1 Department of Physiology and Biophysics, State University of New York, Stony Brook, 11794-8661; 2 Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461; 3 Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612; 4 Department of Chemical Sciences, University of Catania, 95125 Catania, Italy; and 5 Department of Neuroscience, Institut Pasteur, 75015 Paris, France
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ABSTRACT |
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We have identified a novel gap junction
gene by searching the human genome sequence database that encodes a
protein designated as connexin31.9 (Cx31.9). Cx31.9 was most homologous
to human Cx32.4 and did not cluster with either the purported - or
-connexin subfamilies. Expression of Cx31.9 was detected by RT-PCR
in human mRNA from several tissues including cerebral cortex, heart,
liver, lung, kidney, spleen, and testis. A partial Cx31.9 sequence was also represented in the human Expressed Sequence Tag database. Cx31.9
formed intercellular channels in both paired Xenopus oocytes and transfected neuroblastoma N2A cells that were distinguished by an
apparent low unitary conductance (12-15 pS) and a remarkable insensitivity to transjunctional voltage. In contrast, Cx31.9 channels
were gated by cytoplasmic acidification or exposure to halothane like
other connexins. Cx31.9 was able to form heterotypic channels with the
highly voltage-sensitive Xenopus Cx38 (XenCx38), which
provides an opportunity to study gating in heterotypic channels formed
by hemichannels (connexons) composed of connexins with widely divergent
properties. Thus Cx31.9 is a novel human connexin that forms channels
with unique functional properties.
gene family; expression; channel; gap junction
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INTRODUCTION |
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CONNEXINS ARE THE PROTEIN subunits of intercellular channels that constitute vertebrate gap junctions (3). Although gap junctions are ubiquitously found between virtually all cells in contact, they are composed of different connexin isoforms in different tissues. Because intercellular channels require the participation of two neighboring cells, it is possible for each cell to contribute different connexin proteins, thereby increasing the potential for functional diversity (37). A wealth of in vitro expression studies has demonstrated that each type of connexin channel is endowed with distinctive functional properties, which in turn are derived from the unique primary sequences of the constituent connexins (8, 14). Thus whereas the term intercellular communication is widely used to describe gap junction-dependent intercellular signaling, the precise nature of the signals that are exchanged between cells is dictated by the complement of expressed connexins (6). The in vivo significance of this diversity of communication possibilities remains enigmatic, although studies of genetically engineered mice have demonstrated that connexin isoforms are not simply redundant. Targeted deletion of all connexins to date has always resulted in detectable phenotypic changes, although they were often unexpected (14, 26, 40). Furthermore, targeted replacement of connexin genes with different family members in mice has provided a direct demonstration that intrinsic functional properties of unique connexins are required for normal tissue function (24, 36). Taken together, these observations strongly argue that a complete understanding of gap junctional intercellular communication will require documentation of differences in the functional properties of the various connexin isoforms.
It is difficult to envision the complex range of intercellular communication behaviors that are available within any given tissue unless the full connexin complement of its component cells is unambiguously defined. As a prerequisite to this, the identification of all the connexin genes within a given organism's genome is required. The recent completion of the human genome sequencing project makes it possible to seek out and characterize all of the human gap junction genes. In the case of connexins, this search is greatly facilitated by the documented absence of introns within the open reading frame of the majority of connexin genes with the exception of connexin36 (Cx36) and connexin40.1 (2, 22; unpublished results).
In this study, we have combined in silico cloning with standard
molecular biological techniques to identify a novel connexin sequence
in the human genome database that encodes connexin31.9 (Cx31.9). Cx31.9
was most highly related to connexin32.4 (Cx32.4) and clustered on the
third non-/non-
branch of the connexin family tree. Cx31.9 mRNA
was expressed in a variety of tissues and cell types including cerebral
cortex, liver, spleen, and kidney. Cx31.9 mRNA was also represented in
silico in the human Expressed Sequence Tag (EST) database. Functional
characterization of Cx31.9 demonstrated its channel-forming ability in
two different in vitro expression assays. In both assay systems, Cx31.9
channels showed a remarkable insensitivity to voltage-dependent gating,
although the channels displayed normal sensitivity to chemically
induced gating. Thus Cx31.9 is a bona fide member of the human connexin gene family in that it forms intercellular channels with unique functional properties.
