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Uroguanylin and guanylin regulate transport of mouse cortical collecting duct independent of guanylate cyclase C. Background Electrolyte and water homeostasis mostly depend on differentially regulated intestinal and renal transport. Guanylin and uroguanylin were proposed as first hormones linking intestinal with renal electrolyte and water transport, which is disturbed in pathophysiology. Guanylate cyclase C is the intestinal receptor for these peptides, but in guanylate cyclase C-deficient mice renal effects are retained. Unlike for the intestine the sites of renal actions and cellular mechanisms of guanylin peptides are still unclear. Methods After first data on proximal tubular effects in this study their effects are examined in detail in mouse cortical collecting duct (CCD). Effects of guanylin peptides on principal cells of isolated mouse CCD were studied by slow whole-cell patch-clamp analysis, reverse transcription-polymerase chain reaction (RT-PCR), and microfluorimetric measurements of intracellular Ca 2+ . Results Guanylin peptides depolarized or hyperpolarized principal cells. Whereas 8-Br-cyclic guanosine monophosphate (8-Br-cGMP) hyperpolarized, 8-Br-cyclic adenosine monophosphate (8-Br-cAMP) depolarized principal cells. All effects of guanylin peptides were inhibited by Ba 2+ . Hyperpolarizations were blocked by clotrimazole or protein kinase G (PKG) inhibition, suggesting an involvement of basolateral Ca 2+ - and cGMP-dependent K + channels. Effects remained in CCD isolated from guanylate cyclase C-deficient mice. Depolarizations were inhibited by arachidonic acid or inhibition of phospholipase A 2 (PLA 2 ), but not by protein kinase A (PKA) inhibition. Conclusion These results suggest the existence of two signaling pathways for guanylin peptides in principal cells of mouse CCD. One pathway is cGMP- and PKG-dependent but not mediated by guanylate cyclase C, the second involves PLA 2 and arachidonic acid. The first pathway most likely leads to an activation of the basolateral K + -conductance while the latter probably results in decreased activity of ROMK channels in the luminal membrane.

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca 2+ currents (I Ca,L ) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba 2+ currents (I Ba ) through L-type Ca 2+ channels (LTCCs). Under voltage–clamp conditions, holding at –40mV, KMUP-1 (1, 3, 10μM) inhibited I Ba in a concentration-dependent manner and its IC 50 value was 2.27±0.45μM. A high concentration of KMUP-1 (10μM) showed without modifying the I Ba current–voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I Ba was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5μM) intensified KMUP-1-inhibited I Ba . However, the Rho kinase inhibitor Y-27632 (30μM) failed to affect the I Ba inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway.

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier.

Intracellular cAMP injection and negative pressure in the patch-electrode increase the interburst closed time of the same potassium ionic channels in the snail neuron membrane. Sodium channels which were registered as change of background noise are activated both by cAMP injection and by negative pressure. These results are considered in connection with data about the unusual biochemistry of the neuron reaction to cAMP.

Our aim was to observe whether normal human T-cells respond to mitogenic stimulation with large whole-cell inward currents (composed of identifiable single-channel contributions) when [Ca2+]i is not markedly lowered but instead kept normal or moderately low, as has been reported in human leukaemic Jurkat T-cell line and T-cell clones [Kuno et al. (1986) Nature 323, 269–273; Kuno and Gardner (1987) Nature 326, 301–304; Gardner (1990) Annu. Rev. Immunol. 8, 231–252]. Whole-cell patch recordings showed no such currents in cells otherwise normally responding to depolarisation with the macroscopic IK described in T-lymphocytes and thus deemed viable, in agreement with the notion that Ca2+ influx in normal T-cells enterily depends on depletion of internal stores [Putney (1986) Cell Calcium 7, 1–12; Putney (1990) Cell Calcium 11, 611–624].

Non-selective cation channels were detected in membrane patches of cultured human fibroblasts. The channels had a unitary conductance which ranged from 14 to 25 pS in symmetrical 130 mM NACl and were permeable to both sodium and potassium ions. Open channel probability was dependent either on the membrane potential and the Ca2+ concentration on the intracellular side of the membrane. High Ca2+ concentrations in the millimolar range were needed to keep the channel active.

The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.

