scholarly journals Voltage-clamp recordings of light responses from wild-type and mutant mouse cone photoreceptors

2019 ◽  
Vol 151 (11) ◽  
pp. 1287-1299 ◽  
Author(s):  
Norianne T. Ingram ◽  
Alapakkam P. Sampath ◽  
Gordon L. Fain
Keyword(s):  
Voltage Clamp ◽  
Mouse Retina ◽  
Cone Photoreceptors ◽  
Wild Type ◽  
Light Responses ◽  
Α Subunit ◽  
Background Light ◽  
Connexin 36 ◽  
Mouse Lines

We describe the first extensive study of voltage-clamp current responses of cone photoreceptors in unlabeled, dark-adapted mouse retina using only the position and appearance of cone somata as a guide. Identification was confirmed from morphology after dye filling. Photocurrents recorded from wild-type mouse cones were biphasic with a fast cone component and a slower rod component. The rod component could be eliminated with dim background light and was not present in mouse lines lacking the rod transducin-α subunit (Gnat1−/−) or connexin 36 (Cx36−/−). Cones from Gnat1−/− or Cx36−/− mice had resting membrane potentials between −45 and −55 mV, peak photocurrents of 20–25 picoamps (pA) at a membrane potential Vm = −50 mV, sensitivities 60–70 times smaller than rods, and a total membrane capacitance two to four times greater than rods. The rate of activation (amplification constant) was largely independent of the brightness of the flash and was 1–2 s−2, less than half that of rods. The role of Ca2+-dependent transduction modulation was investigated by recording from cones in mice lacking rod transducin (Gnat1), recoverin, and/or the guanylyl-cyclase–activating proteins (GCAPs). In confirmation of previous results, responses of Gnat1−/−;Gcaps−/− cones and triple-mutant Gnat1−/−;Gcaps−/−;Rv−/− cones recovered more slowly both to light flashes and steps and were more sensitive than cones expressing the GCAPs. Cones from all four mouse lines showed significant recovery and escaped saturation even in bright background light. This recovery occurred too rapidly to be caused by pigment bleaching or metaII decay and appears to reflect some modulation of response inactivation in addition to those produced by recoverin and the GCAPs. Our experiments now make possible a more detailed understanding of the cellular physiology of mammalian cone photoreceptors and the role of conductances in the inner and outer segment in producing cone light responses.

Residual photosensitivity in mice lacking both rod opsin and cone photoreceptor cyclic nucleotide gated channel 3 α subunit

2004 ◽  
Vol 21 (5) ◽  
pp. 675-683 ◽  
Author(s):  
ALUN R. BARNARD ◽  
JOANNE M. APPLEFORD ◽  
SUMATHI SEKARAN ◽  
KRISHNA CHINTHAPALLI ◽  
AARON JENKINS ◽  
...  
Keyword(s):  
Ganglion Cells ◽  
Light Exposure ◽  
Early Gene ◽  
Mouse Retina ◽  
Cone Photoreceptors ◽  
Light Responses ◽  
Α Subunit ◽  
Light Reflex ◽  
Pupil Light Reflex ◽  
Retinal Photoreceptors

The mammalian retina contains three classes of photoreceptor. In addition to the rods and cones, a subset of retinal ganglion cells that express the putative sensory photopigment melanopsin are intrinsically photosensitive. Functional and anatomical studies suggest that these inner retinal photoreceptors provide light information for a number of non-image-forming light responses including photoentrainment of the circadian clock and the pupil light reflex. Here, we employ a newly developed mouse model bearing lesions of both rod and cone phototransduction cascades (Rho−/−Cnga3−/−) to further examine the function of these non-rod non-cone photoreceptors. Calcium imaging confirms the presence of inner retinal photoreceptors inRho−/−Cnga3−/−mice. Moreover, these animals retain a pupil light reflex, photoentrainment, and light induction of the immediate early genec-fosin the suprachiasmatic nuclei, consistent with previous findings that pupillary and circadian responses can employ inner retinal photoreceptors.Rho−/−Cnga3−/−mice also show a light-dependent increase in the number of FOS-positive cells in both the ganglion cell and (particularly) inner nuclear layers of the retina. The average number of cells affected is several times greater than the number of melanopsin-positive cells in the mouse retina, suggesting functional intercellular connections from these inner retinal photoreceptors within the retina. Finally, however, while we show that wild types exhibit an increase in heart rate upon light exposure, this response is absent inRho−/−Cnga3−/−mice. Thus, it seems that non-rod non-cone photoreceptors can drive many, but not all, non-image-forming light responses.

