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Lens gap junction proteins of the connexin family
Monday, Sep 21, 1992 La Palms/C
X ICER Abstracts 80
4 LENS GAP JUNCTION PROTEINS OF THE CONNEXIN FAMILY Bever. E.C.. RUD. D.M.. ‘Veenstra. R.D. Department of Pediatrics, Washington Univ., St. Louis, MO 63110 USA and ‘Department of Pharmacology, SUNY Health Science Center, Syracuse, NY 13210 USA. The lens is metabolically coupled by an extensive network of gap junctions. Gap junctions are formed by members of a family of subunit proteins, connexins. Each connexin contains homologous transmembrane and extracellular regions and contains unique cytoplasmic regions which may confer specific physiologic properties. We have been studying the molecular and electrophysiological properties of chick embryo connexins. We demonstrated that connexin43 forms epithelial cell gap junctions. We used PCR amplification and genomic cloning to identify a novel sequence, chick connexin56 (Cx56). The N-terminal region of Cx56 is closely related to rat connexin46 and MP70, which localize to fiber cell junctions. Northern blots of 12 day chick embryo RNA demonstrate that the 6kb Cx56 mRN’A is abundant in lens, but not detectable in other organs. Expression is developmentally regulated with an increase in mRNA levels between 12 and 19 day old embryos. Southern blots show that Cx56 corresponds to a unique single copy gene. We investigated the biophysical properties of different chick connexins (including connexin42, -43, and -45) stably transfected into the noncommunicating cell line N2A. Double whole cell patch clamp recordings demonstrated that these connexins form channels which differ in their transjunctional voltage-dependence and unitary channel conductance. We hope SOOII to study Cx56 channels expressed in the N2A cells, to compare the properties of different lens connexins.
FUNCTIONAL EXPRESSED
Ebihara. Department York, New
PROPERTIES
OF A OOCYTES Steiner. E. of Pharmacology, York
NOVEL
LENS
GAP
JUNCTION
XENOPUS
IN L..
Columbia
University,
Rat connexin46 is a gap junctional cloned from a rat lens cDNA library. cxn46 in solitary Xenopus oocytes development of a large, nonselective,
current.
The
cxn46-induced
New
protein
which was Expression of results in the time and voltage
current
activates
at
potentials positive to -20 mV. On repolarization to 40 mv, a large, inward tail current is observed which decays to zero over a time period of several hundred milliseconds. Removal of external calcium causes a marked increase in the amplitude of the cxn46-induced
current, the left current of
cxn46
shifts along turn-off to
the
the current-voltage voltage axis, and during repolarization.
induce
the
formation
of
channels
was tested in the oocyte pair pairs injected with mRNA for cxn46 oligonucleotides for connexin38 coupled.
In
contrast,
oocyte
relationship slows the The
pairs
ability
cell-to-cell
system. and
were injected
to of
rate Oocyte
antisense
strongly with
antisense alone showed no detectable couolina. The kinetic properties of the gap junctional &ann& could be predicted from the properties of transmembrane current in solitary oocytes using a two gate in series model. These findings suggest that cxn46 is being assembled as gap junctional hemichannels in the nonjunctional membrane of Xenopus oocytes.
RECONSTlTLJTION OF LENS CELL-TO-CELL CHANNELS Klstler. I.. Donaldson. P.. Bond. 1. Department of Cellular 6r Molecular Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand. Cell-to-cell channels (gap junctions) are abundant in the mammalian lens and in the ovine/bovine system contain a connexin related 70kDa polypeptide (MP70) in the outer cortex or its in viva processed 38kDa form deeper in the lens. Structure/function studies using detergent solubilized pore complexes are designed to shed light on the significance of this cleavage. MP70 enriched, preparations were reconstituted into planar lipid bilayers. Two novel channel activities had unitary conductances of 9Op!Zi and 4SpS (150x&i KCl) and were sensitive to the gap junction blocker halothane. The 9OpS channel was asymmetrically voltage dependent consistent with the expected properties of junctional hemichannels. Channel activities of preparations enriched in the 38kDa processed polypeptlde are presently under investigation. In a collaborative project with A. Engel (Basel, Switzerl.) and P. Lampe (St. Paul, USA), processed pore complexes were assembled in vitro into crystalline gap junctions and further into 3-dimensional crystals. Structure analysis and the crystallization of uncleaved pore complexes are aimed at the localization of thi? cleaved peptide segment.
