Evidence For Functional Coupling Research Articles

Pharmacology of the Nav1.1 domain IV voltage sensor reveals coupling between inactivation gating processes

The Nav1.1 voltage-gated sodium channel is a critical contributor to excitability in the brain, where pathological loss of function leads to such disorders as epilepsy, Alzheimer's disease, and autism. This voltage-gated sodium (Nav) channel subtype also plays an important role in mechanical pain signaling by primary afferent somatosensory neurons. Therefore, pharmacologic modulation of Nav1.1 represents a potential strategy for treating excitability disorders of the brain and periphery. Inactivation is a complex aspect of Nav channel gating and consists of fast and slow components, each of which may involve a contribution from one or more voltage-sensing domains. Here, we exploit the Hm1a spider toxin, a Nav1.1-selective modulator, to better understand the relationship between these temporally distinct modes of inactivation and ask whether they can be distinguished pharmacologically. We show that Hm1a inhibits the gating movement of the domain IV voltage sensor (VSDIV), hindering both fast and slow inactivation and leading to an increase in Nav1.1 availability during high-frequency stimulation. In contrast, ICA-121431, a small-molecule Nav1.1 inhibitor, accelerates a subsequent VSDIV gating transition to accelerate entry into the slow inactivated state, resulting in use-dependent block. Further evidence for functional coupling between fast and slow inactivation is provided by a Nav1.1 mutant in which fast inactivation removal has complex effects on slow inactivation. Taken together, our data substantiate the key role of VSDIV in Nav channel fast and slow inactivation and demonstrate that these gating processes are sequential and coupled through VSDIV. These findings provide insight into a pharmacophore on VSDIV through which modulation of inactivation gating can inhibit or facilitate Nav1.1 function.

Acidic Eye‐Drops De‐Swell the Edematous Corneas of Slc4a11‐null Mice; Evidence for Functional Coupling between Slc4a11 and the H+/lactate Cotransporter Mct1

SLC4A11 is the most divergent member of the SLC4 family of acid‐base transporters. Mutations in SLC4A11 are associated with congenital hereditary endothelial dystrophy (CHED): a clouding of the cornea caused by the accumulation of fluid in the corneal stroma. Slc4a11‐null mice have edematous corneas. Slc4a11 is expressed in the basolateral membrane of corneal endothelial cells; these cells normally promote fluid removal from the stroma by absorbing stromal osmolytes such as Na+, HCO3− (via the basolateral Na+/HCO3− cotransporter NBCe1) and lactate (via the basolateral H+/lactate cotransporter MCT1). The contribution of Slc4a11 to endothelial function is not fully understood. In a separate study in Xenopus oocytes, we have observed that mouse Slc4a11 mediates H+ conduction when extracellular pH is raised. At the basolateral membrane of corneal‐endothelial cells, Mct1‐mediated H+/lactate import would tend to raise surface pH (pHs) in the vicinity of Slc4a11. The near‐zero membrane potential reported for actively‐transporting corneal endothelia (Hodson and Wigham, J. Physiol., 1989), predicts that Slc4a11 should mediate H+ efflux in response to elevations in pHs. Thus we hypothesize that Mct1 action promotes Slc4a11‐mediated H+ efflux, which in turn supports Mct1 action and thereby the maintenance of corneal clarity. To investigate coupling between Mct1 and Slc4a11, we expressed Mct1 ± Slc4a11 in Xenopus oocytes. Under voltage clamp at 0 mV, we simultaneously monitored pHs and the Slc4a11‐dependent H+‐current as we applied lactate to the cells. Lactate application caused a sharp rise in pHs in all cells due to Mct action. In cells co‐expressing Mct1+Slc4a11, lactate application also caused a sustained increase in current due to Slc4a11‐mediated H+‐efflux (n=3), consistent with functional coupling between Mct1 and Slc4a11. Our preliminary data suggest that even in the absence of lactate, Slc4a11‐mediated H+‐conduction is significantly enhanced by Mct1 co‐expression (P=0.01, n=3), as if Mct1 also physically enhances the functional expression of Slc4a11. We performed two further assays to investigate the effect of Slc4a11 loss on H+ and lactate handling by mouse corneas. [1] We extracted tissue samples from the anterior section of wild‐type and Slc4a11‐null mouse eyes; Slc4a11‐null samples contained more lactate than wild‐type equivalents (P=0.02, n=4). [2] We measured corneal thickness in wild‐type and Slc4a11‐null mice by pachymetry. The application of acidic eye‐drops to the edematous corneas of Slc4a11‐null mice resulted in their de‐swelling (n=6, P<0.01). The thickness of de‐swollen corneas was not different from the thickness of wild‐type corneas. Together these data indicate that Slc4a11 plays an important role in support of Mct1 action, and thereby endothelial function.Support or Funding InformationSupported by Start‐up Funds from UB:SUNY (to MDP), an Unrestricted Grant from Research to Prevent Blindness (to SPP), and NIH R21‐EY021646 (to MLJ and MDP).

