We describe members of 4 kindreds with a previously unrecognized syndrome characterized by seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (hypokalemia, metabolic alkalosis, and hypomagnesemia). By analysis of linkage we localize the putative causative gene to a 2.5-Mb segment of chromosome 1q23.2-23.3. Direct DNA sequencing of KCNJ10, which encodes an inwardly rectifying K(+) channel, identifies previously unidentified missense or nonsense mutations on both alleles in all affected subjects. These mutations alter highly conserved amino acids and are absent among control chromosomes. Many of these mutations have been shown to cause loss of function in related K(+) channels. These findings demonstrate that loss-of-function mutations in KCNJ10 cause this syndrome, which we name SeSAME. KCNJ10 is expressed in glia in the brain and spinal cord, where it is believed to take up K(+) released by neuronal repolarization, in cochlea, where it is involved in the generation of endolymph, and on the basolateral membrane in the distal nephron. We propose that KCNJ10 is required in the kidney for normal salt reabsorption in the distal convoluted tubule because of the need for K(+) recycling across the basolateral membrane to enable normal activity of the Na(+)-K(+)-ATPase; loss of this function accounts for the observed electrolyte defects. Mice deficient for KCNJ10 show a related phenotype with seizures, ataxia, and hearing loss, further supporting KCNJ10's role in this syndrome. These findings define a unique human syndrome, and establish the essential role of basolateral K(+) channels in renal electrolyte homeostasis.

Natural sugars and artificial sweeteners are sensed by receptors in taste buds. T2R bitter and T1R sweet taste receptors are coupled through G-proteins, alpha-gustducin and transducin, to activate phospholipase C beta2 and increase intracellular calcium concentration. Intestinal brush cells or solitary chemosensory cells (SCCs) have a structure similar to lingual taste cells and strongly express alpha-gustducin. It has therefore been suggested over the last decade that brush cells may participate in sugar sensing by a mechanism analogous to that in taste buds. We provide here functional evidence for an intestinal sensing system based on lingual taste receptors. Western blotting and immunocytochemistry revealed that all T1R members are expressed in rat jejunum at strategic locations including Paneth cells, SCCs or the apical membrane of enterocytes; T1Rs are colocalized with each other and with alpha-gustducin, transducin or phospholipase C beta2 to different extents. Intestinal glucose absorption consists of two components: one is classical active Na+-glucose cotransport, the other is the diffusive apical GLUT2 pathway. Artificial sweeteners increase glucose absorption in the order acesulfame potassium approximately sucralose>> saccharin, in parallel with their ability to increase intracellular calcium concentration. Stimulation occurs within minutes by an increase in apical GLUT2, which correlates with reciprocal regulation of T1R2, T1R3 and alpha-gustducin versus T1R1, transducin and phospholipase C beta2. Our observation that artificial sweeteners are nutritionally active, because they can signal to a functional taste reception system to increase sugar absorption during a meal, has wide implications for nutrient sensing and nutrition in the treatment of obesity and diabetes.

We identify a human mutation (E1053K) in the ankyrin-binding motif of Na(v)1.5 that is associated with Brugada syndrome, a fatal cardiac arrhythmia caused by altered function of Na(v)1.5. The E1053K mutation abolishes binding of Na(v)1.5 to ankyrin-G, and also prevents accumulation of Na(v)1.5 at cell surface sites in ventricular cardiomyocytes. Ankyrin-G and Na(v)1.5 are both localized at intercalated disc and T-tubule membranes in cardiomyocytes, and Na(v)1.5 coimmunoprecipitates with 190-kDa ankyrin-G from detergent-soluble lysates from rat heart. These data suggest that Na(v)1.5 associates with ankyrin-G and that ankyrin-G is required for Na(v)1.5 localization at excitable membranes in cardiomyocytes. Together with previous work in neurons, these results in cardiomyocytes suggest that ankyrin-G participates in a common pathway for localization of voltage-gated Na(v) channels at sites of function in multiple excitable cell types.

A potent (<em>Na</em>,K)-ATPase inhibitor purified from "Sigma Grade* ATP has been identified as vanadium using electron probe microanalysis and confirmed by microwave-induced emission spectroscopy and electron paramagnetic resonance spectroscopy. Sodium orthovanadate (<em>Na</em>3 VO4) is identical with the purified inhibitor with respect to ultraviolet absorbance, migration on thin layer chromatography, and inhibition of (<em>Na</em>,K)-ATPase. The (<em>Na</em>,K)-ATPase is in-inhibited 50% by 40 nM <em>Na</em>3 VO4 under optimal conditions (28 mM Mg2+) and the inhibition is 100% reversible by millimolar concentrations of norepinephrine. The physiological significance of this inhibition is discussed in relation to vanadium concentrations in vivo.