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MATERIALS AND METHODS |
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Molecular cloning of Cx31.9. A BLAST search in the National Center for Biotechnology Information (NCBI, National Library of Medicine, National Institutes of Health) High-Throughput Genome Sequences database yielded a Homo sapiens clone that, contained the intronless open reading frame of a new connexin with a predicted molecular mass of 31.9 kDa. A DNA fragment encompassing the coding sequence of Cx31.9 was amplified from human genomic DNA by PCR using primers corresponding to nucleotides 1-21 (sense: 5'-gatgatGGATCC-atgggggagtgggcgttcctg-3') and 885-865 (antisense: 5'-tagtagTCTAGA-ctagatggccagatctcggcg-3') that contained BamHI (sense) and XbaI (antisense) linkers (capital letters). PCR products were purified using Qiaquick columns (Qiagen, Germantown, MD), digested with BamHI/XbaI (Roche Molecular Biochemicals, Indianapolis, IN), purified by agarose gel electrophoresis, subcloned into the corresponding sites of the pCS2+ expression vector (31), and sequenced on both strands. Pairwise alignment of the translated amino acid sequence of Cx31.9 with 19 other human connexin sequences available in the NCBI database at the time of writing was performed using MegAlign software (DNASTAR, Madison, WI).
RT-PCR analysis of Cx31.9 expression.
Human central nervous system tissues (spinal cord, medulla oblongata,
cerebellum, striatum, hippocampus, and neocortex) were obtained at
autopsy from a 26-yr-old male, frozen in dry ice-cooled isopentane, and
stored at 80°C before total RNA extraction. Additional human total
RNAs were commercially obtained from Ambion (Austin, TX). Total RNA (5 µg) was reverse transcribed with 150 ng of random hexamers and 200 units of RNase H
RT (SuperScript II Invitrogen, Life
Technologies, Carlsbad, CA) in a reaction mixture that contained 20 mM
Tris · HCl (pH 8.4 at 25°C), 50 mM KCl, 5 mM
MgCl2, 0.5 mM dNTP mix, 0.01 M dithiothreitol, and 40 units
of the recombinant RNase inhibitor RNaseOUT (Invitrogen). Samples were
incubated at 25°C for 10 min and then at 42°C for 50 min. The
reaction was terminated by 15 min of incubation at 70°C. After
cooling the samples in ice, 1 µl (2 units) of RNase H
RT was added, and the samples were incubated at 37°C for 20 min. To exclude the significant contribution of genomic DNA to
the final PCR step, incubations with or without RT (RT+ and RT
,
respectively) were run simultaneously for each RNA sample. Only RNA
samples with RT
reactions that did not provide detectable PCR
amplification products were included in the results.
In vitro transcription, oocyte microinjection, and pairing. Cx31.9 in the pCS2+ plasmid was linearized with ApaI, gel purified, and used as template (1 µg of DNA) to produce capped RNA using the mMessage mMachine kit (Ambion). The purity and yield of transcribed cRNA was determined by measuring absorbance at 260/280 nm. Stage V-VI oocytes were collected from adult Xenopus laevis females (Nasco, Fort Atkinson, WI) following previously described protocols (7). Oocytes were isolated and defolliculated by enzymatic digestion and were cultured in modified Barth's (MB) medium at 18°C. For physiological analysis, cells were injected with a total volume of 40 µl of either an antisense oligonucleotide (3 ng/cell) to suppress the endogenous Xenopus connexin38 (XenCx38) or a mixture of the antisense oligonucleotide plus Cx31.9 RNA (40 ng/cell) using a Nanoject II Auto-Nanoliter oocyte injector (Drummond, Broomall, PA). Where specified, oocytes were mock treated with an identical volume of water to determine the ability of Cx31.9 to interact with endogenous XenCx38. After an overnight incubation at 18°C, microinjected oocytes were immersed for a few minutes in hypertonic solution to strip the vitelline envelope (21), transferred to petri dishes containing MB medium, and manually paired with vegetal poles apposed.
Electrical recordings from oocytes.
The functional properties of cell-to-cell channels were assessed by a
dual voltage-clamp procedure that enabled direct quantitation of
junctional conductance (29). Current and voltage
electrodes (1.2- mm diameter, Glass of America, Millville, NJ) were
pulled to a resistance of 1-2 M with a horizontal puller
(Narishige, Tokyo, Japan) and filled with a solution containing 3 M
KCl, 10 mM EGTA, and 10 mM HEPES, pH 7.4. Voltage clamping of oocyte
pairs was performed using two GeneClamp 500 amplifiers (Axon
Instruments, Foster City, CA) controlled by a PC-compatible computer
through a Digidata 1320A interface (Axon Instruments). pCLAMP 8.0 software (Axon Instruments) was used to program stimulus and data
collection paradigms. Current outputs were filtered at 50 Hz and the
sampling interval was 10 ms. For simple measurements of junctional
conductance, both cells of a pair were initially clamped at
40 mV to
ensure zero transjunctional potential (Vj) and
alternating pulses of ±10-20 mV were imposed to one cell. Current
delivered to the cell clamped at
40 mV during the voltage pulse was
equal in magnitude to the junctional current
(Ij) and was divided by the voltage to yield the conductance.
