Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons

Abstract

A new mouse gap junction gene that codes for a protein of 46,551 Da has been identified and designated connexin47 (Cx47). It mapped as a single-copy gene to mouse chromosome 11. In human HeLa cells and Xenopus oocytes, expression of mouse Cx47 or a fusion protein of Cx47 and enhanced green fluorescent protein induced intercellular channels that displayed strong sensitivity to transjunctional voltage. Tracer injections in Cx47-transfected HeLa cells revealed intercellular diffusion of neurobiotin, Lucifer yellow, and 4',6-diamidino-2-phenylindole. Recordings of single channels yielded a unitary conductance of 55 pS main state and 8 pS substate. Cx47 mRNA expression was high in spinal cord and brain but was not found in retina, liver, heart, and lung. A low level of Cx47 expression was detected in ovaries. In situ hybridizations demonstrated high expression in alpha motor neurons of the spinal cord, pyramidal cells of the cortex and hippocampus, granular and molecular layers of the dentate gyrus, and Purkinje cells of the cerebellum as well as several nuclei of the brainstem. This expression pattern is distinct from, although partially overlapping with, that of the neuronally expressed connexin36 gene. Thus, electrical synapses in adult mammalian brain are likely to consist of different connexin proteins depending on the neuronal subtype.

Fig. 1.

The peptide sequences of mouse Cx45 (Hennemann et al., 1992), mouse Cx47 (accession number AJ276435), human Cx46.6 (accession number AF014643), and mouse Cx36 (Condorelli et al., 1998) were aligned. The conserved cysteine residues are printedbold, and the core putative transmembrane domains areboxed. Identical amino acid residues between Cx47 and Cx45 or between Cx47 and Cx36 are marked with anasteriskabove or belowthe sequences, respectively. The nucleotide sequence data of mouse Cx47 are available from European Molecular Biology Laboratory under accession number AJ276435.

Fig. 2.

a, b, Top, Northern blot analysis of Cx47 expression in adult mouse tissues (a) and eight stages of mouse brain development (b) are shown. Bottom, Equal amounts of total RNA were loaded as demonstrated by staining of 18 S ribosomal RNA with ethidium bromide (bottom). The size of the bands is indicated in kilobases on the right.P0, Postnatal day 0; dpc, dies postcoitum.

Fig. 3.

Expression of Cx47 mRNA in the murine CNS. Coronal sections of adult brain and spinal cord were hybridized as described in Materials and Methods. Representative sections from the cerebellum (A–C), forebrain (D–I), hindbrain (J–L), and spinal cord (M–O) are shown. Note that the in situ signal labels preferentially and/or cell layers known to contain specific neuronal phenotypes, such as the Purkinje cell layer and the granule cell layers in the cerebellum (A, B). Within the hippocampal formation (D, G, H), heavy labeling is seen over the dentate gyrus (G) and all subregions of the cornu Ammonis (D, H; the latter micrograph depicts the CA3 region). Within the cerebral cortex (D, E), numerous cells located in all cell layers are positive. In the brainstem, labeling was particularly prominent over nuclei, such as the inferior olive (J, K). The micrographs from the cervical spinal cord (M, N) again show numerous labeled cells within the gray matter and also document that oligodendrocytes and astrocytes within the white matter do not express detectable levels of Cx47 mRNA. Sections shown inA, B, D, E, G, H, J, K, M, and N were hybridized with the Cx47 antisense probe. Controls shown in C, F, I, L, and O were hybridized with a Cx47 sense probe. Rectangles in micrographs A, D, J, and M indicate the areas from which the higher power views shown in B, E, G, H, K, and N, respectively, were taken. Scale bar: A, C, E, G–I, K, N, 100 μm; B, 25 μm; D, F, J, L, M, O, 400 μm.

Fig. 4.

Voltage gating of homotypic Cx47 gap junctions inXenopus oocytes (A, B) and transfected HeLa cells (C, D). A, Graph of initial and steady-state G_j(filled and open triangles, respectively) as a function of V_j. G_j isg_j normalized to its value atV_j = 0. Data represent mean values obtained from five Xenopus oocyte cell pairs with maximum g_j values < 10 μS. Thesolid line is the Boltzmann fit of the steady-state data (see text). B, Representative junctional currents forV_j steps up to ±120 mV in 20 mV increments. Upward and downward currents are elicited by negative and positive V_j values, respectively. Calibration: 500 nA, 2 sec. C, Graph of steady-stateG_j as a function ofV_j in HeLa–Cx47 (filled circles) and HeLa–Cx47-EGFP (open circles) cell pairs. G_j is normalized as described in A. The solid line is a fit of the experimental data to the Boltzmann equation (see text).D, Examples of junctional currents in response to ±30, 50, and 65 mV V_j steps applied to one cell of a HeLa–Cx47 cell pair. Small, repeated positive and negative 20 mV V_j steps were applied to the same cell in between the longer durationV_j steps to regularly monitorI_j. wt, Wild type.

Fig. 5.

Chemical gating and single-channel conductance of Cx47 and Cx47-EGFP expressed in HeLa cells. A, B, Heptanol (2 mm) and CO₂ uncouple Cx47-EGFP homotypic junctions in transfected HeLa cells. Junctional currentI_j was measured by applying repeatedV_j steps to one cell of a pair.A, Application of heptanol (horizontal bar) to a cell pair in whichg_j = 10 nS produced full uncoupling and relatively fast recovery after washout. B, Application of 100% CO₂ (horizontal bar) to a cell pair with g_j = 13 nS fully uncoupled the cells within ∼20 sec, followed by slow recovery after washout. C, Illustration of Cx47 (top) and Cx47-EGFP (bottom) single-channel conductance and gating obtained in cell pairs during the early phase of recovery from full uncoupling with 100% CO₂ is shown. Records are of single-channel currents in response to repeated segments of ±17 mV, 200 msec V_j steps followed by ±65–85 mV, 2 sec V_j ramps. In both cases, a single-channel conductance of ∼55 pS and a substate conductance of ∼8 pS were approximated from the slopes of theI_j–V_jrelations (solid and dashed lines, respectively).

Fig. 6.

Tracer transfer in HeLa wild-type (a, d, g), HeLa–Cx47 (b, e, h), and HeLa–Cx47-EGFP (c, f, i) cells. The cells were injected with Lucifer yellow (a–c), DAPI (d–f), or neurobiotin (g–i). Microphotographs were taken with the appropriate filter set or after fixing and staining in the case of neurobiotin. The injected cells are marked withasterisks. Scale bar: a–g, 50 μm;h, i, 100 μm.

Fig. 7.

Simultaneous tracer transfer and double whole-cell recording between HeLa–Cx47-EGFP cells. A, Illustration of Lucifer yellow transfer between a single cell pair. Lucifer yellow was loaded into one of the cells (cell 1) through the patch pipette. The two panels show (from left to right) a phase-contrast image illustrating the positions of patch pipettes (left pipette was loaded with Lucifer yellow) and a fluorescence image taken with a filter set for Lucifer yellow 4 min after establishing whole-cell recording in cell 1. After establishing a whole-cell recording in cell 2, g_j was determined to be 12 nS. B, Illustration of DAPI transfer between an electrically coupled cell pair. The twopanels are as described in A except that the fluorescence image for DAPI was obtained 10 min after establishing whole-cell recording in cell 1 and a filter set for DAPI was used.g_j was measured to be 40 nS. DAPI preferentially labeled the nuclei of both cells.