The novel product of a five-exon stargazin-related gene abolishes CaV2.2 calcium channel expression
2002; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.1093/emboj/21.7.1514
ISSN1460-2075
Autores Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoArticle1 April 2002free access The novel product of a five-exon stargazin-related gene abolishes CaV2.2 calcium channel expression Fraser J. Moss Fraser J. Moss Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Patricia Viard Patricia Viard Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Anthony Davies Anthony Davies Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Federica Bertaso Federica Bertaso Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Karen M. Page Karen M. Page Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Alex Graham Alex Graham Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Carles Cantí Carles Cantí Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Mary Plumpton Mary Plumpton Bioinformatics Unit, GlaxoSmithKline, Medicines Research Center, Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Christopher Plumpton Christopher Plumpton Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Jeffrey J. Clare Jeffrey J. Clare Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Annette C. Dolphin Corresponding Author Annette C. Dolphin Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Fraser J. Moss Fraser J. Moss Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Patricia Viard Patricia Viard Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Anthony Davies Anthony Davies Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Federica Bertaso Federica Bertaso Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Karen M. Page Karen M. Page Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Alex Graham Alex Graham Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Carles Cantí Carles Cantí Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Mary Plumpton Mary Plumpton Bioinformatics Unit, GlaxoSmithKline, Medicines Research Center, Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Christopher Plumpton Christopher Plumpton Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Jeffrey J. Clare Jeffrey J. Clare Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK Search for more papers by this author Annette C. Dolphin Corresponding Author Annette C. Dolphin Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK Search for more papers by this author Author Information Fraser J. Moss1, Patricia Viard1, Anthony Davies1, Federica Bertaso1, Karen M. Page1, Alex Graham1, Carles Cantí1, Mary Plumpton2, Christopher Plumpton3, Jeffrey J. Clare3 and Annette C. Dolphin 1 1Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT UK 2Bioinformatics Unit, GlaxoSmithKline, Medicines Research Center, Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK 3Gunnels Wood Road, Stevenage, Herts, SG1 2NY UK ‡P.Viard, A.Davies and F.Bertaso contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1514-1523https://doi.org/10.1093/emboj/21.7.1514 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We have cloned and characterized a new member of the voltage-dependent Ca2+ channel γ subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential γ subunits identified by their homology to the stargazin gene, CACNG7 is a five-, and not four-exon gene whose mRNA encodes a protein we have designated γ7. Expression of human γ7 has been localized specifically to brain. N-type current through CaV2.2 channels was almost abolished when co-expressed transiently with γ7 in either Xenopus oocytes or COS-7 cells. Furthermore, immunocytochemistry and western blots show that γ7 has this effect by causing a large reduction in expression of CaV2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed γ7 was observed on pre-existing endogenous N-type calcium channels in sympathetic neurones. Low homology to the stargazin-like γ subunits, different gene structure and the unique functional properties of γ7 imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin. Introduction Voltage-dependent calcium channels (VDCCs) play a fundamental role in the coupling of membrane depolarization to many cellular processes by regulating cytoplasmic Ca2+ concentration in excitable cells. They are hetero-multimers consisting of a pore-forming α1 subunit assembled with auxiliary β, α2δ and possibly γ subunits. These subunits can be encoded by several different genes with alternative splice variants and are expressed in a tissue-specific manner. Much work has concentrated on the characterization of functional properties of the α1, β and α2δ subunits (Birnbaumer et al., 1998; Dolphin, 1998; Jones, 1998; Perez-Reyes, 1998; Catterall, 2000). However, research concerning the role of the γ subunit has not been as extensive. Until recent years, only a single gene, exclusively expressed in skeletal muscle, was believed to encode a VDCC γ subunit (Jay et al., 1990; Powers et al., 1993). Recordings of Ca2+ currents from dihydropyridine receptors (DHPRs) of skeletal myotubes from mice lacking this γ1 subunit suggest that its role is to limit calcium entry through these channels, increase the rate at which the channels inactivate and hyperpolarize the half-maximal potential for the voltage dependence of steady-state inactivation (Freise et al., 2000; Ahern et al., 2001). Subsequently, a second putative VDCC γ subunit, γ2, was identified based on its structural similarity to γ1, despite having only weak protein sequence identity (25%) (Letts et al., 1998). Mutations in the γ2 gene, cacng2, were found to underlie the absence epilepsy phenotype of the allelic stargazer (stg) and waggler (wag) mutant mice. Subsequent studies have identified six further putative γ subunits (γ3–γ8), not all of which have been cloned and expressed (Black and Lennon, 1999; Burgess et al., 1999, 2001; Klugbauer et al., 2000). The γ2, γ3 and γ4 