Dr. John O'Brien
Clinical and Research Interests:
- Molecular mechanisms of gap junction regulation in the vertebrate retina and CNS.
Signal processing in the central nervous system entails a wide range of synaptic interactions. Excitatory and inhibitory chemical synapses carry the bulk of the information flow, however gap junctions also play an integral role in neuronal circuitry. These aqueous intercellular channels provide a pathway for direct transfer of some cytoplasmic constituents between coupled cells. Ionic flow through the gap junctions permits rapid, direct transmission of electrical signals so that the gap junction functions as a synapse. “Electrical synapses” in the retina play important roles in the establishment of receptive field size, noise reduction, synchronizing neuronal firing, and in the transmission of rod signals to the ganglion cells in mammals. Modulation of gap-junctional coupling during light- and dark-adaptation influences the sensitivity and resolution of the retina, and may redirect signal flow in the rod pathway. My lab employs molecular and cell biological methods to study gap junctions in the retina. Our work in this area led to the discovery of two new gap junction proteins, connexin 35 and connexin 34.7, expressed in the retina (Figure 1) and brain. These connexins defined a new subgroup of the gene family called the ? connexins (Figure 2), but now best thought of as the “atypical connexins.” Recently, we have shown that Cx35, and its mammalian homologue Cx36, form gap junctions in many retinal neurons including AII amacrine cells, cone photoreceptors, and rod bipolar cells. A major current focus of the lab is on studying the regulation of intercellular channels formed by the ? connexins. We have found that several protein kinase pathways regulate gap junction function, and that regulation involves several interdependent sites within the connexin protein.
- Patterns of gene expression in the retina.
- Recent research findings
Cx36 in AII amacrine cell gap junctions
Cx35 in Mauthner cell mixed synapses
Gap junctions in photoreceptors
Molecular mechanisms of retinal gap junction regulation
- Rod pathways in the mammalian retina use connexin 36. J Comp Neurol 2001 Jul 30;436(3):336-50. Mills SL, O'Brien JJ, Li W, O'Brien J, Massey SC Department of Ophthalmology and Visual Science, University of Texas at Houston - Health Science Center, Houston, Texas 77030, USA. email@example.com. Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.
- Functional characteristics of skate connexin35, a member of the g subfamily of connexins expressed in the vertebrate retina. Eur J Neurosci 1999 Jun;11(6):1883-90. White TW, Deans MR, O'Brien J, Al-Ubaidi MR, Goodenough DA, Ripps H, Bruzzone R Department of Cell Biology, Harvard Medical School, Boston 02115, USA. firstname.lastname@example.org. Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the g subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage-gated at transjunctional potentials >/= 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the a or b subfamilies. Voltage-activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 mm quinine resulted in a 1.8-fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage-activated currents of Cx46 hemichannels were also enhanced 1. 6-fold following quinine treatment, whereas Cx43-injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.
- Cloning and expression of two related connexins from the perch retina define a distinct subgroup of the connexin family. J Neurosci 1998 Oct 1;18(19):7625-37. O'Brien J, Bruzzone R, White TW, Al-Ubaidi MR, Ripps H Lions of Illinois Eye Research Institute, Department of Ophthalmology and Visual Sciences, University of Illinois College of Medicine, Chicago, Illinois 60612, USA. We have cloned cDNAs for two closely related connexins (Cx), Cx35 and Cx34.7, from a perch retinal cDNA library. Sequencing of PCR products from genomic DNA revealed that both connexins have an intron 71 bp after the translation initiation site; in Cx35, the intron is 900 bp in length, whereas in Cx34.7 it is approximately 20 kb. Southern blots of genomic DNA suggest that the two connexins represent independent single copy genes. In Northern blots, Cx35 and Cx34.7 transcripts were detected in retina and brain; Cx34.7 also showed a weak signal in smooth muscle (gut) RNA. Antibodies against Cx35 labeled a 30 kDa band on a Western blot of retinal membranes, and in histological sections, the pattern of antibody recognition was consistent with labeling of bipolar cells and unidentified processes in the inner plexiform and nerve fiber layers. When expressed in Xenopus oocytes, Cx35 and Cx34.7 formed homotypic gap junctions, but the junctional conductance between paired oocytes expressing Cx35 was 10-fold greater than that recorded for gap junctional channels formed by Cx34.7. The homotypic gap-junctional channels were closed in a voltage-dependent manner but with relatively weak voltage sensitivity. Heterotypic gap junctions formed by Cx35 and Cx34.7 displayed junctional conductances similar to those of Cx34.7 homotypic pairs and showed a slightly asymmetric current-voltage relationship; the side expressing Cx35 exhibited a higher sensitivity to transjunctional potentials. An analysis of the sequence and gene structure of the connexin family revealed that perch Cx35 and Cx34.7, skate Cx35, and mouse Cx36 constitute a novel g subgroup.
