1. We have used dendrite-attached patch-clamp techniques to record single Na+ and Ca2+ channel activity from the apical dendrites (up to 350 microns away from soma) of CA1 pyramidal neurons in rat hippocampal slices (ages: 2-8 weeks). 2. Na+ channels were found in every patch examined (range: 2 to>> 20 channels per patch). Channel openings, which had a slope conductance of 15 +/- 0.3 pS (mean +/- S.E.M.), began with test commands to around -50 mV and consisted of both early transient channel activity and also later occurring prolonged openings of 5-15 ms. All Na+ channel activity was suppressed by inclusion of TTX (1 microM) in the recording pipette. 3. Ca2+ channel activity was recorded in about 80% of the patches examined (range: 1 to>> 10 channels per patch). Several types of channel behaviour were observed in these patches. Single channel recordings in 110 mM BaCl2, revealed an approximately 10 pS channel of small unitary current amplitude (-0.5 pA at -20 mV). These channels began activating at relatively hyperpolarized potentials (-50 mV) and ensemble averages of this low voltage-activated (LVA) channel activity showed rapid inactivation. 4. A somewhat heterogeneous population of high voltage-activated, moderate conductance (HVAm; approximately 17 pS), Ca2+ channel activity was also encountered. These channels exhibited a relatively large unitary amplitude (-0.8 pA at 0 mV) and ensemble averages demonstrated moderate inactivation. The HVAm population of channels could be tentatively subdivided into two separate groups based upon mean channel open times. 5. Less frequently, HVA, large conductance (27 pS) Ca2+ channel activity (HVA1) was also observed. This large unitary amplitude (-1.5 pA at 0 mV) channel activity began with steps to approximately 0 mV and ensemble averages did not show any time-dependent inactivation. The dihydropyridine Ca2+ channel agonist Bay K 8644 (0.5 or 1 microM) was found to characteristically prolong these channel openings. 6. omega-Conotoxin MVIIC (10 microM), did not significantly reduce the amount of channel activity recorded from the LVA, HVAm or HVA1 channel types in dendritic patches. In patches from somata, omega-conotoxin MVIIC was effective in eliminating a significant amount of HVAm Ca2+ channel activity. Inclusion of 50 or 100 microM NiCl2 to the recording solution significantly reduced the amount of channel activity recorded from LVA and HVAm channel types in dendritic patches. A subpopulation of HVAm channels was, however, found to be Ni2+ insensitive. Dendritic HVA, channel activity was unaffected by these low concentrations of Ni2+.(ABSTRACT TRUNCATED AT 400 WORDS)

This article focuses on the regional heterogeneity of the mammalian sinoatrial (SA) node in terms of cell morphology, pacemaker activity, action potential configuration and conduction, densities of ionic currents (i(Na), i(Ca,L), i(to), i(K,r), i(K,s) and i(f)), expression of gap junction proteins (Cx40, Cx43 and Cx45), autonomic regulation, and ageing. Experimental studies on the single SA node cell to the whole animal are reviewed. The heterogeneity is considered in terms of the gradient model of the SA node, in which there is gradual change in the intrinsic properties of SA node cells from periphery to centre, and the alternative mosaic model, in which there is a variable mix of atrial and SA node cells from periphery to centre. The heterogeneity is important for the dependable functioning of the SA node as the pacemaker for the heart, because (i) via multiple mechanisms, it allows the SA node to drive the surrounding atrial muscle without being suppressed electrotonically; (ii) via an action potential duration gradient and a conduction block zone, it promotes antegrade propagation of excitation from the SA node to the right atrium and prevents reentry of excitation; and (iii) via pacemaker shift, it allows pacemaking to continue under diverse pathophysiological circumstances.