Functional expression of Cx31.9 in transfected N2A cells.
N2A cells were cotransfected with Cx31.9 cDNA and enhanced green
fluorescent protein (EGFP) cDNA in separate vectors using the
Lipofectamine 2000 reagent (GIBCO-BRL, Gaithersburg, MD) following previously described procedures (16). Transiently
transfected cells were dissociated 8-12 h after transfection and
plated at low density on 1-cm glass coverslips. Cell cultures were
maintained in a 37°C incubator in a moist 5% CO2-95%
air environment. Cx31.9-expressing cell pairs were identified by the
fluorescent emission of EGFP (excitation, 488 nm; emission, >520 nm)
using a Nikon Diaphot microscope equipped with a xenon arc lamp.
Junctional conductance was measured between cell pairs by using the
dual whole cell voltage-clamp technique with Axopatch 1C or 1D
patch-clamp amplifiers (Axon Instruments) at room temperature. The
solution bathing the cells contained (in mM) 140 NaCl, 5 KCl, 2 CsCl, 2 CaCl2, 1 MgCl2, 5 HEPES, 5 dextrose, 2 pyruvate, and 1 BaCl2; pH 7.4. Patch electrodes had
resistances of 3-5 M when filled with internal solution that contained (in mM) 130 CsCl, 10 EGTA, 0.5 CaCl2, 3 Mg-ATP, 2 Na2-ATP, and 10 HEPES; pH 7.2. Current recordings were
filtered at 0.2-0.5 kHz and sampled at 1-2 kHz. Data were
acquired using pCLAMP 8.0 software and analyzed with either pCLAMP 8.0 or Origin 6.0 software. Each cell of a pair was initially held
at a common holding potential of 0 mV. To evaluate junctional coupling,
hyperpolarizing pulses to various voltages were applied to one cell to
establish a Vj gradient, and
Ij was measured in the second cell (held at 0 mV). Single-channel currents were investigated in weakly coupled cell pairs by applying
140- or
160-mV pulses to one cell of a pair.
Statistical analysis. Results are shown as means ± SE. Comparisons between two populations of data were made with Student's unpaired t-test; P values of 0.05 or less were considered to be significant.
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RESULTS |
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Sequence analysis and tissue distribution of Cx31.9.
We initially identified Cx31.9 from BLAST software searches of human
DNA sequences in the NCBI High-Throughput Genome Sequence database. A
putative connexin open reading frame with a predicted molecular mass of
31.932 kDa (thus Cx31.9) was found in the chromosome 17 draft sequence
(GenBank accession no. AC018629, AF514298). Comparison of the
full-length Cx31.9 amino acid sequence with 19 other human connexin
sequences showed a threshold of 30-40% overall sequence identity
(Fig. 1A), which confirms its
membership in the connexin family of gap junction proteins. Only
connexin Cx32.4 (GenBank accession no. XM064450) showed notable amino
acid identity with Cx31.9 (76%), and an alignment of these two
proteins is shown in Fig. 1B. Cx31.9 has all of the
predicted features of a typical connexin including four transmembrane
domains, cytoplasmic amino and carboxy termini, and the conserved
cysteines at three positions in each of the two extracellular loops.
One unusual feature of Cx31.9 was the relatively high content of
proline in its amino acid composition (33 of 294 amino acids) including
a continuous sequence of 10 prolines in one region of the carboxy terminus. Alignment of a truncated Cx31.9 sequence, which lacks the
highly divergent carboxy terminus, with other truncated human connexins
revealed that it did not cluster within either the - or
-connexin
subgroups, which suggests that it may belong to a new clade that
includes Cx32.4 and several other outlying connexins (Fig.
2).
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Functional expression in Xenopus oocytes and N2A cells.
