among the most widely prescribed drugs worldwide, but their use is associated with glucose intolerance and new-onset diabetes mellitus. The molecular mechanisms remain elusive. Our study reveals that thiazides attenuate insulin secretion through inhibition of the mitochondrial carbonic anhydrase isoform 5b (CA5b) in pancreatic ß cells. We furthermore discovered that pancreatic ß cells express only one functional carbonic anhydrase isoform, CA5b, which is critical in replenishing oxaloacetate in the mitochondrial tricarboxylic acid (TCA) cycle (anaplerosis). These findings explain the mechanism for thiazide-induced glucose intolerance and reveal a fundamental role of CA5b in TCA cycle anaplerosis and insulin secretion in ß cells. BACKGROUND: Thiazide diuretics are associated with glucose intolerance and new-onset diabetes mellitus. Previous studies demonstrated that thiazides attenuate insulin secretion, but the molecular mechanisms remain elusive. We hypothesized that thiazides attenuate insulin secretion via one of the known molecular thiazide targets in ß cells. METHODS: We performed static insulin secretion experiments with islets of wild-type, Sodium/chloride co-transporter (NCC) (SLC12A3), and sodium-driven chloride/bicarbonate exchanger (NDCBE) (SLC4A8) knock-out (KO) mice and with murine Min6 cells with individual knockdown of carbonic anhydrase (CA) isoforms to identify the molecular target of thiazides in ß cells. CA isoform 5b (CA5b) KO mice were then used to assess the role of the putative thiazide target CA5b in ß -cell function and in mediating thiazide sensitivity in vitro and in vivo . RESULTS: Thiazides inhibited glucose- and sulfonylurea-stimulated insulin secretion in islets and Min6 cells at pharmacologically relevant concentrations. Inhibition of insulin secretion by thiazides was CO 2 /HCO 3- -dependent, not additive to unselective CA inhibition with acetazolamide, and independent of extracellular potassium. By contrast, insulin secretion was unaltered in islets of mice lacking the known molecular thiazide targets NCC or NDCBE. CA expression profiling with subsequent knockdown of individual CA isoforms suggested mitochondrial CA5b as a molecular target. In support of these findings, thiazides significantly attenuated Krebs cycle anaplerosis through reduction of mitochondrial oxaloacetate synthesis. CA5b KO mice were resistant to thiazide-induced glucose intolerance, and thiazides did not alter insulin secretion in CA5b KO islets. CONCLUSIONS: Thiazides attenuate insulin secretion via inhibition of the mitochondrial CA5b isoform in ß cells of mice.

Pore-like diffusion barriers in murine cardiac myocytes.

Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses ∼50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10 -7 cm 2 /s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, ∼20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by ß-escin (80 µM) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.

Structural basis for gating mechanism of the human sodium-potassium pump.

P2-type ATPase sodium-potassium pumps (Na+/K+-ATPases) are ion-transporting enzymes that use ATP to transport Na+ and K+ on opposite sides of the lipid bilayer against their electrochemical gradients to maintain ion concentration gradients across the membranes in all animal cells. Despite the available molecular architecture of the Na+/K+-ATPases, a complete molecular mechanism by which the Na+ and K+ ions access into and are released from the pump remains unknown. Here we report five cryo-electron microscopy (cryo-EM) structures of the human alpha3 Na+/K+-ATPase in its cytoplasmic side-open (E1), ATP-bound cytoplasmic side-open (E1•ATP), ADP-AlF4- trapped Na+-occluded (E1•P-ADP), BeF3- trapped exoplasmic side-open (E2P) and MgF42- trapped K+-occluded (E2•Pi) states. Our work reveals the atomically resolved structural detail of the cytoplasmic gating mechanism of the Na+/K+-ATPase.

TMEM16F and dynamins control expansive plasma membrane reservoirs.

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.

The sodium/proton exchanger NHA2 regulates blood pressure through a WNK4-NCC dependent pathway in the kidney.