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

2004 ◽  
Vol 287 (4) ◽  
pp. E739-E743 ◽  
Author(s):  
Burton F. Holmes ◽  
David B. Lang ◽  
Morris J. Birnbaum ◽  
James Mu ◽  
G. Lynis Dohm
Keyword(s):  
Skeletal Muscle ◽  
Dominant Negative ◽  
Treadmill Running ◽  
Wild Type ◽  
Α Subunit ◽  
Ampk Activity ◽  
Denervated Muscles ◽  
Amp Activated Protein Kinase ◽  
Response To Exercise

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK α-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

scholarly journals Subunit Topology of Two 20S Proteasomes from Haloferax volcanii

2003 ◽  
Vol 185 (1) ◽  
pp. 165-174 ◽  
Author(s):  
Steven J. Kaczowka ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Gel Electrophoresis ◽  
Structural Information ◽  
Haloferax Volcanii ◽  
20S Proteasome ◽  
Wild Type ◽  
Epitope Tag ◽  
Α Subunit ◽  
Native Gel ◽  
Recombinant Technology

ABSTRACT Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (α1, α2, and β) which are classified in the 20S proteasome superfamily. The α1 and β proteins alone form active 20S proteasomes; the role of α2, however, is not clear. To address this, α2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The α2 protein copurified with α1 and β in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described α1-β proteasome. Supplementing buffers with 10 mM CaCl2 stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the α1-β and α1-α2-β proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the α1-β proteasome was a symmetrical cylinder with α1 rings on each end. In contrast, the α1-α2-β proteasome appeared to be asymmetrical with homo-oligomeric α1 and α2 rings positioned on separate ends. Inter-α-subunit contacts were only detected when the ratio of α1 to α2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of α proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.

Contribution of Ca and Ca-activated Cl channels to regenerative depolarization and membrane bistability of cone photoreceptors

1992 ◽  
Vol 68 (3) ◽  
pp. 745-755 ◽  
Author(s):  
S. Barnes ◽  
M. C. Deschenes
Keyword(s):  
Voltage Clamp ◽  
Current Injection ◽  
Resting Potential ◽  
Negative Slope ◽  
Equilibrium Potential ◽  
Ca Channels ◽  
Cone Photoreceptors ◽  
Membrane Hyperpolarization ◽  
Cl Channels

1. Cone photoreceptors in several vertebrate species generate Ca-dependent regenerative depolarizations (e.g., Ca spikes lasting up to 2 s) in response to current injection or surround illumination and may remain in a state of prolonged depolarization (e.g., a permanent plateau near 0 mV) after these stimuli. This paper, while confirming the role of Ca channels in the regenerative depolarization, demonstrates that Ca-activated Cl channels either enhance or hinder prolonged depolarization, depending on the value of the chloride equilibrium potential (ECl). 2. Current- and voltage-clamp recordings obtained with the whole-cell patch-clamp technique were compared in 158 isolated tiger salamander cones to determine the contribution of specific ion channel types to the two forms of depolarizing response. Cones dialyzed with CsCl or KCl intracellular solution (such that ECl = 0 mV) that had sustained negative slope regions in their current-voltage (I-V) relations recorded under voltage clamp, were, under current clamp, bistable with respect to their resting potential. Injection of approximately 20-pA steps of depolarizing current resulted in transitions from the negative stable membrane potential (near -50 mV) to a long lasting plateau around 0 mV. Injection of 200–300 pA of hyperpolarizing current could then force a return to the negative stable resting potential, although once repolarization occurred, current injection had to be reduced or terminated to prevent damaging hyperpolarization of the cell. 3. The inward currents accounting for the negative slope region of the I-V relation were carried in Ca and Ca-activated Cl channels. Specific block of Ca-activated Cl current (ICl(Ca)) by 100 microM niflumic acid (NFA) eliminated the prolonged depolarization, even though the negative slope conductance region in the I-V persisted and the cone could still produce the briefer Ca-dependent regenerative depolarizations. Application of 100 microM Cd2+ blocked both forms of depolarization. 4. Substitution of Ba2+, which among other actions did not activate ICl(Ca), usually supported regenerative depolarizations of shortened duration, demonstrating the role of Ca channels in the initial phase of these responses. 5. A difference was observed in the regenerative depolarization when ECl was shifted away from 0 mV, where it had been in the experiments described above. With ECl set to -40 or -60 mV by reduction of [Cl-] in the pipette, steady-state membrane bistability was eliminated and prolonged depolarization did not occur. Under these conditions, application of the Cl channel blocker NFA showed that ICl(Ca) contributes to membrane hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of an amiloride binding region in the α-subunit of ENaC

2003 ◽  
Vol 285 (6) ◽  
pp. F1279-F1290 ◽  
Author(s):  
Ollie Kelly ◽  
Chaomei Lin ◽  
Mohan Ramkumar ◽  
Nina C. Saxena ◽  
Thomas R. Kleyman ◽  
...  
Keyword(s):  
Single Channel ◽  
Point Mutations ◽  
Binding Affinities ◽  
Extracellular Loop ◽  
Wild Type ◽  
Α Subunit ◽  
Open Time ◽  
The Mean ◽  
Α Subunits

