Ching-On Wong

Membrane potential modulates plasma membrane phospholipid dynamics and K-Ras signaling

Membrane potential regulates growth Changes in electrical potential across the plasma membrane can affect cell growth. Zhou et al. discovered that membrane potential influenced the organization of phospholipids in the membrane of cultured mammalian cells and neurons in intact flies (see the Perspective by Accardi). This in turn regulated localization and activity of the small guanine nucleotide binding protein K-Ras, an important regulator of cell proliferation. The cell membrane may thus function analogously to a field-effect transistor by adjusting the strength of mitogenic signaling. Science, this issue p. 873; see also p. 789 Changing the voltage across the plasma membrane causes clustering of a small guanosine triphosphatase. [Also see Perspective by Accardi] Plasma membrane depolarization can trigger cell proliferation, but how membrane potential influences mitogenic signaling is uncertain. Here, we show that plasma membrane depolarization induces nanoscale reorganization of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate but not other anionic phospholipids. K-Ras, which is targeted to the plasma membrane by electrostatic interactions with phosphatidylserine, in turn undergoes enhanced nanoclustering. Depolarization-induced changes in phosphatidylserine and K-Ras plasma membrane organization occur in fibroblasts, excitable neuroblastoma cells, and Drosophila neurons in vivo and robustly amplify K-Ras–dependent mitogen-activated protein kinase (MAPK) signaling. Conversely, plasma membrane repolarization disrupts K-Ras nanoclustering and inhibits MAPK signaling. By responding to voltage-induced changes in phosphatidylserine spatiotemporal dynamics, K-Ras nanoclusters set up the plasma membrane as a biological field-effect transistor, allowing membrane potential to control the gain in mitogenic signaling circuits.

Functional Role of Vanilloid Transient Receptor Potential 4-Canonical Transient Receptor Potential 1 Complex in Flow-Induced Ca2+ Influx

Objective—The present study is aimed at investigating the interaction of TRPV4 with TRPC1 and the functional role of such an interaction in flow-induced Ca2+ influx. Hemodynamic blood flow is an important physiological factor that modulates vascular tone. One critical early event in this process is a cytosolic Ca2+ ([Ca2+]i) rise in endothelial cells in response to flow. Methods and Results—With the use of fluorescence resonance energy transfer, coimmunoprecipitation, and subcellular colocalization methods, it was found that TRPC1 interacts physically with TRPV4 to form a complex. In functional studies, flow elicited a transient [Ca2+]i increase in TRPV4-expressing human embryonic kidney (HEK) 293 cells. Coexpression of TRPC1 with TRPV4 markedly prolonged this [Ca2+]i transient; it also enabled this [Ca2+]i transient to be negatively modulated by protein kinase G. Furthermore, this flow-induced [Ca2+]i increase was markedly inhibited by anti–TRPC1-blocking antibody T1E3 and a dominant-negative construct TRPC1&Dgr;567-793 in TRPV4-C1–coexpressing HEK cells and human umbilical vein endothelial cells. T1E3 also inhibited flow-induced vascular dilation in isolated rat small mesenteric artery segments. Conclusion—This study shows that TRPC1 interacts physically with TRPV4 to form a complex, and this TRPV4-C1 complex may mediate flow-induced Ca2+ influx in vascular endothelial cells. The association of TRPC1 with TRPV4 prolongs the flow-induced [Ca2+]i transient, and it also enables this [Ca2+]i transient to be negatively modulated by protein kinase G. This TRPV4-C1 complex plays a key role in flow-induced endothelial Ca2+ influx.

Heteromeric TRPV4-C1 channels contribute to store-operated Ca(2+) entry in vascular endothelial cells.