The ability of Cx31.9 to form gap junction channels was tested in two
different experimental systems, Xenopus oocytes and N2A
cells (27, 32). To determine the functional
characteristics of Cx31.9 in paired oocytes, cells were injected with
40 ng of in vitro transcribed cRNA and were manually paired after
removal of the vitelline envelope. Oocytes were also injected with
antisense oligonucleotides, against XenCx38 to minimize the
contribution of this endogenous connexin to the recorded conductance
(27). Cx31.9 consistently induced the assembly of
intercellular channels that resulted in levels of gap junctional
conductance that were an order of magnitude greater than those measured
in antisense-treated control pairs (Fig.
4A, 0.54 ± 0.12 vs.
0.03 ± 0.03 µS; P 0.0001, Student's unpaired
t-test) albeit of a lower amplitude than those obtained with
other mammalian connexins in this expression system (1, 27,
41). In addition, we examined the possibility that the human
connexin could interact with endogenous XenCx38 by constructing heterotypic pairs between oocytes injected with Cx31.9 and mock-treated cells that did not receive antisense Cx38 oligonucleotides.
Cx31.9/water pairs developed junctional conductance values that were
greatly suppressed by injection of the antisense oligonucleotides, thus demonstrating the ability of Cx31.9 to recruit endogenous XenCx38 (Fig.
4A). The conductance measurements of these heterotypic pairs were of a magnitude similar to those of homotypic Cx31.9 pairs.
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Voltage-gating behavior of Cx31.9.
To characterize the physiological behavior of channels composed of
Cx31.9, we first analyzed voltage dependence. N2A cells transiently
transfected with Cx31.9 developed junctional conductance values that
exhibited an absence of detectable voltage-dependent channel closure. A
representative family of Ijs evoked by voltage steps of opposite polarities and increasing amplitude shows that Ij did not decrease with time for
Vj values up to ±100 mV (Fig. 5A). This property is unique
to Cx31.9, as no other connexin has previously been shown to be voltage
insensitive within this physiological range of
Vj. Quantification of this unusual
voltage-gating behavior by plotting normalized conductance
(Gj) versus Vj
demonstrated that even at the highest Vj values
tested (±100 mV), Cx31.9 conductance remained insensitive to voltage
in N2A cells (Fig. 5B). To ensure that this unusual property
was not related to the choice of the host cell, we also examined Cx31.9
voltage gating in paired oocytes. Similar to N2A cells, plots of
Gj versus Vj in oocyte
pairs showed a complete lack of sensitivity to voltage at
Vj values of ±100 mV (Fig. 5C).
Because these findings did not rule out the possibility that Cx31.9 may
be endowed with an exquisitely high voltage-gating threshold, we next
performed a limited series of experiments in which depolarizing steps
of up to +180 mV of Vj were imposed from a
holding potential of 90 mV. This protocol showed that when the
driving force was increased to supraphysiological values, Cx31.9
channels began to exhibit a very modest degree of voltage-dependent current decay (13 and 20% reduction in Ij at
150 and 180 mV, respectively,
= 0.49 s at 180 mV,
Fig. 5D). These data indicate that Cx31.9 formed
intercellular channels that were completely voltage insensitive at
Vj values of either polarity
120 mV.
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Pharmacological inhibition of Cx31.9 channels.
The striking absence of voltage gating in Cx31.9 channels led us to
investigate whether these channels were sensitive to chemical gating
induced by intracellular acidification or exposure to volatile anesthetics. The pH sensitivity of Cx31.9 intercellular channels was
tested by perfusing oocyte pairs with MB that had been saturated with
100% CO2, a procedure that typically induces a rapid
cytoplasmic acidification in excess of 1 pH unit (12, 34).
Intracellular acidification triggered a rapid fall in junctional
conductance between oocyte pairs expressing Cx31.9 (>80% closure,
Fig. 6A), which slowly
recovered when the superfusate was switched back to normal MB.
Conductance between pairs of Cx31.9-expressing oocytes was unaffected
by switching superfusates when no change in pH was introduced (data not
shown). Sensitivity of Cx31.9 conductance to the gap junction channel
blocker halothane was determined in N2A cells. Application of halothane
has been shown to produce rapid and reversible decreases in gap
junctional conductance in a wide variety of mammalian cell lines
(9, 25). Figure 6B shows a series of
intercellular currents from a Cx31.9-expressing N2A-cell pair with 1.7 nS of initial conductance. Seven successive sweeps of a 7.7-s ramp
protocol that varied the applied voltage from 100 to +100 mV are
illustrated. Application of 4 mM halothane at the onset of sweep
1 resulted in a 99% decrease in Ij with 1 min. Junctional conductance recovered rapidly on halothane washout (not
shown). These data show that despite the absence of a physiologically relevant voltage gate, Cx31.9 channels were endowed with chemical gating mechanisms indistinguishable from those of other members of the
connexin family.