The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

Murine neocortical cell cultures were transiently deprived of both oxygen and glucose, producing widespread neuronal swelling in less than 60 min, followed by neuronal degeneration over the ensuing several hours, despite return to normal medium. Cultured glia >> 95% astrocytes) were irreversibly injured only by oxygen-glucose deprivation exposures exceeding 4-6 hr. Replacing either Na+ or Cl- with impermeant ions blocked acute neuronal swelling but did not prevent delayed neuronal degeneration. While neuronal swelling and death were increased by removing Ca2+ from the exposure medium, combined removal of extracellular Ca2+ together with Na+ or Cl- substitution blocked both acute and delayed injury. If acute swelling was limited by a hyperosmolar medium, then neuronal loss depended on extracellular [Ca2+]. Oxygen-glucose deprivation was associated with a large increase in extracellular glutamate concentration. Both early swelling and later neuronal degeneration were blocked by addition of NMDA receptor antagonists to the exposure medium but not by the AMPA/kainate receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione (CNQX), dihydropyridines nifedipine or nimodipine, or TTX. Oxygen-glucose deprivation induced substantial neuronal uptake of tracer 45Ca2+ from the exposure medium that was reduced by NMDA receptor antagonists and closely paralleled the degree of subsequent neuronal loss. These observations suggest the presence of two distinct components of hypoxic injury, each involving NMDA receptor activation and each capable of leading to neuronal death. Acute swelling is mediated by influx of Na+, Cl-, and water, and is enhanced by removal of extracellular Ca2+. Delayed neuronal degeneration depends on the presence of extracellular Ca2+ and correlates closely with cellular uptake of 45Ca2+.

1 Amiloride inhibits Na transport and short-circuit current (SCC) across the toad bladder. It is 1000 times more active at the mucosal than serosal surface. The lowest effective concentration was 10(-7)M.2. The inhibition was non-competitive with the sodium on the mucosal side of the bladder.3. Vasopressin, cyclic adenosine monophosphate (AMP) and aldosterone increased Na transport and SCC across the bladder and these effects were inhibited by amiloride.4. The antagonism of amiloride for vasopressin was non-competitive.5. Amphotericin B also increases Na transport across the bladder but its action was not changed by amiloride.6. Amiloride was without effects on SCC and diffusion potentials in bladders metabolically inhibited with CN(-) and iodoacetic acid (IAA).7. Neither plasma albumin, Ca(2+) nor adenosine triphosphate (ATP) altered the effects of amiloride.8. The only structural analogue of amiloride found to reduce SCC similarly was guanidine which was 1000 times less active. Pyrazine and a substituted pyrazine analogue were without effect. Neither guanidine nor the substituted pyrazine compound were competitive with amiloride.9. Amiloride had no effect on the osmotic permeability of the toad bladder either in the presence or absence of vasopressin.10. Na transport across the toad colon was also reduced by 10(-5)M amiloride at the mucosal surface.11. The possible mechanism of action of amiloride is discussed.

The membrane rotor ring from the vacuolar-type (V-type) sodium ion-pumping adenosine triphosphatase (Na+-ATPase) from Enterococcus hirae consists of 10 NtpK subunits, which are homologs of the 16-kilodalton and 8-kilodalton proteolipids found in other V-ATPases and in F1Fo- or F-ATPases, respectively. Each NtpK subunit has four transmembrane alpha helices, with a sodium ion bound between helices 2 and 4 at a site buried deeply in the membrane that includes the essential residue glutamate-139. This site is probably connected to the membrane surface by two half-channels in subunit NtpI, against which the ring rotates. Symmetry mismatch between the rotor and catalytic domains appears to be an intrinsic feature of both V- and F-ATPases.

In the crystal structure of the membrane-embedded rotor ring of the sodium ion-translocating adenosine 5'-triphosphate (ATP) synthase of Ilyobacter tartaricus at 2.4 angstrom resolution, 11 c subunits are assembled into an hourglass-shaped cylinder with 11-fold symmetry. Sodium ions are bound in a locked conformation close to the outer surface of the cylinder near the middle of the membrane. The structure supports an ion-translocation mechanism in the intact ATP synthase in which the binding site converts from the locked conformation into one that opens toward subunit a as the rotor ring moves through the subunit a/c interface.

BACKGROUND

DNA variants appearing to predispose to drug-associated "acquired" long-QT syndrome (aLQTS) have been reported in congenital long-QT disease genes. However, the incidence of these genetic risk factors has not been systematically evaluated in a large set of patients with aLQTS. We have previously identified functionally important DNA variants in genes encoding K+ channel ancillary subunits in 11% of an aLQTS cohort.