There is controversy as to whether TRP channels participate in mediating store-operated current (I(SOC)) and store-operated Ca(2+) entry (SOCE). Our recent study has demonstrated that TRPC1 forms heteromeric channels with TRPV4 in vascular endothelial cells and that Ca(2+) store depletion enhances the vesicle trafficking of heteromeric TRPV4-C1 channels, causing insertion of more channels into the plasma membrane in vascular endothelial cells. In the present study, we determined whether the enhanced TRPV4-C1 insertion to the plasma membrane could contribute to SOCE and I(SOC). We found that thapsigargin-induced SOCE was much lower in aortic endothelial cells derived from trpv4(-/-) or trpc1(-/-) knockout mice when compared to that of wild-type mice. In human umbilical vein endothelial cells (HUVECs), thapsigargin-induced SOCE was markedly reduced by knocking down the expression of TRPC1 and/or TRPV4 with respective siRNAs. Brefeldin A, a blocker of vesicular translocation, inhibited the SOCE. These results suggest that an enhanced vesicular trafficking of heteromeric TRPV4-C1 channels contributes to SOCE in vascular endothelial cells. Vascular tension studies suggest that such an enhanced trafficking of TRPV4-C1 channels may play a role in thapsigargin-induced vascular relaxation in rat small mesenteric arteries.

The role of TRPMLs in endolysosomal trafficking and function.

Members of the Transient Receptor Potential-Mucolipin (TRPML) constitute a family of evolutionarily conserved cation channels that function predominantly in endolysosomal vesicles. Whereas loss-of-function mutations in human TRPML1 were first identified as being causative for the lysosomal storage disease, Mucolipidosis type IV, most mammals also express two other TRPML isoforms called TRPML2 and TRPML3. All three mammalian TRPMLs as well as TRPML related genes in other species including Caenorhabditis elegans and Drosophila exhibit overlapping functional and biophysical properties. The functions of TRPML proteins include roles in vesicular trafficking and biogenesis, maintenance of neuronal development, function, and viability, and regulation of intracellular and organellar ionic homeostasis. Biophysically, TRPML channels are non-selective cation channels exhibiting variable permeability to a host of cations including Na(+), Ca(2+), Fe(2+), and Zn(2+), and are activated by a phosphoinositide species, PI(3,5)P2, that is mostly found in endolysosomal membranes. Here, we review the functional and biophysical properties of these enigmatic cation channels, which represent the most ancient and archetypical TRP channels.

Feast or famine: Role of TRPML in preventing cellular amino acid starvation

Lysosomal storage diseases are metabolic disorders characterized by the accumulation of acidic vacuoles, and are usually the consequence of the deficiency of an enzyme responsible for the metabolism of vesicular lipids, proteins or carbohydrates. In contrast, mucolipidosis type IV (MLIV), results from the absence of a vesicular Ca2+ release channel called mucolipin 1/transient receptor potential mucolipin 1 (MCOLN1/TRPML1) which is required for the fusion of amphisomes with lysosomes. In Drosophila, ablation of the MCOLN1 homolog (trpml) leads to diminished viability during pupation when the animals rely on autophagy for nutrients. This pupal lethality results from decreased target of rapamycin complex 1 (TORC1) signaling, and is reversed by reactivating TORC1. Our findings indicate that one of the primary causes of toxicity in the absence of TRPML is cellular amino acid starvation, and the resulting decrease in TORC1 activity. Furthermore, our findings raise the intriguing possibility that the neurological dysfunction in MLIV patients may arise from amino acid deprivation in neurons. Therefore, future studies evaluating the levels of amino acids and TORC1 activity in MLIV neurons may aid in the development of novel therapeutic strategies to combat the severe manifestations of MLIV.