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Unitary conductance of Cx31.9 channels.
We routinely failed to observe current transitions between open and
closed states of Cx31.9 channels at Vj values up
to ±120 mV in N2A cell pairs. The absence of Cx31.9 voltage gating
within this physiological range suggested that this result could be due to the channels residing predominantly in the open state.
Alternatively, Cx31.9 channels might have a very small unitary
conductance (j) with transitions that would be poorly
resolved at these voltages. To further examine this issue, we performed
a limited series of experiments where supraphysiological driving
voltages were applied to Cx31.9 cell pairs that displayed low levels of
coupling. Figure 7 shows two
representative traces derived from cell pairs that contain two active
gap junction channels. At a Vj of 160 mV, both channels remained open for most of the duration of the pulse with one
channel closing briefly (Fig. 7A). In a second trace
acquired at 140 mV, one of the two channels transited to the closed
state twice during the voltage pulse (Fig. 7B). Because such
transitions were only rarely observed, we were unable to obtain
sufficient data to accurately calculate either open probability or
values of
j. We did qualitatively measure
j in all traces where clear transitions could be
resolved, and we consistently obtained values between 12 and 15 pS (in
130 mM CsCl, Fig. 7C). Thus Cx31.9 appears to form channels
with a relatively low
j value that remain predominantly open at physiological voltages.
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Comparison of Cx31.9 and XenCx38 voltage gating.
Several studies using both oocyte pairs and transfected cells have
documented that connexons do not invariably maintain their electrical
"fingerprints" when they pair heterotypically with a different
connexon; thus distinct functional properties may arise from
connexon-connexon interactions in heterotypic channels (37). Heterotypic pairing of Cx31.9 to XenCx38 offered a
unique possibility to evaluate whether altered gating properties would result from interactions between voltage-sensitive XenCx38 and voltage-insensitive Cx31.9 connexons. As previously reported (7, 13), coupling between homotypic pairs of oocytes that expressed XenCx38 displayed a marked sensitivity to voltage with symmetrical decreases of Ij at Vj
values greater than ±20 mV (Fig.
8A). Furthermore, the plot of
Gjss versus Vj (Fig.
8B) confirmed that the voltage-dependent component of
XenCx38 gating could not be precisely fit by a simple Boltzmann
equation (7, 43). In contrast, analysis of heterotypic Cx31.9/XenCx38 Ijs showed a clear asymmetric
voltage closure with the XenCx38 side closing for
Vj values >40 mV, whereas the Cx31.9 side was
unaffected by voltage (Fig. 8C). Plotting the
Gj/Vj relationship revealed a slight reduction in the Gjss value
when the Cx31.9 side of the channel was relatively positive with regard
to Vj, albeit 90% of the initial conductance
remained at the largest Vj value tested (120 mV,
Fig. 8D). This deviation from the behavior of homotypic
Cx31.9 channels could represent either a novel property imparted by the
allosteric interaction of Cx31.9 with a highly voltage-gated connexon
or alternatively could result from a minimal contribution of homotypic
XenCx38 channels that escaped antisense blockade.
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DISCUSSION |
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We have identified and functionally characterized a new member of
the human connexin gene family, Cx31.9. The Cx31.9 sequence clustered
within a subgroup of connexins distinct from the previously described
- or
-subfamilies (19). Cx31.9 mRNA was distributed in the brain and several other organs and was present in the human EST
database. In two different functional expression assays, Cx31.9 formed
intercellular channels that were distinguished by a low
j and a remarkable insensitivity to
Vj. Thus Cx31.9 represents a novel human
connexin that forms channels with unique functional properties.
The new human Cx31.9 gene is closely related to the human Cx32.4 gene with which it shares 76% sequence identity and localization on chromosome 17. Of all other connexin genes, only connexin26 and connexin30, which also reside on the same chromosome (human 13, mouse 14), share as high a level of amino acid identity (10, 18). The main differences between Cx31.9 and Cx32.4 are an 18-amino acid insertion into the cytoplasmic loop of Cx31.9 and a general loss of homology in the second half of the cytoplasmic tail including the absence of the proline-rich domain in Cx32.4. Thus it appears that the Cx31.9 and Cx32.4 genes may have arisen by gene duplication.