Anatomical and functional gonadotrope networks in the teleost pituitary.

Mammalian pituitaries exhibit a high degree of intercellular coordination; this enables them to mount large-scale coordinated responses to various physiological stimuli. This type of communication has not been adequately demonstrated in teleost pituitaries, which exhibit direct hypothalamic innervation and expression of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in distinct cell types. We found that in two fish species, namely tilapia and zebrafish, LH cells exhibit close cell–cell contacts and form a continuous network throughout the gland. FSH cells were more loosely distributed but maintained some degree of cell–cell contact by virtue of cytoplasmic processes. These anatomical differences also manifest themselves at the functional level as evidenced by the effect of gap-junction uncouplers on gonadotropin release. These substances abolished the LH response to gonadotropin-releasing hormone stimulation but did not affect the FSH response to the same stimuli. Dye transfer between neighboring LH cells provides further evidence for functional coupling. The two gonadotropins were also found to be differently packaged within their corresponding cell types. Our findings highlight the evolutionary origin of pituitary cell networks and demonstrate how the different levels of cell–cell coordination within the LH and FSH cell populations are reflected in their distinct secretion patterns.

Evidence for functional coupling of cGMP/cGKI signalling and TRPC channels in endothelium but not in vascular smooth muscle

Background Signaling via cGMP-dependent protein kinase I (cGKI) is the major pathway in vascular smooth muscle (SM), by which endothelial NO regulates vascular tone. Recent evidence suggests that canonical transient receptor potential (TRPC) channels are targets of cGKI in SM and mediate the relaxant effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on vascular tone and peripheral resistance using Trpc6, Trpc3, Trpc3/6, Trpc1/3/6, and SM-specific cGKI (sm-cGKI) mice.

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.

Coordinated Leading and Lagging Strand DNA Synthesis by Using the Herpes Simplex Virus 1 Replication Complex and Minicircle DNA Templates

The origin-specific replication of the herpes simplex virus 1 genome requires seven proteins: the helicase-primase (UL5-UL8-UL52), the DNA polymerase (UL30-UL42), the single-strand DNA binding protein (ICP8), and the origin-binding protein (UL9). We reconstituted these proteins, excluding UL9, on synthetic minicircular DNA templates and monitored leading and lagging strand DNA synthesis using the strand-specific incorporation of dTMP and dAMP. Critical features of the assays that led to efficient leading and lagging stand synthesis included high helicase-primase concentrations and a lagging strand template whose sequence resembled that of the viral DNA. Depending on the nature of the minicircle template, the replication complex synthesized leading and lagging strand products at molar ratios varying between 1:1 and 3:1. Lagging strand products (∼0.2 to 0.6 kb) were significantly shorter than leading strand products (∼2 to 10 kb), and conditions that stimulated primer synthesis led to shorter lagging strand products. ICP8 was not essential; however, its presence stimulated DNA synthesis and increased the length of both leading and lagging strand products. Curiously, human DNA polymerase α (p70-p180 or p49-p58-p70-p180), which improves the utilization of RNA primers synthesized by herpesvirus primase on linear DNA templates, had no effect on the replication of the minicircles. The lack of stimulation by polymerase α suggests the existence of a macromolecular assembly that enhances the utilization of RNA primers and may functionally couple leading and lagging strand synthesis. Evidence for functional coupling is further provided by our observations that (i) leading and lagging strand synthesis produce equal amounts of DNA, (ii) leading strand synthesis proceeds faster under conditions that disable primer synthesis on the lagging strand, and (iii) conditions that accelerate helicase-catalyzed DNA unwinding stimulate decoupled leading strand synthesis but not coordinated leading and lagging strand synthesis.

Splicing-Dependent RNA Polymerase Pausing in Yeast

SummaryIn eukaryotic cells, there is evidence for functional coupling between transcription and processing of pre-mRNAs. To better understand this coupling, we performed a high-resolution kinetic analysis of transcription and splicing in budding yeast. This revealed that shortly after induction of transcription, RNA polymerase accumulates transiently around the 3′ end of the intron on two reporter genes. This apparent transcriptional pause coincides with splicing factor recruitment and with the first detection of spliced mRNA and is repeated periodically thereafter. Pausing requires productive splicing, as it is lost upon mutation of the intron and restored by suppressing the splicing defect. The carboxy-terminal domain of the paused polymerase large subunit is hyperphosphorylated on serine 5, and phosphorylation of serine 2 is first detected here. Phosphorylated polymerase also accumulates around the 3′ splice sites of constitutively expressed, endogenous yeast genes. We propose that transcriptional pausing is imposed by a checkpoint associated with cotranscriptional splicing.