Drosophila TRPML Is Required for TORC1 Activation

Loss-of-function mutations in TRPML1 (transient receptor potential mucolipin 1) cause the lysosomal storage disorder, mucolipidosis type IV (MLIV). Here, we report that flies lacking the TRPML1 homolog displayed incomplete autophagy and reduced viability during the pupal period--a phase when animals rely on autophagy for nutrients. We show that TRPML was required for fusion of amphisomes with lysosomes, and its absence led to accumulation of vesicles of significantly larger volume and higher luminal Ca(2+). We also found that trpml(1) mutant cells showed decreased TORC1 (target of rapamycin complex 1) signaling and a concomitant upregulation of autophagy induction. Both of these defects in the mutants were reversed by genetically activating TORC1 or by feeding the larvae a high-protein diet. The high-protein diet also reduced the pupal lethality and the increased volume of acidic vesicles. Conversely, further inhibition of TORC1 activity by rapamycin exacerbated the mutant phenotypes. Finally, TORC1 exerted reciprocal control on TRPML function. A high-protein diet caused cortical localization of TRPML, and this effect was blocked by rapamycin. Our findings delineate the interrelationship between the TRPML and TORC1 pathways and raise the intriguing possibility that a high-protein diet might reduce the severity of MLIV.

cAMP activates TRPC6 channels via the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB)-mitogen-activated protein kinase kinase (MEK)-ERK1/2 signaling pathway.

cAMP is an important second messenger that executes diverse physiological function in living cells. In this study, we investigated the effect of cAMP on canonical TRPC6 (transient receptor potential channel 6) channels in TRPC6-expressing HEK293 cells and glomerular mesangial cells. The results showed that 500 μm 8-Br-cAMP, a cell-permeable analog of cAMP, elicited [Ca2+]i increases and stimulated a cation current at the whole-cell level in TRPC6-expressing HEK293 cells. The effect of cAMP diminished in the presence of the PI3K inhibitors wortmannin and LY294002 or the MEK inhibitors PD98059, U0126, and MEK inhibitor I. 8-Br-cAMP also induced phosphorylation of MEK and ERK1/2. Conversion of serine to glycine at an ERK1/2 phosphorylation site (S281G) abolished the cAMP activation of TRPC6 as determined by whole-cell and cell-attached single-channel patch recordings. Experiments based on a panel of pharmacological inhibitors or activators suggested that the cAMP action on TRPC6 was not mediated by PKA, PKG, or EPAC (exchange protein activated by cAMP). Total internal fluorescence reflection microscopy showed that 8-Br-cAMP did not alter the trafficking of TRPC6 to the plasma membrane. We also found that, in glomerular mesangial cells, glucagon-induced [Ca2+]i increases were mediated through the cAMP-PI3K-PKB-MEK-ERK1/2-TRPC6 signaling pathway. In summary, this study uncovered a novel TRPC6 activation mechanism in which cAMP activates TRPC6 via the PI3K-PKB-MEK-ERK1/2 signaling pathway.

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on beta-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca(2+) ([Ca(2+)](i)) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca(2+) influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca(2+) dye, it was found that epinephrine and isoprenaline, a beta-adrenoceptor agonist, induced endothelial Ca(2+) influx in the presence of bradykinin. This Ca(2+) influx was inhibited by L-cis-diltiazem and LY-83583, and by a beta(2)-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca(2+) influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca(2+) influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate beta-adrenoceptor agonist-induced endothelial Ca(2+) influx and subsequent vascular dilation.

Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma Membrane

Background: Drosophila trpml mutants reproduced many defects associated with mucolipidosis type IV, but the fly TRPML channel remains uncharacterized. Results: Drosophila TRPML is a phosphoinositide-regulated cation channel on endolysosome and plasma membranes. Conclusion: Fly TRPML largely resembles mammalian TRPML1, but exhibits differences in subcellular localization and pH dependence. Significance: The data support using Drosophila for assessing TRPML1 function. Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca2+ and Fe2+ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca2+, Mn2+, and Fe2+, but not Fe3+. The TRPML currents were inhibited by trivalent cations Fe3+, La3+, and Gd3+. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.

Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinases

Background and purpose:  TRPC5 is a Ca2+‐permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant‐derived isoflavone, on TRPC5 activity, and the mechanism(s) involved.