The plasma membrane potential (Vm) is key to many physiological processes; however, its ionic etiology in white fat adipocytes is poorly characterized. To address this question, we employed the perforated patch current clamp and cell-attached patch clamp methods in isolated primary white fat adipocytes and their cellular model 3T3-L1. The resting Vm of primary and 3T3-L1 adipocytes were Ϫ32.1 Ϯ 1.2 mV (n ϭ 95) and Ϫ28.8 Ϯ 1.2 mV (n ϭ 87), respectively. Vm was independent of cell size and fat content. Elevation of extracellular K ϩ to 50 mM by equimolar substitution of bath Na ϩ did not affect Vm, whereas substitution of bath Na ϩ with the membrane-impermeant cation N-methyl-D-glucamine ϩ -hyperpolarized Vm by 16 mV, data indicative of a nonselective cation permeability. Substitution of 133 mM extracellular Cl Ϫ with gluconate-depolarized Vm by 25 mV, whereas Cl Ϫ substitution with I Ϫ caused a Ϫ9 mV hyperpolarization. Isoprenaline (10 M), but not insulin (100 nM), significantly depolarized Vm. Single-channel ion activity was voltage independent; currents were indicative for Cl Ϫ with an inward slope conductance of 16 Ϯ 1.3 pS (n ϭ 11) and a reversal potential close to the Cl Ϫ equilibrium potential, Ϫ29 Ϯ 1.6 mV. Although the reduction of extracellular Cl Ϫ elevated the intracellular Ca 2ϩ of adipocytes, this was not as large as that produced by elevation of extracellular K ϩ . In conclusion, the Vm of white fat adipocytes is well described by the Goldman-HodgkinKatz equation with a predominant permeability to Cl Ϫ , where its biophysical and single-channel properties suggest a volume-sensitive anion channel identity. Consequently, changes in serum Cl Ϫ homeostasis or the adipocyte's permeability to this anion via drugs will affect its Vm, intracellular Ca 2ϩ , and ultimately its function and its role in metabolic control. adipocyte; white fat; 3T3-L1 cells; chloride; membrane potential THE PLASMA MEMBRANE POTENTIAL (Vm) is a fundamental biological property for the survival and function of virtually every eukarocytic cell. In excitable tissues of animalia, such as the heart, muscles, and nerves, excursions in Vm in the form of action potentials represent the fastest known biological means of intraorganism communication. The action potential often acts to mediate intracellular signaling; for example, an associated influx of extracellular Ca 2ϩ controls functions as diverse as contraction to secretion. In nonexcitable tissues, subtle changes in Vm are involved in the control of the transmembrane and transcellular fluxes of many solutes. Moreover, the membrane potential per se also has an important role to play in basic cell homoeostasis: the regulation of intracellular ionic composition and the maintenance of osmotic equilibrium. A thorough understanding of the etiology of Vm permits us to accurately control it experimentally, a process that allows us to investigate associated physiological and pathological sequelae.

L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.

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