Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating

The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel’s selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating

Regulation of the Non-image Forming Photopigment Melanopsin by Phosphorylation

Melanopsin is an atypical vertebrate visual pigment expressed in a subset of retinal ganglion cells in the mammalian retina. Melanopsin-based phototransduction is involved in non-image forming light responses including circadian photo-entrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep in vertebrates. Phosphorylation is the most common post-translational modification of proteins. The addition of a phosphate group to either a serine, threonine or tyrosine can cause a variety of changes in a protein. These include activation or inhibition of protein function, changes in protein structure and changes in the protein's interactions with other molecules. This work explores some of the ways in which melanopsin is regulated by phosphorylation. I have determined that mouse melanopsin is phosphorylated in a light-dependent manner and that this phosphorylation is involved in the deactivation of the signaling response. This phosphorylation was demonstrated in vitro, in heterologous expression systems and in vivo. I have also shown that the G protein coupled receptor kinase 2 (GRK2) is the best candidate kinase for phosphorylation in a heterologous expression system. I characterized the members of the GRK family expressed in melanopsin-expressing retinal ganglion cells to determine the endogenous kinase candidates. I then identified six sites in the cytoplasmic carboxy tail that are important for this phosphorylation by mutational analysis. The importance of these sites was also shown by characterization of the naturally diverse zebrafish melanopsins. Additionally, I demonstrated that protein kinase A (PKA) mediated phosphorylation of melanopsin occurs in both a heterologous expression system and in vivo. These phosphorylations were localized to the intracellular loops of melanopsin by mutational and functional analysis. Phosphorylation of these sites was shown to be involved in inhibiting melanopsin signaling in HEK293 cells. I hypothesize that phosphorylation of melanopsin by PKA is a form of circadian regulation of melanopsin function

Regulation of the Non-image Forming Photopigment Melanopsin by Phosphorylation

Melanopsin is an atypical vertebrate visual pigment expressed in a subset of retinal ganglion cells in the mammalian retina. Melanopsin-based phototransduction is involved in non-image forming light responses including circadian photo-entrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep in vertebrates. Phosphorylation is the most common post-translational modification of proteins. The addition of a phosphate group to either a serine, threonine or tyrosine can cause a variety of changes in a protein. These include activation or inhibition of protein function, changes in protein structure and changes in the protein's interactions with other molecules. This work explores some of the ways in which melanopsin is regulated by phosphorylation. I have determined that mouse melanopsin is phosphorylated in a light-dependent manner and that this phosphorylation is involved in the deactivation of the signaling response. This phosphorylation was demonstrated in vitro, in heterologous expression systems and in vivo. I have also shown that the G protein coupled receptor kinase 2 (GRK2) is the best candidate kinase for phosphorylation in a heterologous expression system. I characterized the members of the GRK family expressed in melanopsin-expressing retinal ganglion cells to determine the endogenous kinase candidates. I then identified six sites in the cytoplasmic carboxy tail that are important for this phosphorylation by mutational analysis. The importance of these sites was also shown by characterization of the naturally diverse zebrafish melanopsins. Additionally, I demonstrated that protein kinase A (PKA) mediated phosphorylation of melanopsin occurs in both a heterologous expression system and in vivo. These phosphorylations were localized to the intracellular loops of melanopsin by mutational and functional analysis. Phosphorylation of these sites was shown to be involved in inhibiting melanopsin signaling in HEK293 cells. I hypothesize that phosphorylation of melanopsin by PKA is a form of circadian regulation of melanopsin function

3-dimenstional model of mouse melanopsin highlighting the predicted PKA phosphorylation sites found in intracellular loops.

<p>The sites in the C-tail are not depicted. Model constructed by LOMETS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#pone.0045387-Wu1" target="_blank">[30]</a> modeling server, and sites identified by Group-based Prediction System (GPS 2.0) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#pone.0045387-Xue2" target="_blank">[20]</a>.</p

Effect of 8-Br cAMP on mutant and wild type melanopsin calcium signaling.

<p>A) Graph of the peak calcium response of melanopsin and melanopsin mutants as measured by fluorescent calcium assay. In black is the average maximum fluorescence for untreated cells, while 200 µM 8-Br cAMP treated response is shown in grey. Error bars represent standard deviation. B) The average percent 8-Br cAMP induced inhibition in signaling is shown.</p

List of primers used to create single and double PKA mutants.

<p>List of primers used to create single and double PKA mutants.</p

List of PKA phosphorylation sites predicted by GPS2.0.

<p>List of the predicted PKA phosphoryation sites in mouse melanopsin. The score is a measure of the similarity of a peptide sequence centered on a phosphorylatable residue to a known phosphorylation site for a given kinase family.</p

Effect of 8-Br cAMP on light induced calcium mobilization in melanopsin-transfected cells.

<p>A) Time course of the calcium response of HEK-293 cells in the presence and absence of 8-Br cAMP. HEK-293 cells were transiently transfected with DNA for wild-type melanopsin. Some cells were treated with 200 µM 8-Br cAMP. Melanopsin signaling was monitored by measuring intracellular calcium levels as described in Methods. B) HEK-293 cells transfected with melanopsin were treated with varying concentrations of 8-Br cAMP to show a concentration dependent decrease in melanopsin signaling. The peak response of the time course is plotted. C) Pre-treatment of transfected cells with the specific PKA inhibitor KT5720 removed the effect of 8-Br cAMP treatment also in a concentration dependent manner. Error bars represent standard deviation.</p

Proximity-dependent ligation assay.

<p>Melanopsin-transfected HEK cells were fixed with 4% PFA for 30 min. with or without pretreatment with 200 µM 8-Br cAMP for 30 minutes before fixation. Melanopsin phosphorylation was assayed with the PLA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045387#s4" target="_blank">Materials and Methods</a>. The red fluorescence puncta indicates that the antibody bound to melanopsin’s intracellular C-terminal domain is within 40 nm of the phospho-serine antibody when bound to phosphorylation sites in the intracellular loops. Cells visualized by confocal microscopy. Blue staining indicates DAPI staining of cell nuclei. Images represent Z-stacks of images taken through entire cell.</p

Dopamine agonist induces phosphorylation <i>in vivo</i>.

<p>PLA was performed on mouse retinal section following treatment with the D1 agonist A68930. Retinal sections (16 mm) were taken from dark-adapted wild type C57/B6 mice (panels labeled Dark, or Dark + D1 agonist) or dark-adapted melanopsin knockout mice (opn4 ^LacZ/LacZ) (panel labeled Melanopsin knock out + D1 agonisit). Before fixation retina were treated with the dopamine D1 agonist A68930 (labled +D1 agonist) or left untreated (Dark). Outer nuclear layer (ONL): Inner nuclear layer (INL) and Ganglion cell layer (GCL).</p