Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens

Abstract

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Keywords: TRPV1/4; cell volume; lens; mechanosensors; osmotic stress; zonular tension.

Figure 1

The dual-feedback control system that maintains hydrostatic pressure in the lens. Lens surface pressure (p_set) is maintained by the competing activities of the two arms of a dual-feedback system that regulate ion transporters that control the intracellular osmolarity of cells at the lens surface. Increases in pressure (D_pi), hypoosmotic stress, increased zonular tension, or the TRPV4 agonist GSK1016790A (GSK), all work via TRPV4 to activate a signaling pathway that involves the release of ATP via hemichannel, the subsequent activation of purinergic P2Y receptors, and the Src family of protein tyrosine kinases (SFK) to increase the activity of the Na⁺/K^- ATPase and decrease lens pressure. Decreases in pressure (D_pi), hyperosmotic stress, decreased zonular tension or the TRPV1 agonist capsaicin all work via TRPV1 to activate the extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K/Akt) and the WNK (Kinase with no lysine (K)), and SPAK (Ste20-related proline-alanine-rich kinase)/OSR1(oxidative stress-responsive kinase-1) signaling pathway to directly activate the sodium potassium dichloride cotransporter (NKCC) and to eventually reduce the decrease in the activity of the Na⁺/K^- ATPase to effect an increase in surface pressure. This scheme is based on earlier model [7,18].

Figure 2

Effect of cutting lens zonules on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex of the mouse lens. (A). Overview montage of the mouse lens sectioned axially and labelled with the membrane marker wheatgerm agglutin (WGA, red) and nuclei marker 4,6-Diamidino-2-phenylindole (DAPI, blue), showing the three regions (white boxes) in the outer cortex from where the high-magnification images shown in (B–E) were captured. (B–E) High-power images from the three regions defined in panel (A) obtained from lenses organ-cultured with (B,C) or without (D,E) their zonules attached, which were labelled with DAPI and either TRPV1 (B,D) or TRPV4 (C,E) antibodies (green).

Figure 3

Time course showing the effect of cutting lens zonules on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex mouse lens. Images captured from axial sections obtained from region II of the outer cortex of mouse lenses organ-cultured with or without their zonules attached for 3 min (A,E), 20 min (B,F), 45 min (C,G) or 120 min (D,H). (A–D) TRPV1 labelling (green) in region II is cytoplasmic and unaffected by changes in zonular tension. (E–H) TRPV4 labelling (green) in region II is membranous (left) in lenses cultured in the presence of zonular tension but is removed from the membrane over time in lenses organ-cultured after cutting the zonules. Nuclei are labelled with DAPI (blue).

Figure 4

Effect of pharmacological modulation of zonular tension on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex of the mouse lens. (A). Overview montage of the mouse lens sectioned axially and labelled with the membrane marker WGA (red) and nuclei marker DAPI (blue), showing the three regions (white boxes) in the outer cortex from where the high-magnification images shown in (B–G) were captured. (B–G) High-power images from the three regions defined in panel (A) obtained from lenses organ-cultured in situ to maintain zonular tension in the absence (B,E) or presence tropicamide (C,F) or pilocarpine (D,G) showing TRPV1 (B–D) and TRPV4 (E–G) labelling (green). Nuclei are labelled with DAPI (blue).

Figure 5

Effect of pharmacological modulation of zonular tension on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex of the mouse lenses organ-cultured with and without zonules. Images captured from axial sections obtained from region II of the outer cortex of mouse lenses organ-cultured either in situ (+Zonules) or ex vitro (−Zonules) in the absence (A,D) or presence tropicamide (B,E) or pilocarpine (C,F). (A–C) The addition of tropicamide (B) or pilocarpine (C) in the presence (left panels) or absence (right panels) zonular tension had no effect on the cytoplasmic labelling of TRPV1 (green) in region II of the outer cortex. (D–F) The addition of tropicamide (E) in the presence (left panels) or absence (right panels) of zonular tension had no effect on the subcellular distribution of TRPV4 (green). However, pilocarpine addition to lenses organ-cultured with their zonules intact ((F), left panel) induced a shift of TRPV4 labelling to the cytoplasm. Nuclei are labelled with DAPI (blue).