RESULTS

The coding regions of the genes encoding the pore-forming channel proteins KvLQT1, HERG, and SCN5A were screened in (1) the same aLQTS cohort (n=92) and (2) controls, drawn from patients tolerating QT-prolonging drugs (n=67) and cross sections of the Middle Tennessee (n=71) and US populations (n=90). The frequency of three common nonsynonymous coding region polymorphisms was no different between aLQTS and control subjects, as follows: 24% versus 19% for H558R (SCN5A), 3% versus 3% for R34C (SCN5A), and 14% versus 14% for K897T (HERG). Missense mutations (absent in controls) were identified in 5 of 92 patients. KvLQT1 and HERG mutations (one each) reduced K+ currents in vitro, consistent with the idea that they augment risk for aLQTS. However, three SCN5A variants did not alter I(Na), which argues that they played no role in the aLQTS phenotype.

CONCLUSIONS

DNA variants in the coding regions of congenital long-QT disease genes predisposing to aLQTS can be identified in approximately 10% to 15% of affected subjects, predominantly in genes encoding ancillary subunits.

OBJECTIVE

To characterize the voltage-gated ion channels in human beta-cells from nondiabetic donors and their role in glucose-stimulated insulin release.

METHODS

Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated beta-cells. The ion channel complement was determined by quantitative PCR.

RESULTS

Human beta-cells express two types of voltage-gated K(+) currents that flow through delayed rectifying (K(V)2.1/2.2) and large-conductance Ca(2+)-activated K(+) (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K(V)2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents (Na(V)1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na(+) currents with TTX reduced glucose-stimulated (6-20 mmol/l) insulin secretion by 55-70%. Human beta-cells are equipped with L- (Ca(V)1.3), P/Q- (Ca(V)2.1), and T- (Ca(V)3.2), but not N- or R-type Ca(2+) channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca(2+) channels reduced glucose-induced (6 mmol/l) secretion by 60-70%. Membrane potential recordings suggest that L- and T-type Ca(2+) channels participate in action potential generation. Blockade of P/Q-type Ca(2+) channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca(2+) channels only had a minor effect.

CONCLUSIONS

Voltage-gated T-type and L-type Ca(2+) channels as well as Na(+) channels participate in glucose-stimulated electrical activity and insulin secretion. Ca(2+)-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca(2+) influx through P/Q-type Ca(2+) channels.

1. The inward facing membranes of in vitro frog skin epithelium were depolarized with solutions of high K concentration. The electrical properties of the epithelium are then expected to be governed by the outward facing, Na-selective membrane.2. In this state, the transepithelial voltage (V) was clamped to zero and step-changes of Na activity in the outer solution ((Na)(o)) were performed with a fast-flow chamber at constant ionic strength, while the short-circuit current was recorded.3. At pre-selected times after a step-change of (Na)(o) the current response (I) to a fast voltage staircase was recorded. This procedure was repeated after blocking the Na channels with amiloride to obtain the current-voltage curve of transmembrane and paracellular shunt pathways. The current-voltage curve of the Na channels was computed by subtracting the shunt current from the total current.4. The instantaneous I(Na)-V curve thus obtained at a given (Na)(o) could easily be fitted with the constant field equation in the range between -50 and zero mV. This fit yielded approximate estimates of P(Na), the Na- permeability of the Na-selective membrane (at this (Na)(o)) and the cellular Na activity, (Na)(c). As residual properties of the serosal membrane were ignored the computed values are expected to underestimate the true ones.5. At constant (Na)(c), the steady-state value of 1/P(Na) increases linearly with (Na)(o). Error analysis and the effect of drugs show that the dependence is not due to the residual properties of the inward facing membranes but reflects the true behaviour of P(Na).6. The steady-state P(Na) at a given (Na)(o) is smaller than the transient P(Na) observed right after a stepwise increase of (Na)(o) to this value. The time constant of P(Na)-relaxation is in the order of seconds.7. In conclusion, Na transport through open Na-selective channels of the outward facing membrane of the stratum granulosum cells can be described as an electrodiffusion process which as such does not saturate with increasing (Na)(o). However, when added to the outer border of the membrane Na causes a decrease of P(Na) within several seconds. It is considered that binding of Na results in closure of Na channels.

The plasma membrane Na+-H+ exchanger is a ubiquitous transport system that participates in diverse cell functions involving the cellular uptake of Na+ or extrusion of H+. It has a tightly coupled 1:1 stoichiometry, has affinity for Li+ and NH+4 in addition to Na+ and H+, and can function in multiple amiloride-sensitive exchange modes involving these cations. These general transport properties may be explained by kinetic models involving either cation-hydroxyl cotransport or actual cation-proton exchange. The most important kinetic property of the Na+-H+ exchanger is its greater than first-order dependence on [H+]i. This property enables the Na+-H+ exchanger to play an important role in the regulation of intracellular pH.