NHA2 is a sodium/proton exchanger associated with arterial hypertension in humans, but the role of NHA2 in kidney function and blood pressure homeostasis is currently unknown. Here we show that NHA2 localizes almost exclusively to distal convoluted tubules in the kidney. NHA2 knock-out mice displayed reduced blood pressure, normocalcemic hypocalciuria and an attenuated response to the thiazide diuretic hydrochlorothiazide. Phosphorylation of the thiazide-sensitive sodium/chloride cotransporter NCC and its upstream activating kinase Ste20/SPS1-related proline/alanine rich kinase (SPAK), as well as the abundance of with no lysine kinase 4 (WNK4), were significantly reduced in the kidneys of NHA2 knock-out mice. In vitro experiments recapitulated these findings and revealed increased WNK4 ubiquitylation and enhanced proteasomal WNK4 degradation upon loss of NHA2. The effect of NHA2 on WNK4 stability was dependent from the ubiquitylation pathway protein Kelch-like 3 (KLHL3). More specifically, loss of NHA2 selectively attenuated KLHL3 phosphorylation and blunted protein kinase A- and protein kinase C-mediated decrease of WNK4 degradation. Phenotype analysis of NHA2/NCC double knock-out mice supported the notion that NHA2 affects blood pressure homeostasis by a kidney-specific and NCC-dependent mechanism. Thus, our data show that NHA2 as a critical component of the WNK4-NCC pathway and is a novel regulator of blood pressure homeostasis in the kidney.

On the existence of endocytosis driven by membrane phase separations.

Large endocytic responses can occur rapidly in diverse cell types without dynamins, clathrin, or actin remodeling. Our experiments suggest that membrane phase separations are crucial with more ordered plasma membrane domains being internalized. Not only do these endocytic processes rely on coalescence of membrane domains, they are promoted by participation of membrane proteins in such domains, one important regulatory influence being palmitoylation. Membrane actin cytoskeleton in general resists membrane phase transitions, and its remodeling may play many roles. Besides membrane 'caging' and 'pinching' roles, typically ascribed to clathrin and dynamins, cytoskeleton remodeling may modify local membrane tension and buckling, as well as the presence and location of actin- and tension-free membrane patches. Endocytosis that depends on membrane phase separations becomes activated in metabolic stress and in response to Ca and PI3 kinase signaling. Internalized membrane traffics normally, and the secretory pathway eventually resupplies membrane to the plasmalemma or directs internalized membrane to other locations, including the extracellular space as exosomes. We describe here that endocytosis driven by membrane phase transitions is regulated by the same signaling mechanisms that regulate macropinocytosis, and it may play diverse roles in cells from nutrient assimilation to membrane recycling, cell migration, and the initiation of quiescent or hibernating cell states. Membrane ordering and phase separations have been shown to promote endocytosis in diverse cell types, including fibroblasts, myocytes, glial cells, and immune cells. We propose that clathrin/dynamin-independent endocytosis represents a continuum of related mechanisms with variable but universal dependence on membrane ordering and actin remodeling. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Hypertrophy of human embryonic stem cell-derived cardiomyocytes supported by positive feedback between Ca2+ and diacylglycerol signals.