Until now the only reported effect of depolarization on the intracellular pH (pHi) of excitable cells in an acidification of the cell cytoplasm. It seems unlikely that this could be a direct effect of membrane potential because pHi is known to be regulated by an electroneutral mechanism and in most cells H+ ions are not in equilibrium with the membrane potential (Em). In any case the membrane conductance to H+ ions would be expected to be small because they are at such low concentrations on either side of the cell membrane. But it is possible that the H+ ion permeability of the membrane increases on depolarization just like that of other ions in the bathing medium depolarization just like that of other ions in the bathing medium (Na+, K+ and Ca2+ for example). To test this idea we have made pHi measurements on molluscan neurones under voltage-clamp. Our findings, presented here, provide evidence for a large increase in H+ ion permeability in depolarized cells. We suggest that this increase in proton conductance may be the basis for the "nonspecific' currents previously described in perfused molluscan neurones and we assess the physiological significance of this newly discovered pathway.

Mutations increasing WNK1 kinase expression in humans cause the pseudohypoaldosteronism type II hypertension syndrome. This condition is treated effectively by thiazide diuretics, which exert their effects by inhibiting the Na+-Cl(-) cotransporter (NCC), suggesting a link between WNK1 and NCC. Here, we demonstrate that the SPAK and OSR1 kinases that are activated by WNK1 phosphorylate human NCC at three conserved residues (Thr46, Thr55 and Thr60). Activation of the WNK1-SPAK/OSR1 signalling pathway by treatment of HEK293 or mpkDCT kidney distal-convoluted-tubule-derived cells with hypotonic low-chloride conditions induced phosphorylation of NCC at residues phosphorylated by SPAK/OSR1. Efficient phosphorylation of NCC was dependent upon a docking interaction between an RFXI motif in NCC and SPAK/OSR1. Mutation of Thr60 to Ala in NCC markedly inhibited phosphorylation of Thr46 and Thr55 as well as NCC activation induced by hypotonic low-chloride treatment of HEK293 cells. Our results establish that the WNK1-SPAK/OSR1 signalling pathway plays a key role in controlling the phosphorylation and activity of NCC. They also suggest a mechanism by which increased WNK1 overexpression could lead to hypertension and that inhibitors of SPAK/OSR1 might be of use in reducing blood pressure by suppressing phosphorylation and hence activity of NCC.

Cardiotonic steroids (CTS), long used to treat heart failure, are endogenously produced in mammals. Among them are the hydrophilic cardenolide ouabain and the more hydrophobic cardenolide digoxin, as well as the bufadienolides marinobufagenin and telecinobufagin. The physiological effects of endogenous ouabain on blood pressure and cardiac activity are consistent with the "Na(+)-lag" hypothesis. This hypothesis assumes that, in cardiac and arterial myocytes, a CTS-induced local increase of Na(+) concentration due to inhibition of Na(+)/K(+)-ATPase leads to an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)) via a backward-running Na(+)/Ca(2+) exchanger. The increase in [Ca(2+)](i) then activates muscle contraction. The Na(+)-lag hypothesis may best explain short-term and inotropic actions of CTS. Yet all data on the CTS-induced alteration of gene expression are consistent with another hypothesis, based on the Na(+)/K(+)-ATPase "signalosome," that describes the interaction of cardiac glycosides with the Na(+) pump as machinery activating various signaling pathways via intramembrane and cytosolic protein-protein interactions. These pathways, which may be activated simultaneously or selectively, elevate [Ca(2+)](i), activate Src and the ERK1/2 kinase pathways, and activate phosphoinositide 3-kinase and protein kinase B (Akt), NF-kappaB, and reactive oxygen species. A recent development indicates that new pharmaceuticals with antihypertensive and anticancer activities may be found among CTS and their derivatives: the antihypertensive rostafuroxin suppresses Na(+) resorption and the Src-epidermal growth factor receptor-ERK pathway in kidney tubule cells. It may be the parent compound of a new principle of antihypertensive therapy. Bufalin and oleandrin or the cardenolide analog UNBS-1450 block tumor cell proliferation and induce apoptosis at low concentrations in tumors with constitutive activation of NF-kappaB.