In this study, high-conductance Ca2+-activated K+ channels from rat skeletal muscle were incorporated into planar phospholipid bilayers, and discrete blockade of single channels by Ba2+ was studied. With 150 mM K+ held constant in the internal solution, increasing external K+ over the range 100-1,000 mM raises the rate of Ba2+ dissociation. This "enhancement effect," which operates at K+ concentrations 3-4 orders of magnitude higher than those required for the "lockin" effect described previously, depends on applied voltage, saturates with K+ concentration, and is not observed with Na+. The voltage dependence of the Ba2+ off-rate varies with external K+ in a way suggesting that K+, entering the channel from the external side, forces Ba2+ dissociation to the internal solution. With K+ held fixed in the external solution, the Ba2+ off-rate decreases as internal K+ is raised over the range 0-50 mM. This "lock-in" effect is similar to that seen on the external side (Neyton and Miller, 1988), except that the internal lock-in site is of lower affinity and shows only a fivefold preference for K+ over Na+. All the results taken together argue strongly that this channel's conduction pathway contains four sites of very high affinity for K+, all of which may be simultaneously occupied under normal conducting conditions. According to this view, the mutual destabilization resulting from this high ionic occupancy leads to the unusually high conductance of this K+-specific channel.

The combination of the specificity provided by fluorescence microscopy and the ability to quantitatively analyze specimens in three dimensions allows the fundamental organization of cells to be probed as never before. Key features in this emergent technology have been the development of a wide variety of fluorescent dyes or fluorescently labeled probes to provide the requisite specificity. High-quality, cooled charge-coupled devices have recently become available. Functioning as nearly ideal imagers or "electronic film," they are more sensitive than photomultipliers and provide extraordinarily accurate direct digital readout from the microscope. Not only is this precision crucial for accurate quantitative imaging such as that required for the ratioing necessary to determine intracellular ion concentrations, but it also opens the way for sophisticated image processing. It is important to realize that image processing isn't simply a means to improve image aesthetics, but can directly provide new, biologically important information. The impact of modern video microscopy techniques (Allen, 1985; Inoué, 1986) attests to the fact that many biologically relevant phenomena take place at the limits of conventional microscopy. Image processing can be used to substantially enhance the resolution and contrast obtainable in two dimensions, enabling the invisible to be seen and quantitated. Cells are intrinsically three-dimensional. This can simply be a nuisance because of limited depth of focus of the microscope or it could be a fundamental aspect of the problem being studied. In either case, image processing techniques can be used to rapidly provide the desired representation of the data. In this chapter we have discussed the nature of image formation in three dimensions and dealt with several means to remove contaminating out-of-focus information. The most straightforward of these methods uses only information from adjacent focal planes to correct the central one. This approach can be readily applied to virtually any problem and with most commonly available image processing hardware to provide a substantially deblurred image in almost real time. In addition to covering more sophisticated algorithms where the utmost in three-dimensional imaging is required, we have developed a method for extremely rapidly and accurately producing an in-focus, high-resolution "synthetic projection" image from a thick specimen. This is equivalent to that produced by a microscope having the impossible combination of a high-NA objective lens and an infinite depth of focus. A variation on this method allows efficient calculation of stereo pairs.(ABSTRACT TRUNCATED AT 400 WORDS)