Human embryonic stem cell-derived cardiomyocytes develop pronounced hypertrophy in response to angiotensin-2, endothelin-1, and a selected mix of three fatty acids. All three of these responses are accompanied by increases in both basal cytoplasmic Ca2+ and diacylglycerol, quantified with the Ca2+ sensor Fluo-4 and a FRET-based diacylglycerol sensor expressed in these cardiomyocytes. The heart glycoside, ouabain (30 nM), and a recently developed inhibitor of diacylglycerol lipases, DO34 (1 µM), cause similar hypertrophy responses, and both responses are accompanied by equivalent increases of basal Ca2+ and diacylglycerol. These results together suggest that basal Ca2+ and diacylglycerol form a positive feedback signaling loop that promotes execution of cardiac growth programs in these human myocytes. Given that basal Ca2+ in myocytes depends strongly on the Na+ gradient, we also tested whether nanomolar ouabain concentrations might stimulate Na+/K+ pumps, as described by others, and thereby prevent hypertrophy. However, stimulatory effects of nanomolar ouabain (1.5 nM) were not verified on Na+/K+ pump currents in stem cell-derived myocytes, nor did nanomolar ouabain block hypertrophy induced by endothelin-1. Thus, low-dose ouabain is not a "protective" intervention under the conditions of these experiments in this human myocyte model. To summarize, the major aim of this study has been to characterize the progression of hypertrophy in human embryonic stem cell-derived cardiac myocytes in dependence on diacylglycerol and Na+ gradient changes, developing a case that positive feedback coupling between these mechanisms plays an important role in the initiation of hypertrophy programs.

Lipid signaling to membrane proteins: From second messengers to membrane domains and adapter-free endocytosis.

Lipids influence powerfully the function of ion channels and transporters in two well-documented ways. A few lipids act as bona fide second messengers by binding to specific sites that control channel and transporter gating. Other lipids act nonspecifically by modifying the physical environment of channels and transporters, in particular the protein-membrane interface. In this short review, we first consider lipid signaling from this traditional viewpoint, highlighting innumerable Journal of General Physiology publications that have contributed to our present understanding. We then switch to our own emerging view that much important lipid signaling occurs via the formation of membrane domains that influence the function of channels and transporters within them, promote selected protein-protein interactions, and control the turnover of surface membrane.

Loss of Sodium/Hydrogen Exchanger NHA2 Exacerbates Obesity- and Aging-Induced Glucose Intolerance in Mice.

We previously demonstrated that the sodium/hydrogen exchanger NHA2, also known as NHEDC2 or SLC9B2, is critical for insulin secretion by ß-cells. To gain more insights into the role of NHA2 on systemic glucose homeostasis, we studied the impact of loss of NHA2 during the physiological aging process and in the setting of diet-induced obesity. While glucose tolerance was normal at 2 months of age, NHA2 KO mice displayed a significant glucose intolerance at 5 and 12 months of age, respectively. An obesogenic high fat diet further exacerbated the glucose intolerance of NHA2 KO mice. Insulin levels remained similar in NHA2 KO and WT mice during aging and high fat diet, but fasting insulin/glucose ratios were significantly lower in NHA2 KO mice. Peripheral insulin sensitivity, measured by insulin tolerance tests and hyperinsulinemic euglycemic clamps, was unaffected by loss of NHA2 during aging and high fat diet. High fat diet diminished insulin secretion capacity in both WT and NHA2 KO islets and reduced expression of NHA2 in WT islets. In contrast, aging was characterized by a gradual increase of NHA2 expression in islets, paralleled by an increasing difference in insulin secretion between WT and NHA2 KO islets. In summary, our results demonstrate that loss of the sodium/hydrogen exchanger NHA2 exacerbates obesity- and aging-induced glucose intolerance in mice. Furthermore, our data reveal a close link between NHA2 expression and insulin secretion capacity in islets.

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes.

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes.

Mutation in the Monocarboxylate Transporter 12 Gene Affects Guanidinoacetate Excretion but Does Not Cause Glucosuria.