Mutations in the gene encoding the K+ channel KCNQ2 cause neonatal epilepsy and myokymia, indicating that KCNQ2 regulates the excitability of CNS neurons and motor axons, respectively. We show here that KCNQ2 channels are functional components of axon initial segments and nodes of Ranvier, colocalizing with ankyrin-G and voltage-dependent Na+ channels throughout the CNS and PNS. Retigabine, which opens KCNQ channels, diminishes axonal excitability. Linopirdine, which blocks KCNQ channels, prolongs the repolarization of the action potential in neonatal nerves. The clustering of KCNQ2 at nodes and initial segments lags that of ankyrin-G during development, and both ankyrin-G and KCNQ2 can be coimmunoprecipitated in the brain. KCNQ3 is also a component of some initial segments and nodes in the brain. The diminished activity of mutant KCNQ2 channels accounts for neonatal epilepsy and myokymia; the cellular locus of these effects may be axonal initial segments and nodes.

An algorithm is presented for the calculation of metal ion concentrations from given total metal concentrations (and vice versa) in physiological media containing metal-chelating compounds. In such media, conditions differ from those used for stability constant determination of metal-chelator equilibria; therefore calculated metal ion concentrations are incorrect. We recompute stability constants to reflect the effects of ionic strength and temperature of physiological solutions. Twelve different equilibria can be considered per metal-chelator pair. The computer program also calculates the contribution of ionized species of metals, chelator, complexes and pH buffers to ionic strength. Measurements with a Ca-selective electrode and with fura-2 show that calculated ionic Ca2+ concentrations are correct from 10 nM up to the millimolar range. The importance of the correct calculation of metal ion concentrations in physiological experiments is demonstrated by data, and derived kinetic parameters, on Na+/Ca2+ exchange and the ATP-dependent Ca2+ pump of enterocyte plasma membrane vesicles. The program is written in Turbo Pascal and will run on IBM-compatible computers. It is menu-driven and supports the use of a Microsoft mouse.

Salinity is a major abiotic stress affecting approximately 7% of the world's total land area resulting in billion dollar losses in crop production around the globe. Recent progress in molecular genetics and plant electrophysiology suggests that the ability of a plant to maintain a high cytosolic K+/Na+ ratio appears to be critical to plant salt tolerance. So far, the major efforts of plant breeders have been aimed at improving this ratio by minimizing Na+ uptake and transport to shoot. In this paper, we discuss an alternative approach, reviewing the molecular and ionic mechanisms contributing to potassium homeostasis in salinized plant tissues and discussing prospects for breeding for salt tolerance by targeting this trait. Major K+ transporters and their functional expression under saline conditions are reviewed and the multiple modes of their control are evaluated, including ameliorative effects of compatible solutes, polyamines and supplemental calcium. Subsequently, the genetic aspects of inheritance of K+ transport 'markers' are discussed in the general context of salt tolerance as a polygenic trait. The molecular identity of 'salt tolerance' genes is analysed, and prospects for future research and breeding are examined.

The SLC4 family consists of ten genes. All appear to encode integral membrane proteins with very similar hydropathy plots-consistent with the presence of 10-14 transmembrane segments. At least eight SLC4 members encode proteins that transport HCO(3)(-) (or a related species, such as CO(3)(2-)) across the plasma membrane. Functionally, these eight proteins fall into two major groups: three Cl-HCO(3) exchangers (AE1-3) and five Na(+)-coupled HCO(3)(-) transporters (NBCe1, NBCe2, NBCn1, NDCBE, NCBE). Two of the Na(+)-coupled HCO(3)(- )transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO(3)(-) transporters and all three AEs are electroneutral. At least NDCBE transports Cl(-) in addition to Na(+) and HCO(3)(-). Whether NCBE transports Cl(-)-in addition to Na(+) and HCO(3)(-)-is unsettled. In addition, two other SLC4 members (AE4 and BTR1) do not yet have a firmly established function; on the basis of homology, they fall between the two major groups. A characteristic of many, though not all, SLC4 members is inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 gene products play important roles in the carriage of CO(2) by erythrocytes, the absorption or secretion of H(+) or HCO(3)(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.