One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the α-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278–283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type α-subunits or α-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities ( Po) of wild-type channels were distributed into two populations: one with a high Po and one with a low Po. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high- Po) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type α-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating.

scholarly journals Role of Two G-Protein Alpha Subunits, TgaA and TgaB, in the Antagonism of Plant Pathogens by Trichoderma virens

2004 ◽  
Vol 70 (1) ◽  
pp. 542-549 ◽  
Author(s):  
Prasun K. Mukherjee ◽  
Jagannathan Latha ◽  
Ruthi Hadar ◽  
Benjamin A. Horwitz
Keyword(s):  
G Protein ◽  
Plant Pathogens ◽  
Sclerotium Rolfsii ◽  
Trichoderma Virens ◽  
Loss Of Function ◽  
Wild Type ◽  
Protein Subunit ◽  
Α Subunit ◽  
Germ Tubes

ABSTRACT G-protein α subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein α subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Gαi class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of Rhizoctonia solani and Sclerotium rolfsii in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The tgaA mutants germinated by straight unbranched germ tubes, while tgaB mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both tgaA and tgaB mutants and the wild type overgrew, coiled, and lysed the mycelia of R. solani, but tgaA mutants had reduced ability to colonize S. rolfsii colonies. In the soil plate assay, both mutants parasitized the sclerotia of R. solani, but tgaA mutants were unable to parasitize the sclerotia of S. rolfsii. Thus, tgaA is involved in antagonism against S. rolfsii, but neither G protein subunit is involved in antagonism against R. solani. T. virens, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.

scholarly journals Cardiac Gsα overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway

1998 ◽  
Vol 95 (16) ◽  
pp. 9669-9674 ◽  
Author(s):  
Alan S. Lader ◽  
Yong-Fu Xiao ◽  
Yoshihiro Ishikawa ◽  
Yanning Cui ◽  
Dorothy E. Vatner ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Transgenic Mice ◽  
Protein Kinase A ◽  
Adenylyl Cyclase ◽  
Cardiac Myocytes ◽  
Receptor Activation ◽  
Wild Type ◽  
Α Subunit ◽  
Kinase A

The α subunit of the stimulatory heterotrimeric G protein (Gsα) is critical for the β-adrenergic receptor activation of the cAMP messenger system. The role of Gsα in regulating cardiac Ca2+ channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac Gsα were used to assess the role of Gsα on the whole-cell Ca2+ currents (ICa). Cardiac myocytes from transgenic mice had a 490% higher peak ICa compared with those of either wild-type controls or Gsα-nonexpressing littermates. The effect of Gsα overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPγS-activated Gsα. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated Gsα. The data indicate that Gsα overexpression is associated with a constitutive enhancement of ICa which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.

Physiological role of the α1- and α2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site

2006 ◽  
Vol 290 (3) ◽  
pp. R524-R528 ◽  
Author(s):  
Iva Dostanic-Larson ◽  
John N. Lorenz ◽  
James W. Van Huysse ◽  
Jon C. Neumann ◽  
Amy E. Moseley ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Binding Site ◽  
Cardiac Glycoside ◽  
Biological Significance ◽  
Physiological Role ◽  
Wild Type ◽  
Α Subunit ◽  
Specific Distribution ◽  
Induced Hypertension

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic α-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the α1- and α2-isoforms. While knockout mice demonstrated the importance of the α1- and α2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant α2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive α1-isoform and a ouabain-resistant α2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.

scholarly journals The regulation of AMP-activated protein kinase by phosphorylation

2000 ◽  
Vol 345 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Silvie C. STEIN ◽  
Angela WOODS ◽  
Neil A. JONES ◽  
Matthew D. DAVISON ◽  
David CARLING
Keyword(s):  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Mutant Complex

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.

scholarly journals S-cone photoreceptors in the primate retina are functionally distinct from L and M cones

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Jacob Baudin ◽  
Juan M Angueyra ◽  
Raunak Sinha ◽  
Fred Rieke
Keyword(s):  
Visual Perception ◽  
Perceptual Sensitivity ◽  
Cone Photoreceptors ◽  
Human Visual Perception ◽  
Light Responses ◽  
Background Light ◽  
Light Levels ◽  
Psychophysical Studies

Daylight vision starts with signals in three classes of cone photoreceptors sensitive to short (S), middle (M), and long (L) wavelengths. Psychophysical studies show that perceptual sensitivity to rapidly varying inputs differs for signals originating in S cones versus L and M cones; notably, S-cone signals appear perceptually delayed relative to L- and M-cone signals. These differences could originate in the cones themselves or in the post-cone circuitry. To determine if the cones could contribute to these and related perceptual phenomena, we compared the light responses of primate S, M, and L cones. We found that S cones generate slower light responses than L and M cones, show much smaller changes in response kinetics as background-light levels increase, and are noisier than L and M cones. It will be important to incorporate these differences into descriptions of how cone signaling shapes human visual perception.

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