Ca2+ inward currents evoked by membrane depolarization have been studied by the intracellular dialysis technique in the somatic membrane of isolated dorsal root ganglion neurones of new-born rats. In about 20% of the investigated cells a hump has been detected on the descending branch of the current-voltage curve, indicating the presence of two populations of Ca2+ channels differing in their potential-dependent characteristics. An initial less regular component of the Ca2+ current was activated at membrane potentials from -75 to -70 mV. Its amplitude reached 0.2-0.9 nA at 14.6 mM-extracellular Ca2+. The activation kinetics of this component could be approximated by the Hodgkin-Huxley equation using the square of the m variable. tau m varied in the range from 8 to 1 ms at potentials between -60 and -25 mV ('fast' Ca2+ current). The second component of the Ca2+ current was activated at membrane depolarizations to between -55 and -50 mV. It could be recorded in all cells investigated and reached a maximum value of 1-7 nA at the same extracellular Ca2+ concentration. This component decreased rapidly during cell dialysis with saline solutions. The decrease could be slowed down by cooling and accelerated by warming the extracellular solution. Intracellular introduction of 3',5'-cAMP together with ATP and Mg2+ not only prevented the decrease but often restored the maximal current amplitude to its initial level. The activation kinetics of this component could also be approximated by a square function, tau m being in the range 16-2.5 ms at membrane potentials between -20 and +3 mV ('slow' Ca2+ current). The fast Ca2+ current inactivated exponentially at sustained depolarizations in a potential-dependent manner, tau h varying from 76 to 35 ms at potentials between -50 and -30 mV. The inactivation of the slow Ca2+ current studied in double-pulse experiments was current-dependent and developed very slowly (time constant of several hundreds of milliseconds). It slowed down even more at low temperature or after substitution of Ba2+ for Ca2+ in the extracellular solution. Both currents could also be carried by Ba2+ and Sr2+, although the ion-selecting properties of the two types of channels showed quantitative differences. Specific blockers of Ca2+ channels (Co2+, Mn2+, Cd2+, Ni2+ or verapamil) exerted similar effects on them. The existence of metabolically dependent and metabolically independent Ca2+ channels in the neuronal membrane and their possible functional role are discussed.

Spleen cells from a BALB/c mouse that had been immunized with human thymocytes were fused with the myeloma line P3-NS 1/1 Ag 4.1. One of the resulting hybrid clones (NA 1/34) secreted an antibody that was highly specific for human thymocytes. Eighty-five % of thymocytes expressed the antigen designated HTA1. There were an estimated 15 x 10(4) molecules of HTA 1 per cell, and it is therefore a major surface molecule. The expression of this antigen on thymocytes appears to be reciprocal to HLA, as recognized by another monoclonal antibody W6/32. Immunoprecipitated material from [125I]-labeled thymocyte membranes was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate which disclosed a single component of 45,000 molecular weight.

Emotional states can be transferred to others via emotional contagion, leading people to experience the same emotions without their awareness. Emotional contagion is well established in laboratory experiments, with people transferring positive and negative emotions to others. Data from a large real-world social network, collected over a 20-y period suggests that longer-lasting moods (e.g., depression, happiness) can be transferred through networks [Fowler JH, Christakis NA (2008) BMJ 337:a2338], although the results are controversial. In an experiment with people who use Facebook, we test whether emotional contagion occurs outside of in-person interaction between individuals by reducing the amount of emotional content in the News Feed. When positive expressions were reduced, people produced fewer positive posts and more negative posts; when negative expressions were reduced, the opposite pattern occurred. These results indicate that emotions expressed by others on Facebook influence our own emotions, constituting experimental evidence for massive-scale contagion via social networks. This work also suggests that, in contrast to prevailing assumptions, in-person interaction and nonverbal cues are not strictly necessary for emotional contagion, and that the observation of others' positive experiences constitutes a positive experience for people.

Acid sensing is associated with nociception, taste transduction, and perception of extracellular pH fluctuations in the brain. Acid sensing is carried out by the simplest class of ligand-gated channels, the family of H(+)-gated Na(+) channels. These channels have recently been cloned and belong to the acid-sensitive ion channel (ASIC) family. Toxins from animal venoms have been essential for studies of voltage-sensitive and ligand-gated ion channels. This paper describes a novel 40-amino acid toxin from tarantula venom, which potently blocks (IC(50) = 0.9 nm) a particular subclass of ASIC channels that are highly expressed in both central nervous system neurons and sensory neurons from dorsal root ganglia. This channel type has properties identical to those described for the homomultimeric assembly of ASIC1a. Homomultimeric assemblies of other members of the ASIC family and heteromultimeric assemblies of ASIC1a with other ASIC subunits are insensitive to the toxin. The new toxin is the first high affinity and highly selective pharmacological agent for this novel class of ionic channels. It will be important for future studies of their physiological and physio-pathological roles.