We have designed and synthesized benzo[c]quinolizinium derivatives and evaluated their effects on the activity of G551D cystic fibrosis transmembrane conductance regulator (CFTR) expressed in Chinese hamster ovary and Fisher rat thyroid cells. We demonstrated, using iodide efflux, whole cell patch clamp, and short-circuit recordings, that 5-butyl-6-hydroxy-10-chlorobenzo[c]quinolizinium chloride (MPB-91) restored the activity of G551D CFTR (EC50= 85 μM) and activated CFTR in Calu-3 cells (EC50= 47 μM). MPB-91 has no effect on the ATPase activity of wild-type and G551D NBD1/R/GST fusion proteins or on the ATPase, GTPase, and adenylate kinase activities of purified NBD2. The activation of CFTR by MPB-91 is independent of phosphorylation because 1) kinase inhibitors have no effect and 2) the compound still activated CFTR having 10 mutated protein kinase A sites (10SA-CFTR). The new pharmacological agent MPB-91 may be an important candidate drug to ameliorate the ion transport defect associated with CF and to point out a new pathway to modulate CFTR activity.

Recurrent epileptiform activity occurs spontaneously in cultured CNS neurons and in brain slices in which GABA inhibition has been blocked. We demonstrate here that pharmacological treatments resulting in either the block of glutamine production by astrocytes or the inhibition of glutamine uptake by neurons suppress or markedly decrease the frequency of spontaneous epileptiform discharges both in primary hippocampal cultures and in disinhibited hippocampal slices. These data point to an important role for the neuron-astrocyte metabolic interaction in sustaining episodes of intense rhythmic activity in the CNS, and thereby reveal a new potential target for antiepileptic treatments.

Calcium-dependent chloride channels are required for normal electrolyte and fluid secretion, olfactory perception, and neuronal and smooth muscle excitability. The molecular identity of these membrane proteins is still unclear. Treatment of bronchial epithelial cells with interleukin-4 (IL-4) causes increased calcium-dependent chloride channel activity, presumably by regulating expression of the corresponding genes. We performed a global gene expression analysis to identify membrane proteins that are regulated by IL-4. Transfection of epithelial cells with specific small interfering RNA against each of these proteins shows that TMEM16A, a member of a family of putative plasma membrane proteins with unknown function, is associated with calcium-dependent chloride current, as measured with halide-sensitive fluorescent proteins, short-circuit current, and patch-clamp techniques. Our results indicate that TMEM16A is an intrinsic constituent of the calcium-dependent chloride channel. Identification of a previously unknown family of membrane proteins associated with chloride channel function will improve our understanding of chloride transport physiopathology and allow for the development of pharmacological tools useful for basic research and drug development.

Purpose: The murine homeobox-containing Otx gene is required for correct nervous system and sense organ development. Otx l -/- mice obtained by replacing Otx with the lac Z gene show developmental abnormalities of the cerebellum, mesencephalon, and cerebral cortex associated with spontaneous epileptic seizures (1). The epileptogenic mechanisms accounting for these seizures were investigated by means of electrophysiological recordings made from neocortical slices. Methods: The 400-μm slices were prepared from the somatosensory cortex of Otx l -/- and Otx l +/+ mice, and the current clamp intracellular recordings were obtained from layer V pyramidal neurons by means of pipettes containing K + acetate 1.5 mol/L and biocytin 2% (pH 7.3). Results: Synaptic responses could be evoked in the neocortical pyramidal neurons by electrically stimulating the underlying white matter. γ-Aminobutyric acid A/B-mediated inhibitory postsynaptic potentials were more pronounced in the Otx l -/- than in the control pyramidal neurons from the earliest postnatal period; multisynaptic excitatory postsynaptic potentials were significantly more expressed in the Otx l -/- mice also at the end of the first postnatal month, when they were only rarely encountered in controls. Conclusion: Excessive excitatory amino acid-mediated synaptic driving may lead to a hyperexcitable condition that is responsible for the epileptic manifestations occurring in Otx l -/- mice. This excess of excitation is not counteracted by well-developed γ-aminobutyric acid activity, which seems to be involved in the synchronization of cell discharges. Our ongoing and more extensive comparative analysis of the mutants and controls should help to clarify the way in which the putative rearrangement taking place in Otx l -/- neocortex may lead to the excitatory hyperinnervation of layer V pyramidal neurons.