Co-operative Effect Between γ-Aminobutyric Acid A Receptors and Central-Type Benzodiazepine Receptors on Amylase Release in Rat Parotid Acinar Cells

We investigated the inhibitory role of γ-aminobutyric acid A (GABA A ) receptors on amylase release and the evidence for functional coupling with central-type benzodiazepine receptors in rat parotid acinar cells. Muscimol and GABA decreased isoprenaline-induced amylase release. This effect was blocked by bicuculline, a GABA A-receptor antagonist, and enhanced by clonazepam, a central-type benzodiazepine-receptor agonist, and diazepam, a central- and peripheral-type benzodiazepine-receptor agonist. Although bicuculline completely blocked the combination effect of GABA A-receptor agonist and clonazepam, it did not completely block the combination effect with diazepam. These results suggest that protein secretion is suppressively regulated by GABA A receptors coupled with central-type benzodiazepine receptors.

Transmembrane Helices 3 and 4 Are Involved in Substrate Recognition by the Na+/Dicarboxylate Cotransporter, NaDC1

The Na(+)/dicarboxylate cotransporters (NaDC1) from mouse (m) and rabbit (rb) differ in their ability to handle glutarate. Substrate-dependent inward currents, measured using two-electrode voltage clamp, were similar for glutarate and succinate in Xenopus oocytes expressing mNaDC1. In contrast, currents evoked by glutarate in rbNaDC1 were only about 5% of the succinate-dependent currents. To identify domains involved in glutarate transport, we constructed a series of chimeric transporters between mouse and rabbit NaDC1. Although residues found in multiple transmembrane helices (TM) participate in glutarate transport, the most important contribution is made by TM 3 and 4 and the associated loops. The R(M3-4) chimera, consisting of rbNaDC1 with substitution of TM 3-4 from mNaDC1, had a decreased K(0.5)(glutarate) of 4 mM compared with 15 mM in wild-type rbNaDC1 without any effect on K(0.5)(succinate). The chimeras were also characterized using dual-label competitive uptakes with (14)C-glutarate and (3)H-succinate to calculate the transport specificity ratio (TSR), a measure of relative catalytic efficiency with the two substrates. The TSR analysis provides evidence for functional coupling in the transition state between TM 3 and 4. We conclude that TM 3 and 4 contain amino acid residues that are important determinants of substrate specificity and catalytic efficiency in NaDC1.

Functional expression and properties of a nicotinic alpha9/5-HT3A chimeric receptor.

We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.

Direct evidence for functional coupling between human placental folate transporter and folate receptor in a heterologous expression system

Direct evidence for functional coupling between human placental folate transporter and folate receptor in a heterologous expression system

Basolateral membrane Na+/H+ exchange enhances HCO3- absorption in rat medullary thick ascending limb: evidence for functional coupling between basolateral and apical membrane Na+/H+ exchangers.

The role of basolateral membrane Na+/H+ exchange in transepithelial HCO3- absorption (JHCO3) was examined in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. In Na(+)-free solutions, addition of Na+ to the bath resulted in a rapid, amiloride-sensitive increase in intracellular pH. In MTALs perfused and bathed with solutions containing 146 mM Na+ and 25 mM HCO3-, bath addition of amiloride (1 mM) or 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 50 microM) reversibly inhibited JHCO3 by 50%. Evidence that the inhibition of JHCO3 by bath amiloride was the result of inhibition of Na+/H+ exchange included the following: (i) the IC50 for amiloride was 5-10 microM, (ii) EIPA was a 50-fold more potent inhibitor than amiloride, (iii) the inhibition by bath amiloride was Na+ dependent, and (iv) significant inhibition was observed with EIPA as low as 0.1 microM. Fifty micromolar amiloride or 1 microM EIPA inhibited JHCO3 by 35% when added to the bath but had no effect when added to the tubule lumen, indicating that addition of amiloride to the bath did not directly inhibit apical membrane Na+/H+ exchange. In experiments in which apical Na+/H+ exchange was assessed from the initial rate of cell acidification following luminal EIPA addition, bath EIPA secondarily inhibited apical Na+/H+ exchange activity by 46%. These results demonstrate basolateral membrane Na+/H+ exchange enhances transepithelial HCO3- absorption in the MTAL. This effect appears to be the result of cross-talk in which an increase in basolateral membrane Na+/H+ exchange activity secondarily increases apical membrane Na+/H+ exchange activity.