How a naive human neuroepithelial cell becomes an electrophysiologically active neuron remains unknown. Here, we describe the early physiological development of neurons differentiating from naive human embryonic stem (hES) cells. We found that differentiating neuronal cells progressively decrease their resting membrane potential, gain characteristic Na+ and K+ currents, and fire mature action potentials by 7 weeks of differentiation. This is similar to the maturation pattern observed in animals, albeit on a greatly expanded time scale. An additional 3 weeks of differentiation resulted in neurons that could fire repetitive trains of action potentials in response to depolarizing current pulses. The onset of spontaneous synaptic activity also occurred after 7 weeks of differentiation, in association with the differentiation of astrocytes within the culture. Cocultures of hES cell-derived neuroepithelial cells with exogenous astrocytes significantly accelerated the onset of synaptic currents but did not alter action potential generation. These findings suggest that the development of membrane characteristics and action potentials depend on the intrinsic maturation of Na+ and K+ currents, whereas synaptic transmission is enhanced by astrocytes, which may be achieved independently of the maturation of action potentials. Furthermore, we found that although astrocyte-conditioned medium accelerated synaptic protein localization, it did not increase synaptic activity, suggesting a contact-dependent mechanism by which astrocytes augment synaptic activity. These results lay the foundation for future studies examining the functional development of human neurons and provide support for the potential application of human cells in restorative neuronal therapies.

Swelling of primary astrocyte cultures by exposing them to hypotonic media caused release of label after the cells had been allowed to accumulate 3H-L-glutamate, 3H-D-aspartate, or 3H-taurine. Comparable release of endogenous L-glutamate or taurine, as measured by high-pressure liquid chromatography (HPLC), was also found. Release of label was not affected by treating the cells with cytochalasin B, indicating that microfilament polymerization was not significantly involved. Hypotonic-induced release did not appear to principally involve reversal of the Na(+)-dependent uptake system since increasing external K+ to depolarize the cells by replacement of external Na+, thus maintaining isotonic conditions, increased release to a lesser extent. Threo beta-hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, added externally stimulated efflux of 3H-L-glutamate independently of the swelling-induced efflux. Upon restoration of swollen cells to isotonic medium they showed an unimpaired ability to take up 3H-L-glutamate. The swelling-induced release of label was inhibited by a number of anion transport inhibitors, one of which has been shown to significantly improve outcome in an experimental brain trauma/hypoxia model in which astrocyte swelling is an early event.

NaCl-sensitive yeast mutants were isolated to identify genes essential for NaCl tolerance. Complementation of a mutant highly sensitive to Na+ and Li+ led to the isolation of the CNB1 gene. This gene encodes the regulatory subunit (CNB) of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Cells deficient in CNB accumulated Li+ due to reduced expression of ENA1, a gene encoding a P-type ATPase involved in Na+ and Li+ efflux. In addition, the K+ transport system of cnb1 delta cells was not converted to the high affinity state that facilitates better discrimination of K+ over Na+. Thus the cnb1 delta strain resembled a trk1 mutant. These results indicate that adaptation to NaCl stress in Saccharomyces cerevisiae requires a signal transduction pathway involving Ca2+ and protein phosphorylation-dephosphorylation. In this pathway, calcineurin would coordinate gene expression and activity of ion transporters to facilitate ion homeostasis.

The effects of tetrodotoxin, procaine, and manganese ions were examined on the Ca spike of the barnacle muscle fiber injected with Ca-binding agent as well as on the action potential of the ventricular muscle fiber of the frog heart. Although tetrodotoxin and procaine very effectively suppress the "Na spike" of other tissues, no suppressing effects are found on "Ca spike" of the barnacle fiber, while the initiation of the Ca spike is competitively inhibited by manganese ions. The initial rate of rise of the ventricular action potential is suppressed by tetrodotoxin and procaine but the plateau phase of the action potential is little affected. In contrast the suppressing effect of manganese ions is mainly on the plateau phase. The results suggest that the plateau phase of the ventricular action potential is related to the conductance increase in the membrane to Ca ions even though Na conductance change may also contribute to the plateau.

The SD0 mutant of influenza virus A/WSN/33 (WSN), characterized by a 24-amino-acid deletion in the neuraminidase (NA) stalk, does not grow in embryonated chicken eggs because of defective NA function. Continuous passage of SD0 in eggs yielded 10 independent clones that replicated efficiently. Characterization of these egg-adapted viruses showed that five of the viruses contained insertions in the NA gene from the PB1, PB2, or NP gene, in the region linking the transmembrane and catalytic head domains, demonstrating that recombination of influenza viral RNA segments occurs relatively frequently. The other five viruses did not contain insertions in this region but displayed decreased binding affinity toward sialylglycoconjugates, compared with the binding properties of the parental virus. Sequence analysis of one of the latter viruses revealed mutations in the hemagglutinin (HA) gene, at sites in close proximity to the sialic acid receptor-binding pocket. These mutations appear to compensate for reduced NA function due to stalk deletions. Thus, balanced HA-NA functions are necessary for efficient influenza virus replication.