5.26
X ICER Abstracts 80
4 LENS GAP JUNCTION PROTEINS OF THE CONNEXIN FAMILY Bever. E.C.. RUD. D.M.. ‘Veenstra. R.D. Department of Pediatrics, Washington Univ., St. Louis, MO 63110 USA and ‘Department of Pharmacology, SUNY Health Science Center, Syracuse, NY 13210 USA. The lens is metabolically coupled by an extensive network of gap junctions. Gap junctions are formed by members of a family of subunit proteins, connexins. Each connexin contains homologous transmembrane and extracellular regions and contains unique cytoplasmic regions which may confer specific physiologic properties. We have been studying the molecular and electrophysiological properties of chick embryo connexins. We demonstrated that connexin43 forms epithelial cell gap junctions. We used PCR amplification and genomic cloning to identify a novel sequence, chick connexin56 (Cx56). The N-terminal region of Cx56 is closely related to rat connexin46 and MP70, which localize to fiber cell junctions. Northern blots of 12 day chick embryo RNA demonstrate that the 6kb Cx56 mRN’A is abundant in lens, but not detectable in other organs. Expression is developmentally regulated with an increase in mRNA levels between 12 and 19 day old embryos. Southern blots show that Cx56 corresponds to a unique single copy gene. We investigated the biophysical properties of different chick connexins (including connexin42, -43, and -45) stably transfected into the noncommunicating cell line N2A. Double whole cell patch clamp recordings demonstrated that these connexins form channels which differ in their transjunctional voltage-dependence and unitary channel conductance. We hope SOOII to study Cx56 channels expressed in the N2A cells, to compare the properties of different lens connexins.
FUNCTIONAL EXPRESSED
Ebihara. Department York, New
PROPERTIES
OF A OOCYTES Steiner. E. of Pharmacology, York
NOVEL
LENS
GAP
JUNCTION
XENOPUS
IN L..
Columbia
University,
Rat connexin46 is a gap junctional cloned from a rat lens cDNA library. cxn46 in solitary Xenopus oocytes development of a large, nonselective,
current.
The
cxn46-induced
New
protein
which was Expression of results in the time and voltage
current
activates
at
potentials positive to -20 mV. On repolarization to 40 mv, a large, inward tail current is observed which decays to zero over a time period of several hundred milliseconds. Removal of external calcium causes a marked increase in the amplitude of the cxn46-induced
current, the left current of
cxn46
shifts along turn-off to
the
the current-voltage voltage axis, and during repolarization.
induce
the
formation
of
channels
was tested in the oocyte pair pairs injected with mRNA for cxn46 oligonucleotides for connexin38 coupled.
In
contrast,
oocyte
relationship slows the The
pairs
ability
cell-to-cell
system. and
were injected
to of
rate Oocyte
antisense
strongly with
antisense alone showed no detectable couolina. The kinetic properties of the gap junctional &ann& could be predicted from the properties of transmembrane current in solitary oocytes using a two gate in series model. These findings suggest that cxn46 is being assembled as gap junctional hemichannels in the nonjunctional membrane of Xenopus oocytes.
RECONSTlTLJTION OF LENS CELL-TO-CELL CHANNELS Klstler. I.. Donaldson. P.. Bond. 1. Department of Cellular 6r Molecular Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand. Cell-to-cell channels (gap junctions) are abundant in the mammalian lens and in the ovine/bovine system contain a connexin related 70kDa polypeptide (MP70) in the outer cortex or its in viva processed 38kDa form deeper in the lens. Structure/function studies using detergent solubilized pore complexes are designed to shed light on the significance of this cleavage. MP70 enriched, preparations were reconstituted into planar lipid bilayers. Two novel channel activities had unitary conductances of 9Op!Zi and 4SpS (150x&i KCl) and were sensitive to the gap junction blocker halothane. The 9OpS channel was asymmetrically voltage dependent consistent with the expected properties of junctional hemichannels. Channel activities of preparations enriched in the 38kDa processed polypeptlde are presently under investigation. In a collaborative project with A. Engel (Basel, Switzerl.) and P. Lampe (St. Paul, USA), processed pore complexes were assembled in vitro into crystalline gap junctions and further into 3-dimensional crystals. Structure analysis and the crystallization of uncleaved pore complexes are aimed at the localization of thi? cleaved peptide segment.
5.26