The dendrogram of all known human connexin sequences (see Fig. 2)
supports the idea of a common origin for these genes and their
assignment to a third subgroup distinct from the - or
- connexin
genes. Many reports had previously noted that the sequence divergence
between connexin36 (Cx36) or connexin45 (Cx45) and the
-connexins
was greater than the sequence divergence between the
- and
-subfamilies themselves (2, 6, 22, 23, 28). Despite
this, Cx45 (variously called either
-6 or
-7) and Cx36 (
-9)
were erroneously assigned to the
-subgroup (20). Our analysis clearly indicates the existence of a third subfamily that is
equal in magnitude to the previously described groups. It should be
noted that the original division into two groups has always been poorly
defined (17, 19), and an alternative view would be that
there is simply one broad family of connexin genes.
Several observations suggested that Cx31.9 was a functional member of the connexin family. First, the Cx31.9 sequence displayed features consistent with other well-characterized connexins, including four potential transmembrane domains and conserved extracellular cysteine residues required for hemichannel docking (11). Second, transcription of the Cx31.9 gene has been confirmed by RT-PCR analysis and a search of the human EST database. Finally, when Cx31.9 was expressed in Xenopus oocytes or N2A cells, it formed gap junction channels that lacked voltage dependence but retained normal chemical gating, which indicates that the two gates were independent. This observation was in keeping with previous studies that have used chimerical constructs and connexin mutants to show that the two mechanisms could be independently modulated (33, 35). Taken together, these data demonstrate that Cx31.9 is an actively transcribed and functional connexin.
Heterotypic pairing of Cx31.9 to XenCx38 offered a unique opportunity
to evaluate alterations in gating properties that result from
interactions between voltage-sensitive and -insensitive connexons. In
light of the fact that Cx31.9 did not display physiologically relevant
voltage gating, it was surprising to find that the voltage-gating threshold and kinetics of XenCx38 shifted when it was paired with Cx31.9 in heterotypic channels. Historically, Vj
sensitivity of gap junctional channels was considered an intrinsic
property of the component hemichannels (4, 5, 14, 15).
However, in many examples, connexins did not invariably maintain
electrical fingerprints when paired with a different partner, and these
distinct gating properties may have arisen from connexon-connexon
interactions in heterotypic channels (7, 16, 38, 39). Our
data indicate that the voltage-insensitive Cx31.9 imparted a higher
voltage threshold to XenCx38 connexons that also gated with much slower values.
The absence of voltage sensitivity in Cx31.9 may be a reflection of the unusually high proline content of this connexin (11%) relative to voltage-sensitive connexins like XenCx38 (5% proline, 17 of 334 amino acids). The high proline content of Cx31.9 might result in a more rigid structure that is incapable of undergoing the conformational changes associated with gating at physiological voltages. It is not clear how pairing the insensitive Cx31.9 with XenCx38 resulted in a >20 mV shift in the voltage-gating threshold and a significant slowing of the kinetics of channel closure. The number of predicted gating charges did not change between heterotypic and homotypic channels when the XenCx38 side was positive, which is consistent with conservation of this property in the heterotypic channels. Further studies are required to elucidate the exact mechanisms by which Cx31.9 is able to modulate the voltage gating of XenCx38. Finally, this unique characteristic of Cx31.9 offers a novel approach to future mutational analysis of gap junction gating by screening for amino acid substitutions that will introduce voltage sensitivity.
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ACKNOWLEDGEMENTS |
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We thank M.-M. Gabellec for excellent technical assistance.
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FOOTNOTES |
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This study was supported by funding from National Institutes of Health Grants EY-13163, AR-47102, and DC-005491 (to T. W. White); EY-013869 (to M. Srinivas); EY-06516 (to H. Ripps); the Telethon Foundation Grant E.1283 and the Italian Ministero dell' Università e della Ricerca Scientifica e Tecnologica (to D. F. Condorelli); the Association RETINA France (to R. Bruzzone); and by fellowships from the Marine Biological Laboratory at Woods Hole, MA (to T. W. White, M. Srinivas, and R. Bruzzone).
Address for reprint requests and other correspondence: T. W. White, Dept. of Physiology and Biophysics, State Univ. of New York, T5-147, Basic Science Tower, Stony Brook, NY 11794-8661 (E-mail: thomas.white{at}sunysb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 29, 2002;10.1152/ajpcell.00163.2002
Received 10 April 2002; accepted in final form 14 May 2002.
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