- Isolation and characterization of a skate retinal GABA transporter cDNA. Mol Vis 1998 Mar 6;4:6. Qian X, Malchow RP, O'Brien J, Al-Ubaidi MR Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA. PURPOSE: The inhibitory neurotransmitter g-aminobutyric acid (GABA) is believed to play a crucial role in the processing of information within the vertebrate retina. Extracellular concentrations of GABA are thought to be tightly regulated by carrier-mediated transport proteins in neurons and glial cells. The purpose of this work was to isolate the gene that encodes one of these transport proteins in the skate retina. METHODS: cDNA clones were isolated from a skate retinal cDNA library using a mouse retinal GABA transporter (GAT1) cDNA as a probe. The PCR technique was used to fill sequence gaps, and 5' and 3' RACE were employed to amplify the 5' and 3' untranslated regions. The amplified fragments were subcloned into a T-vector. Blots containing RNA from 10 different tissues were probed to determine the size of the transcript and the tissue distribution. RESULTS: Sequence analysis revealed that the skate retinal GABA transporter cDNA shared 72% identity with the mouse GABA transporter-1 at the DNA level and 80% identity at the amino acid level. Multiple sequence alignments showed that our sequence is closest to the Torpedo GABA transporter-1. Two transcripts, 4.5 and 7 kb, were detected in retina and possibly brain by RNA blot analysis. Fourteen introns were detected in the skate GABA transporter gene. CONCLUSIONS: We successfully isolated a full length GABA transporter cDNA from the retina of the skate. The size of the full length sequence of the skate retinal GABA transporter is in agreement with the size of the smaller transcript detected on RNA blots. The larger transcript observed on the RNA blot may be the result of either alternative splicing or utilization of a downstream poly A signal.
- Molecular cloning of a rod opsin cDNA from the skate retina. Gene 1997 Jul 9;193(2):141-50. O'Brien J, Ripps H, Al-Ubaidi MR Department of Ophthalmology and Visual Sciences, Univeristy of Illinois College of Medicine, Chicago 60612, USA. Skates (Raja erinacea and R. ocellata) are among the few animals that have an exclusively rod retina. However, skate rods are unusual in that they are capable of adapting to extremely high levels of illumination that initially saturate the rod photocurrent. This adaptive process restores the ability of the visual cells to respond to incremental photic stimuli and enables them to function under ambient conditions that are subserved by the cone mechanism in mixed (rod/cone) retinae. As a first step towards exploring the molecular basis of visual adaptation in the skate retina, we have cloned and analyzed the opsin cDNA from a skate retina library. The cDNA codes for a protein 354 amino acids (aa) long and 39.7 kDa predicted molecular mass, and labels a single abundant transcript of 1.7 kb in retinal RNA. Amino acid alignments and a parsimony analysis of nucleotide alignments show the skate opsin to be homologous to other rod opsins. An analysis of the aa sequence reveals a high degree of conservation of those residues thought to be important for most aspects of rhodopsin function. However, a few critical aa replacements may indicate alterations in the interactions of skate rhodopsin with other proteins in the phototransduction cascade. In particular, replacements of Glu150 with serine and Cys323 with leucine are in cytoplasmic domains thought to interact with transducin and rhodopsin kinase. The latter change eliminates one of the conserved acylation sites in the carboxyl terminal tail. These substitutions increase the similarity of the cytoplasmic domains of skate opsin to those of blue-sensitive visual pigments.
- Effects of Ca2+ on oxidative phosphorylation in mitochondria from the thermogenic organ of marlin. J Exp Biol 1996 Dec;199 (12):2679-87. O'Brien J, Block BA Department of Organismal Biology and Anatomy, University of Chicago, IL 60637, USA. Mitochondria from the muscle-derived thermogenic (heater) organ and oxidative red muscle of the blue marlin (Makaira nigricans) were studied in order to evaluate aspects of the mechanism of thermogenesis in heater tissue. We investigated whether short-term Ca2+-induced uncoupling of mitochondria contributes to the thermogenic cycle of the heater organ by enhancing the respiration rate. Specific electrodes were used to obtain simultaneous measurements of oxygen consumption and Ca2+ fluxes on isolated mitochondria, and the effects of various concentrations of Ca2+ on respiration rates and the ADP phosphorylated/atomic oxygen consumed (P/O) ratio were examined. Addition of Ca2+ in excess of 10 µmol l-1 to respiring heater organ or red muscle mitochondria partially inhibited state 3 respiration and reduced the P/O ratio, indicating that the mitochondria were partially uncoupled. These effects were blocked by 2 µmol l-1 Ruthenium Red. In heater organ mitochondria, state 3 respiration rate and the P/O ratio were not significantly reduced by 1 µmol l-1 free Ca2+, a concentration likely to be near the maximum achieved in a stimulated cell. This indicates that transient increases in cytosolic Ca2+ concentration may not significantly reduce the P/O ratio of heater organ mitochondria. The activity of 2-oxoglutarate dehydrogenase in heater organ mitochondria was stimulated by approximately 15% by Ca2+ concentrations between 0.2 and 1 µmol l-1. These results suggest that heater organ mitochondria are able to maintain a normal P/O ratio and should maintain ATP output during transient increases in Ca2+ concentration, supporting a model in which an ATP-consuming process drives thermogenesis. Activation of mitochondrial dehydrogenases by low levels of Ca2+ may also enhance respiration and contribute to thermogenesis.