A heterozygous mutation (c.643C>A; p.Q215X) in the monocarboxylate transporter 12-encoding gene MCT12 (also known as SLC16A12) that mediates creatine transport was recently identified as the cause of a syndrome with juvenile cataracts, microcornea, and glucosuria in a single family. Whereas the MCT12 mutation cosegregated with the eye phenotype, poor correlation with the glucosuria phenotype did not support a pathogenic role of the mutation in the kidney. Here, we examined MCT12 in the kidney and found that it resides on basolateral membranes of proximal tubules. Patients with MCT12 mutation exhibited reduced plasma levels and increased fractional excretion of guanidinoacetate, but normal creatine levels, suggesting that MCT12 may function as a guanidinoacetate transporter in vivo However, functional studies in Xenopus oocytes revealed that MCT12 transports creatine but not its precursor, guanidinoacetate. Genetic analysis revealed a separate, undescribed heterozygous mutation (c.265G>A; p.A89T) in the sodium/glucose cotransporter 2-encoding gene SGLT2 (also known as SLC5A2) in the family that segregated with the renal glucosuria phenotype. When overexpressed in HEK293 cells, the mutant SGLT2 transporter did not efficiently translocate to the plasma membrane, and displayed greatly reduced transport activity. In summary, our data indicate that MCT12 functions as a basolateral exit pathway for creatine in the proximal tubule. Heterozygous mutation of MCT12 affects systemic levels and renal handling of guanidinoacetate, possibly through an indirect mechanism. Furthermore, our data reveal a digenic syndrome in the index family, with simultaneous MCT12 and SGLT2 mutation. Thus, glucosuria is not part of the MCT12 mutation syndrome.

Role of Na/H exchange in insulin secretion by islet cells.

PURPOSE OF REVIEW: Sodium/hydrogen exchangers (NHEs) are a large family of transport proteins catalyzing the exchange of cations for protons across lipid bilayer membranes. Several isoforms are expressed in ß cells of the endocrine pancreas, including the recently discovered and poorly characterized isoform NHA2. This review will summarize advances in our understanding of the roles of NHEs in the regulation of insulin secretion in ß cells. RECENT FINDINGS: Plasmalemmal full-length NHE1 defends ß cells from intracellular acidification, but has no role in stimulus-secretion coupling and is not causally involved in glucose-induced alkalinization of the ß cell. The function of a shorter NHE1 splice variant, which localizes to insulin-containing large dense core vesicles, remains currently unknown. In contrast, in-vitro and in-vivo studies indicate that the NHA2 isoform is required for insulin secretion and clathrin-mediated endocytosis in ß cells. SUMMARY: Recent data highlight the importance of NHEs in the regulation of cellular pH, clathrin-mediated endocytosis and insulin secretion in ß cells. Based on these studies, a pathophysiological role of NHEs in human disorders of the endocrine pancreas seems likely and should be investigated.

Sodium/hydrogen exchanger NHA2 is critical for insulin secretion in ß-cells.

NHA2 is a sodium/hydrogen exchanger with unknown physiological function. Here we show that NHA2 is present in rodent and human ß-cells, as well as ß-cell lines. In vivo, two different strains of NHA2-deficient mice displayed a pathological glucose tolerance with impaired insulin secretion but normal peripheral insulin sensitivity. In vitro, islets of NHA2-deficient and heterozygous mice, NHA2-depleted Min6 cells, or islets treated with an NHA2 inhibitor exhibited reduced sulfonylurea- and secretagogue-induced insulin secretion. The secretory deficit could be rescued by overexpression of a wild-type, but not a functionally dead, NHA2 transporter. NHA2 deficiency did not affect insulin synthesis or maturation and had no impact on basal or glucose-induced intracellular Ca(2+) homeostasis in islets. Subcellular fractionation and imaging studies demonstrated that NHA2 resides in transferrin-positive endosomes and synaptic-like microvesicles but not in insulin-containing large dense core vesicles in ß-cells. Loss of NHA2 inhibited clathrin-dependent, but not clathrin-independent, endocytosis in Min6 and primary ß-cells, suggesting defective endo-exocytosis coupling as the underlying mechanism for the secretory deficit. Collectively, our in vitro and in vivo studies reveal the sodium/proton exchanger NHA2 as a critical player for insulin secretion in the ß-cell. In addition, our study sheds light on the biological function of a member of this recently cloned family of transporters.