In liver and intestine, transporters play a critical role in maintaining the enterohepatic circulation and bile acid homeostasis. Over the past two decades, there has been significant progress toward identifying the individual membrane transporters and unraveling their complex regulation. In the liver, bile acids are efficiently transported across the sinusoidal membrane by the Na(+) taurocholate cotransporting polypeptide with assistance by members of the organic anion transporting polypeptide family. The bile acids are then secreted in an ATP-dependent fashion across the canalicular membrane by the bile salt export pump. Following their movement with bile into the lumen of the small intestine, bile acids are almost quantitatively reclaimed in the ileum by the apical sodium-dependent bile acid transporter. The bile acids are shuttled across the enterocyte to the basolateral membrane and effluxed into the portal circulation by the recently indentified heteromeric organic solute transporter, OSTalpha-OSTbeta. In addition to the hepatocyte and enterocyte, subgroups of these bile acid transporters are expressed by the biliary, renal, and colonic epithelium where they contribute to maintaining bile acid homeostasis and play important cytoprotective roles. This article will review our current understanding of the physiological role and regulation of these important carriers.

NHE3 is the Na+/H+ exchanger located on the intestinal and renal brush border membrane, where it functions in transepithelial Na+ absorption. The brush border Na+ absorptive process is acutely inhibited by activation of cAMP-dependent protein kinase, but the molecular mechanism of this inhibitory effect is poorly understood. We have identified two regulatory proteins, E3KARP and NHERF, that interact with NHE3 to enable cAMP to inhibit NHE3. The two regulatory proteins are structurally related, sharing approximately 50% identity in amino acid sequences. It has been previously shown that when NHE3 is transfected into PS120 fibroblasts or Caco-2 cells, cAMP failed to inhibit NHE3 activity. Northern blot analysis showed that both PS120 and Caco-2 cells lacked the expression of both E3KARP and NHERF. In contrast, other cell lines in which cAMP inhibits NHE3, including OK, CHO, and LLC-PK1 cells, expressed NHERF-related regulatory proteins. To determine their functions in cAMP-dependent inhibition of NHE3, E3KARP and NHERF were transfected into PS120/NHE3 fibroblasts. Transfection in PS120/NHE3 fibroblasts with either NHERF or E3KARP reconstituted cAMP-induced inhibition of NHE3, resulting in 25-30% inhibition in these cells.

Astrocyte glutamate release can modulate synaptic activity and participate in brain intercellular signaling. P2X7 receptors form large ion channels when activated by ATP or other ligands. Here we show that P2X7 receptors provide a route for excitatory amino acid release from astrocytes. Studies were performed using murine cortical astrocyte cultures. ATP produced an inward current in patch-clamped astrocytes with properties characteristic of P2X7 receptor activation: the current was amplified in low divalent cation medium, blocked by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and more potently activated by 3'-O-(4-benzoyl)benzoyl ATP (BzATP) than by ATP itself. Measurement of current reversal potentials showed the relative BzATP-induced permeabilities to different substrates to be Na+, 1>> Cl-, 0.34>> N-methyl-D-glucamine, 0.27>> L-glutamate, 0.15 approximately D-aspartate, 0.16. Astrocytes exposed to BzATP also became permeable to Lucifer yellow, indicating a large channel opening. Release of L-glutamate and D-aspartate through P2X7 channels was confirmed using radiolabeled tracers. As with the inward current, release of glutamate and D-aspartate was induced by BzATP more potently than ATP, amplified in Ca2+/Mg2+-free medium, and blocked by PPADS or oxidized ATP. Efflux through P2X7 channels is a previously unrecognized route of ligand-stimulated, nonvesicular astrocyte glutamate release.

