Nefeli Slavi

Thyronamine induces TRPM8 channel activation in human conjunctival epithelial cells

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca(2+) ([Ca(2+)]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20μM), a TRPM8 agonist, increased Ca(2+) influx as well as whole-cell currents whereas BCTC (10μM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23°C or T1AM (1μM) induced Ca(2+) transients that were blocked by this antagonist. TRPM8 activation by both 1µM T1AM and 20μM icilin prevented capsaicin (CAP) (20μM) from inducing increases in Ca(2+) influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20μM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20μM capsazepine this rise was completely blocked. Similarly, T1AM (1μM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Background: Delivery of the anti-oxidant, glutathione, to the lens nucleus is vital for its transparency. Results: Gap junction channels, which couple central fiber cells to outer lens cells, are permeable to glutathione. Conclusion: Glutathione diffuses from cortical cells to the nucleus via gap junctions. Significance: The age-dependent decline of glutathione levels, an initiating factor in cataract formation, might be due to alterations in coupling. Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (PNa/PCl ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (PNa/PGSH ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome.

Background: Aberrantly functioning Cx26 hemichannels are a common feature of GJB2 mutations causing syndromic deafness. Results: pH and Zn2+, factors that inhibit hemichannels, are less effective in the A40V KID syndrome mutant. Conclusion: Impaired inhibition by pH and Zn2+ can contribute to the pathogenesis of KID syndrome. Significance: Data provide new insights into Cx26 hemichannel function and possible contributions to tissue function. Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca2+. This study presents two new possible contributing factors, pH and Zn2+. Plasma pH levels and micromolar concentrations of Zn2+ inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H+ flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn2+. N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn2+ responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn2+. From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.

Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels.

Mutations in connexin 26 hemichannels are the most common cause of congenital sensorineural hearing loss. Sanchez et al. investigate two mutations with disparate effects, N14K and N14Y, and find that the open state is stabilized in N14K channels.

Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens

Purpose Many proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements. Methods Human lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)–electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells. Results Cleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels. Conclusions Accumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.

Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy

Significance Vascular regeneration during retinal ischemia is critical for curtailing hypoxia-driven aberrant neovascularization and neuronal damage. Apoptotic cell death of astrocytes is a key initiating factor for improper vascular growth. Here we report that astrocytic connexin (Cx43) gap junction (GJ) channels are major contributors to astrocyte degeneration and vascular remodeling that follow tissue ischemia. Astrocyte apoptosis is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ), which in turn leads to an increase in GJ coupling and amplification of injury. Deletion of Cx43 or inhibition of its phosphorylation by CK1δ rescues astrocytes and leads to restoration of a functional vasculature in the retina, while reducing neovascularization and improving neuroretinal function, thereby providing viable options for the treatment of ischemic retinopathies. Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.