The oscillation of membrane potential in mammalian central neurons is of interest because it relates to the role of oscillations in brain function. It has been proposed that the entorhinal cortex (EC), particularly the stellate cells of layer II (ECIIscs), plays an important part in the genesis of the theta rhythm. These neurons occupy a key position in the neocortex-hippocampus-neocortex circuit, a crucial crossroad in memory functions. Neuronal oscillations typically rely on the activation of voltage-dependent Ca2+ conductances and the Ca2+ -dependent K+ conductance that usually follows, as seen in other limbic subcortical structures generating theta rhythmicity. Here we report, however, that similar oscillations are generated in ECIIscs by a Na+ conductance. The finding of a subthreshold, voltage-gated, Na+ -dependent rhythmic membrane oscillation in mammalian neurons indicates that rhythmicity in heterogeneous neuronal networks may be supported by different sets of intrinsic ionic mechanisms in each of the neuronal elements involved.

To clarify the physiological role of Na(+)-D-glucose cotransporter SGLT1 in small intestine and kidney, Sglt1(-/-) mice were generated and characterized phenotypically. After gavage of d-glucose, small intestinal glucose absorption across the brush-border membrane (BBM) via SGLT1 and GLUT2 were analyzed. Glucose-induced secretion of insulinotropic hormone (GIP) and glucagon-like peptide 1 (GLP-1) in wild-type and Sglt1(-/-) mice were compared. The impact of SGLT1 on renal glucose handling was investigated by micropuncture studies. It was observed that Sglt1(-/-) mice developed a glucose-galactose malabsorption syndrome but thrive normally when fed a glucose-galactose-free diet. In wild-type mice, passage of D-glucose across the intestinal BBM was predominantly mediated by SGLT1, independent the glucose load. High glucose concentrations increased the amounts of SGLT1 and GLUT2 in the BBM, and SGLT1 was required for upregulation of GLUT2. SGLT1 was located in luminal membranes of cells immunopositive for GIP and GLP-1, and Sglt1(-/-) mice exhibited reduced glucose-triggered GIP and GLP-1 levels. In the kidney, SGLT1 reabsorbed ∼3% of the filtered glucose under normoglycemic conditions. The data indicate that SGLT1 is 1) pivotal for intestinal mass absorption of d-glucose, 2) triggers the glucose-induced secretion of GIP and GLP-1, and 3) triggers the upregulation of GLUT2.

Stimulation of beta2-adrenergic receptors on the cell surface by adrenaline or noradrenaline leads to alterations in the metabolism, excitability, differentiation and growth of many cell types. These effects have traditionally been thought to be mediated exclusively by receptor activation of intracellular G proteins. However, certain physiological effects of beta2-adrenergic receptor stimulation, notably the regulation of cellular pH by modulation of Na+/H+ exchanger (NHE) function, do not seem to be entirely dependent on G-protein activation. We report here a direct agonist-promoted association of the beta2-adrenergic receptor with the Na+/H+ exchanger regulatory factor (NHERF), a protein that regulates the activity of the Na+/H+ exchanger type 3 (NHE3). NHERF binds to the beta2-adrenergic receptor by means of a PDZ-domain-mediated interaction with the last few residues of the carboxy-terminal cytoplasmic domain of the receptor. Mutation of the final residue of the beta2-adrenergic receptor from leucine to alanine abolishes the receptor's interaction with NHERF and also markedly alters beta2-adrenergic receptor regulation of NHE3 in cells without altering receptor-mediated activation of adenylyl cyclase. Our findings indicate that agonist-dependent beta2-adrenergic receptor binding of NHERF plays a role in beta2-adrenergic receptor-mediated regulation of Na+/H+ exchange.

A low affinity receptor for IgG immune complexes, Fc gamma RIII(CD16), is expressed on human NK cells as an integral membrane glycoprotein anchored through a transmembrane peptide; on polymorphonuclear neutrophils (PMN) the receptor is anchored through a phosphatidylinositol (PI) linkage. The protein on NK cells has a molecular mass 6-10 kD larger than that on PMN, and, unlike the latter, is resistant to PI-specific phospholipase C (PI-PLC). Fc gamma RIII(CD16) transcripts isolated from PMN and NK cells of single donors revealed multiple single nucleotide differences, one of which converts an in frame UGA termination codon to a CGA codon. The resulting open reading frame encodes a longer cytoplasmic domain for Fc gamma RIII(CD16) in NK cells, contributing to its transmembrane anchor. Two nearly identical, linked genes that encode these transcripts have been cloned for Fc gamma RIII(CD16), one of which (III-1) is allelic for NA-1 and NA-2. The allelic sites have been mapped to two single nucleotides in the extracellular domain. These genes are transcribed in a cell type-specific fashion to generate the alternatively anchored forms of this receptor.

The retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that lies in close association with the rod and cone photoreceptors. This epithelium has diverse features, three of which are discussed in some detail in this review, namely the daily phagocytosis of rod and cone outer segment fragments that are shed from their distal ends; the uptake, processing, transport and release of vitamin A (retinol) and some of its visual cycle intermediates (retinoids); and some of the aspects of its apical and basolateral membrane polarity that are the reverse of most other epithelia. Phagocytosis takes place at the apical surface via membrane receptor-mediated processes that are not yet well defined. Retinol uptake occurs at both the basolateral and apical surfaces by what appear to be separate receptor-mediated processes. The release of a crucial retinoid, 11-cis retinaldehyde (11-cis retinal), occurs solely across the apical membrane. Delivery of retinol across the basolateral membrane is mediated by a retinol binding protein (RBP) that is secreted by the liver as a complex with retinol (vitamin A). Within the cell, retinol and its derivatives are solubilized by intracellular retinoid binding proteins that are selective for retinol (cellular retinol binding protein, CRBP) and 11-cis retinoids (cellular retinal binding protein, CRALBP). Release of 11-cis retinal across the apical membrane and re-uptake of retinol from the photoreceptors during the visual cycle is promoted by an intercellular retinoid binding protein (IRBP). Na,K-ATPase, the membrane-integrated enzyme required to set up the ion gradients that drive other ion transporters, is largely localized to the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Epithelia separate tissue spaces by regulating the passage of ions, solutes, and water through both the transcellular and paracellular pathways. Paracellular permeability is defined by intercellular tight junctions, which vary widely among tissues with respect to solute flux, electrical resistance, and ionic charge selectivity. To test the hypothesis that members of the claudin family of tight junction proteins create charge selectivity, we assessed the effect of reversing the charge of selected extracellular amino acids in two claudins using site-directed mutagenesis. Claudins were expressed in cultured Madin-Darby canine kidney cell monolayers under an inducible promoter, and clones were compared with and without induction for transmonolayer electrical resistance and dilution potentials. Expression and localization of claudins were determined by immunoblotting, immunofluorescence microscopy, and freeze-fracture electron microscopy. We observed that substituting a negative for a positive charge at position 65 in the first extracellular domain of claudin-4 increased paracellular Na+ permeability. Conversely, substituting positive for negative charges at three positions in the first extracellular domain of claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na+ to Cl-. These results support a model where claudins create charge-selective channels in the paracellular space.

Nociceptive sensory neurons are unusual in expressing voltage-gated inward currents carried by sodium channels resistant to block by tetrodotoxin (TTX) as well as currents carried by conventional TTX-sensitive sodium channels and voltage-dependent calcium channels. To examine how currents carried by each of these helps to shape the action potential in small-diameter dorsal root ganglion cell bodies, we voltage clamped cells by using the action potential recorded from each cell as the command voltage. Using intracellular solutions of physiological ionic composition, we isolated individual components of current flowing during the action potential with the use of channel blockers (TTX for TTX-sensitive sodium currents and a mixture of calcium channel blockers for calcium currents) and ionic substitution (TTX-resistant current measured by the replacement of extracellular sodium by N-methyl-D-glucamine in the presence of TTX, with correction for altered driving force). TTX-resistant sodium channels activated quickly enough to carry the largest inward charge during the upstroke of the nociceptor action potential (approximately 58%), with TTX-sensitive sodium channels also contributing significantly ( approximately 40%), especially near threshold, and high voltage-activated calcium currents much less (approximately 2%). Action potentials had a prominent shoulder during the falling phase, characteristic of nociceptive neurons. TTX-resistant sodium channels did not inactivate completely during the action potential and carried the majority (58%) of inward current flowing during the shoulder, with high voltage-activated calcium current also contributing significantly (39%). Unlike calcium current, TTX-resistant sodium current is not accompanied by opposing calcium-activated potassium current and may provide an effective mechanism by which the duration of action potentials (and consequently calcium entry) can be regulated.