Ion channel clustering enhances weak electric field detection by neutrophils: apparent roles of SKF96365-sensitive cation channels and myeloperoxidase trafficking in cellular responses

We have tested Galvanovskis and Sandblom’s prediction that ion channel clustering enhances weak electric field detection by cells as well as how the elicited signals couple to metabolic alterations. Electric field application was timed to coincide with certain known intracellular chemical oscillators (phase-matched conditions). Polarized, but not spherical, neutrophils labeled with anti-K v 1.3, FL-DHP, and anti-TRP1, but not anti-T-type Ca 2+ channels, displayed clusters at the lamellipodium. Resonance energy transfer experiments showed that these channel pairs were in close proximity. Dose-field sensitivity studies of channel blockers suggested that K + and Ca 2+ channels participate in field detection, as judged by enhanced oscillatory NAD(P)H amplitudes. Further studies suggested that K + channel blockers act by reducing the neutrophil’s membrane potential. Mibefradil and SKF93635, which block T-type Ca 2+ channels and SOCs, respectively, affected field detection at appropriate doses. Microfluorometry and high-speed imaging of indo-1-labeled neutrophils was used to examine Ca 2+ signaling. Electric fields enhanced Ca 2+ spike amplitude and triggered formation of a second traveling Ca 2+ wave. Mibefradil blocked Ca 2+ spikes and waves. Although 10 μM SKF96365 mimicked mibefradil, 7 μM SKF96365 specifically inhibited electric field-induced Ca 2+ signals, suggesting that one SKF96365-senstive site is influenced by electric fields. Although cells remained morphologically polarized, ion channel clusters at the lamellipodium and electric field sensitivity were inhibited by methyl-β-cyclodextrin. As a result of phase-matched electric field application in the presence of ion channel clusters, myeloperoxidase (MPO) was found to traffic to the cell surface. As MPO participates in high amplitude metabolic oscillations, this suggests a link between the signaling apparatus and metabolic changes. Furthermore, electric field effects could be blocked by MPO inhibition or removal while certain electric field effects were mimicked by the addition of MPO to untreated cells. Therefore, channel clustering plays an important role in electric field detection and downstream responses of morphologically polarized neutrophils. In addition to providing new mechanistic insights concerning electric field interactions with cells, our work suggests novel methods to remotely manipulate physiological pathways.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46726/1/249_2005_Article_1.pd

Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for β(2)-adrenergic regulation

L-type Ca(2+) channels play a critical role in regulating Ca(2+)-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca(2+) channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca(2+) channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for β(2)-adrenergic receptor (AR) regulation of I(Ca,L). Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Ca(v)1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca(v)1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca(v)1.2, β(2)-AR (not β(1)-AR), G protein α(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized β(2)-AR/Ca(v)1.2 signaling complex, β(2)-AR stimulation (salbutamol plus atenolol) of I(Ca,L) was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I(Ca,L) in response to β(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl β-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas β(1)-AR stimulation (norepinephrine plus prazosin) of I(Ca,L) was not altered. These findings demonstrate that subcellular localization of L-type Ca(2+) channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways

A distinct pool of phosphatidylinositol 4,5-bisphosphate in caveolae revealed by a nanoscale labeling technique

Multiple functionally independent pools of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] have been postulated to occur in the cell membrane, but the existing techniques lack sufficient resolution to unequivocally confirm their presence. To analyze the distribution of PI(4,5)P2 at the nanoscale, we developed an electron microscopic technique that probes the freeze-fractured membrane preparation by the pleckstrin homology domain of phospholipase C-δ1. This method does not require chemical fixation or expression of artificial probes, it is applicable to any cell in vivo and in vitro, and it can define the PI(4,5)P2 distribution quantitatively. By using this method, we found that PI(4,5)P2 is highly concentrated at the rim of caveolae both in cultured fibroblasts and mouse smooth muscle cells in vivo. PI(4,5)P2 was also enriched in the coated pit, but only a low level of clustering was observed in the flat undifferentiated membrane. When cells were treated with angiotensin II, the PI(4,5)P2 level in the undifferentiated membrane decreased to 37.9% within 10 sec and then returned to the initial level. Notably, the PI(4,5)P2 level in caveolae showed a slower but more drastic change and decreased to 20.6% at 40 sec, whereas the PI(4,5)P2 level in the coated pit was relatively constant and decreased only to 70.2% at 10 sec. These results show the presence of distinct PI(4,5)P2 pools in the cell membrane and suggest a unique role for caveolae in phosphoinositide signaling

TRPC1 forms the stretch-activated cation channel in vertebrate cells

The mechanosensitive cation channel (MscCa) transduces membrane stretch into cation (Na+, K+, Ca2+ and Mg2+) flux across the cell membrane, and is implicated in cell-volume regulation(1), cell locomotion(2), muscle dystrophy(3) and cardiac arrhythmias(4). However, the membrane protein(s) that form the MscCa in vertebrates remain unknown. Here, we use an identification strategy that is based on detergent solubilization of frog oocyte membrane proteins, followed by liposome reconstitution and evaluation by patch-clamp(5). The oocyte was chosen because it expresses the prototypical MscCa (greater than or equal to 10(7) MscCa/oocyte)(6) that is preserved in cytoskeleton-deficient membrane vesicles(7). We identified a membrane-protein fraction that reconstituted high MscCa activity and showed an abundance of a protein that had a relative molecular mass of 80,000 (M-r 80K). This protein was identified, by immunological techniques, as the canonical transient receptor potential channel 1 (TRPC1)(8-10). Heterologous expression of the human TRPC1 resulted in a > 1,000% increase in MscCa patch density, whereas injection of a TRPC1-specific antisense RNA abolished endogenous MscCa activity. Transfection of human TRPC1 into CHO-K1 cells also significantly increased MscCa expression. These observations indicate that TRPC1 is a component of the vertebrate MscCa, which is gated by tension developed in the lipid bilayer, as is the case in various prokaryotic mechanosensitive (Ms) channels(11)