Figure 6

Effect of osmotic challenge on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex of the mouse lens. (A). Overview montage of the mouse lens sectioned axially and labelled with the membrane marker WGA (red) and nuclei marker DAPI (blue), showing the three regions (white boxes) in the outer cortex from where the high-magnification images shown in (B–G) were captured. (B–G) High-power images from the three regions defined in panel A obtained from lenses organ-cultured in situ to maintain zonular tension in the presence of isotonic (B,E), hypotonic (C,F) or hypertonic (D,G), AAH showing TRPV1 (B–D) and TRPV4 (E–G) labelling (green). Nuclei are labelled with DAPI (blue).

Figure 7

Effect of osmotic challenge on the subcellular localization of TRPV1 and TRPV4 labelling in the outer cortex of the mouse lenses organ-cultured with and without zonules. Images captured from axial sections obtained from region II of the outer cortex of mouse lenses organ-cultured either in situ (+Zonules) or ex vitro (−Zonules) in the presence of isotonic (A,D), hypotonic (B,E) or hypertonic (C,F) AAH. (A–C) TRPV1 was found cytoplasmic in lenses incubated in isotonic (A) and hypotonic (B) AAH, while it was found membranous in lenses treated with hypertonic (C) AAH regardless of the presence (left panels) or absence (right panels) of zonular tension. (D–F) TRPV4 membrane labelling in lenses organ-cultured in situ with (left panels) or without (right panels) zonules attached was unchanged by hypotonic challenge (E); however, a shift of TRPV4 labelling to the cytoplasm was observed following hypertonic exposure (F, left panel), which was not dependent on the presence of zonular tension (F, right panel). Nuclei are labelled with DAPI (blue).

Figure 8

Effects of pharmacological activators of TRPV1 and TRPV4 activity on the subcellular localization of TRPV1 and TRPV4 in the outer cortex of the mouse lens. (A) Overview montage of the mouse lens sectioned axially and labelled with the membrane marker WGA (red) and nuclei marker DAPI (blue), showing the three regions (white boxes) in the outer cortex from where the high-magnification images shown in (B–G) were captured. (B–G) High-power images from the three regions defined in panel (A) obtained from lenses organ-cultured in situ to maintain zonular tension in the absence ((B,E) no modulator) or presence of either the TRPV1-activator capsaicin (C,F) or the TRPV4-activator GSK (D,G) showing TRPV1 (B–D) and TRPV4 (E–G) labelling (green). Nuclei are labelled with DAPI (blue).

Figure 9

Effects of pharmacological activators of TRPV1 and TRPV4 activity on the subcellular localization of TRPV1 and TRPV4 in the outer cortex of the mouse lens organ-cultured with and without zonules. Images captured from axial sections obtained from region II of the outer cortex of mouse lenses organ-cultured either in situ (+Zonules) or ex vitro (−Zonules) in absence ((A,D) no modulator) or presence of either the TRPV1-activator capsaicin (B,E) or the TRPV4-activator GSK (C,F). (A–C) TRPV1 labelling is cytoplasmic in lenses treated with capsaicin (B) but exposure to GSK induces a shift in TRPV1 labelling to the membrane in lenses organ-cultured with and without zonules attached. (D–F) TRPV4 labelling in lenses cultured in situ with their zonules attached remained membranous following exposure to capsaicin (E, left panel), but TRPV4 labelling shifted to the cytoplasm by exposure to GSK (F, left panel), and these changes were unaffected by cutting the zonules (E,F, right panels). Nuclei are labelled with DAPI (blue).