The inhibitory GABAergic projection of thalamic nucleus reticularis (nRt) neurons onto thalamocortical relay cells (TCs) is important in generating the normal thalamocortical rhythmicity of slow wave sleep, and may be a key element in the production of abnormal rhythms associated with absence epilepsy. Both TCs and nRt cells can generate prominent Ca(2+)-dependent low-threshold spikes, which evoke bursts of Na(+)-dependent fast spikes, and are influential in rhythm generation. Substantial differences in the pattern of burst firing in TCs versus nRt neurons led us to hypothesize that there are distinct forms of transient Ca2+ current (I(T)) underlying burst discharges in these two cell types. Using whole-cell voltage-clamp recordings, we analyzed I(T) in acutely isolated TCs and nRt neurons and found three key differences in biophysical properties. (1) The transient Ca2+ current in nRt neurons inactivated much more slowly than I(T) in TCs. This slow current is thus termed I(Ts). (2) The rate of inactivation for I(Ts) was nearly voltage independent. (3) Whole-cell I(Ts) amplitude was increased when Ba2+ was substituted for Ca2+ as the charge carrier. In addition, activation kinetics were slower for I(Ts) and the activation range was depolarized compared to that for I(T). Other properties of I(Ts) and I(T) were similar, including steady-state inactivation and sensitivities to blockade by divalent cations, amiloride, and antiepileptic drugs. Our findings demonstrate that subtypes of transient Ca2+ current are present in two different classes of thalamic neurons. The properties of I(Ts) lead to generation of long-duration calcium-dependent spike bursts in nRt cells. The resultant prolonged periods of GABA release onto TCs would play a critical role in maintaining rhythmicity by inducing TC hyperpolarization and promoting generation of low-threshold calcium spikes within relay nuclei.

STIM1 in the endoplasmic reticulum and CRACM1 in the plasma membrane are essential molecular components for controlling the store-operated CRAC current. CRACM1 proteins multimerize and bind STIM1, and the combined overexpression of STIM1 and CRACM1 reconstitutes amplified CRAC currents. Mutations in CRACM1 determine the selectivity of CRAC currents, demonstrating that CRACM1 forms the CRAC channel's ion-selective pore, but the CRACM1 homologs CRACM2 and CRACM3 are less well characterized. Here, we show that both CRACM2 and CRACM3, when overexpressed in HEK293 cells stably expressing STIM1, potentiate I(CRAC) to current amplitudes 15-20 times larger than native I(CRAC). A nonconducting mutation of CRACM1 (E106Q) acts as a dominant negative for all three CRACM homologs, suggesting that they can form heteromultimeric channel complexes. All three CRACM homologs exhibit distinct properties in terms of selectivity for Ca(2+) and Na(+), differential pharmacological effects in response to 2-APB, and strikingly different feedback regulation by intracellular Ca(2+). Each of the CRAC channel proteins' specific functional features and the potential heteromerization provide for flexibility in shaping Ca(2+) signals, and their characteristic biophysical and pharmacological properties will aid in identifying CRAC-channel species in native cells that express them.

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100-200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.

Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In beta-cells, ACh binds to muscarinic M(3) receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA(2) produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca(2+) concentration ([Ca(2+)](c)) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca(2+)](c) by mobilizing Ca(2+) from the endoplasmic reticulum; the resulting fall in Ca(2+) in the organelle produces a small capacitative Ca(2+) entry. ACh also depolarizes the plasma membrane of beta-cells by a Na(+)- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca(2+)](c). Surprisingly, ACh can also inhibit voltage-dependent Ca(2+) channels and stimulate Ca(2+) efflux when [Ca(2+)](c) is elevated. However, under physiological conditions, the net effect of ACh on [Ca(2+)](c) is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca(2+)](c) and the other involves a marked, PKC-mediated increase in the efficiency of Ca(2+) on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.

The serum- and glucocorticoid-regulated kinase (sgk1) is induced by mineralocorticoids and, in turn, upregulates heterologously expressed renal epithelial Na(+) channel (ENaC) activity in Xenopus oocytes. Accordingly, Sgk1 is considered to mediate the mineralocorticoid stimulation of renal ENaC activity and antinatriuresis. Here we show that at standard NaCl intake, renal water and electrolyte excretion is indistinguishable in sgk1-knockout (sgk1(-/-)) mice and wild-type (sgk1(+/+)) mice. In contrast, dietary NaCl restriction reveals an impaired ability of sgk1(-/-) mice to adequately decrease Na(+) excretion despite increases in plasma aldosterone levels and proximal-tubular Na(+) and fluid reabsorption, as well as decreases in blood pressure and glomerular filtration rate.