- Connexin35: a gap-junctional protein expressed preferentially in the skate retina. Mol Biol Cell 1996 Feb;7(2):233-43. O'Brien J, Al-Ubaidi MR, Ripps H Department of Ophthalmology and Visual Sciences, University of Illinois College of Medicine, Chicago 60612, USA. We have used low stringency hybridization to clone a novel connexin from a skate retinal cDNA library. A rat connexin 32 clone was used to isolate a single partial clone that was subsequently used to isolate seven more overlapping clones of the same cDNA. Two clones containing the entire open reading frame have a consensus sequence of 1456 bp and predict a protein of 302 amino acids length and molecular mass of 35,044 daltons, referred to as connexin35 or Cx35. Southern blot analysis suggests that the cloned sequence lies in a single gene with one intron. Polymerase chain reaction amplification from genomic DNA and partial sequencing of this intron showed that it was approximately 950 bp in length, and located within the coding region 71 bp after the translation start site. Hydropathy analysis of the predicted protein and alignments with previously cloned connexins indicate that Cx35 has a long cytoplasmic loop and a relatively short carboxyl terminal tail. Multiple sequence alignments show that Cx35 has similarities to both a and b groups of connexins and suggests that its origins may be near the divergence point for the two groups. Consensus sequences consistent with sites for phosphorylation by protein kinase C and by cAMP - or cGMP -dependent protein kinase were identified. Two transcripts were detected in Northern blot analysis: a 1.95-kb primary transcript and a 4.6-kb minor transcript. In RNA samples from 10 tissues, transcripts were detected only in the retina.
- Physiological differences between the a and b ryanodine receptors of fish skeletal muscle. Biophys J 1995 Feb;68(2):471-82. O'Brien J, Valdivia HH, Block BA Department of Organismal Biology and Anatomy, University of Chicago, Illinois 60637. Two isoforms of the sarcoplasmic reticulum Ca2+ release channel (ryanodine receptor or RYR) are expressed together in the skeletal muscles of most vertebrates. We have studied physiological properties of the two isoforms (a and b) by comparing SR preparations from specialized fish muscles that express the a isoform alone to preparations from muscles containing both a and b. Regulation of channel activity was assessed through [3H]ryanodine binding and reconstitution into planar lipid bilayers. Distinct differences were observed in the calcium-activation and -inactivation properties of the two isoforms. The fish a isoform, expressed alone in extraocular muscles, closely resembled the rabbit skeletal muscle RYR. Maximum [3H]ryanodine binding and maximum open probability (Po) of the a RYR were achieved from 1 to 10 mm free Ca2+. Millimolar Ca2+ reduced [3H]ryanodine binding and Po close to zero. The b isoform more closely resembled the fish cardiac RYR in Ca2+ activation of [3H]ryanodine binding. The most prominent difference of the b and cardiac isoforms from the a isoform was the lack of inactivation of [3H]ryanodine binding and Po by millimolar free Ca2+. Differences in activation of [3H]ryanodine binding by adenine nucleotides and inhibition by Mg2+ suggest that the b and cardiac RYRs are not identical, however. [3H]ryanodine binding by the a RYR was selectively inhibited by 100 mm tetracaine, whereas cardiac and b RYRs were much less affected. Tetracaine can thus be used to separate the properties of the a and b RYRs in preparations in which both are present. The distinct physiological properties of the a and b RYRs that are present together in most vertebrate muscles support models of EC coupling incorporating both directly coupled and Ca2+-coupled channels within a single triad junction.
- The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor. Biophys J 1993 Dec;65(6):2418-27. O'Brien J, Meissner G, Block BA Department of Organismal Biology and Anatomy, University of Chicago, Illinois 60637. The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (a and b) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the a isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the a (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the a RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the a isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the a RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the a isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.
Location & Contact
6431 Fannin, MSB 7.024
Houston, Texas 77030
- Dr. John O'Brien
- Department of Ophthalmology
- University of Texas-Houston Medical School
6431 Fannin, MSB 7.024 Houston, TX 77030
- phone: (713) 500-5983
fax: (713) 500-0682