subunits form a subfamily exclusively localized to the central nervous system (CNS) (Letts et al., 1998; Klugbauer et al., 2000) whose interaction with VDCCs has been investigated in several studies (Letts et al., 1998; Klugbauer et al., 2000; Kang et al., 2001; Sharp et al., 2001). The γ5 and γ7 subunits (Burgess et al., 1999, 2001) are predicted to represent another subfamily of stargazin-related proteins, with extremely low sequence identity to γ1 and ∼25% identity to γ2. These subunits, like other members of the putative γ subunit superfamily, are proteins predicted to have four transmembrane segments with intracellular N- and C-termini, and were reported to be encoded by a gene assembled from four exons (Burgess et al., 2001). However, assembly of the full-length γ5 and γ7 cDNAs has not been described and there are no functional data for either of these γ subunits. In the present study, we report the identification, cloning and functional characterization of a novel protein we have named the γ7 subunit. The first four exons of the gene encoding this protein are identical to those encoding the predicted γ7 subunit gene previously described by Burgess et al. (2001), but the transcription and translation of a final fifth exon results in the γ7 described in the present study having a very different and much longer C-terminus. Our results show that the co-expression of the γ7 subunit almost abolishes the functional expression and markedly suppresses the level of CaV2.2 subunit protein. We also report the identification of the γ5 subunit which, like γ7, is predicted to be encoded by a five-exon gene. Results Cloning of the γ5 and γ7 genes The full-length mouse stargazin sequence (Letts et al., 1998) was used as a query sequence to search the DNA databases. A short 487 bp sequence was assembled from three expressed sequence tags (ESTs) and was found to contain an open reading frame (ORF) with 26% identity to stargazin, although it contained no in-frame start or stop codons. A 487 bp fragment corresponding to this in silico sequence was amplified from human whole brain cDNA, and sequence analysis confirmed the computer predictions. Primers specific for each end of this fragment were used to amplify the missing parts of the ORF using 5′ and 3′ RACE. A 330 bp fragment containing the missing 5′ sequence and start ATG codon, and a 400 bp band containing the missing 3′ sequence and stop codon were obtained. These three DNA fragments were then used to assemble the full-length 828 bp stargazin-like ORF in a single 'splice-overlap' PCR. When this full-length sequence was used to search the human high-throughput genomic sequences (HTGS) using the BLASTn algorithm, a bacterial artificial chromosome (BAC), clone AC008440, derived from human chromosome 19 was identified. Analysis of this BAC using the gene prediction program Genscan (Burge and Karlin, 1997) predicts that the 828 bp ORF is assembled from five exons and encodes a 275 amino acid protein. When compared with all of the previously published γ subunits, this 828 bp cDNA clone exhibited 100% identity to the previously predicted human γ7 subunit over the first four exons (Burgess et al., 2001). However, the sequence of the cDNA clone described here diverges from the γ7 sequence (Burgess et al., 2001), due to the presence of the fifth exon (Figure 1A and B). It would therefore appear that CACNG7 is in fact a five-exon gene that encodes the γ7 subunit, and the previously predicted four-exon γ7 sequence results from read-through into intron four (Figure 1B). Figure 1.Protein sequences, proposed splicing mechanism and hydropathy plots of a family of low homology stargazin-related genes. (A) Alignment of the five-exon γ5 and γ7 subunit sequences with the previously predicted four-exon γ5 and γ7 subunits using the Clustal_W algorithm. Dotted lines indicate consensus N-glycosylation sites and solid triangles beneath residues mark consensus sites for phosphorylation by cAMP- and cGMP-dependent protein kinase, protein kinase C, casein kinase II or tyrosine kinase. The exon–intron boundaries are marked by solid dots above the residue whose codon is interrupted by the adjacent intron. Note that the sequence identity between γ5 and γ7 is 80% conserved throughout the additional fifth exon, whereas the predicted four-exon γ5 and γ7 subunits differ greatly in sequence identities and length. The two large open triangles designate a pair of cysteine residues that are conserved amongst the putative VDCC γ subunits and may be involved in the formation of disulfide bridges. The transmembrane-spanning segments, as predicted by the TMpred program (Hofmann and Stoffel, 1993), are indicated by solid underlining. The residues highlighted in bold in the C-terminus of γ7 identify the epitope for the anti-γ7 antibody. (B) A schematic diagram of the γ5 and γ7 gene structure. The full-length genes are encoded by five exons interrupted by four introns, I1–I4. If the previously predicted four-exon γ5 and γ7 subunits were expressed, the extent of predicted read-through into intron 4 is displayed above the gene structure. The start codon, terminal amino acids of exon 4, and both normally spliced and intron-retained γ5 or γ7 are shown below the gene structure. (C) Hydropathy plots of the five-exon γ5 and γ7 subunits predicted by the TMpred program (Hofmann and Stoffel, 1993) compared with the previously predicted four-exon γ5 and γ7. Amino acid position is shown on the x-axis, and positive TMpred values indicate putative membrane-spanning regions. γ7 and γ5 are predicted to have four transmembrane-spanning α-helices with intracellular N- and C-termini. Download figure Download PowerPoint BLAST searches of mouse HTGS identified cacng7, the mouse orthologue of the γ7 gene within BAC AC079557, which, like its human counterpart, contained five exons. Confirmation that the mouse γ7 mRNA is expressed was obtained by amplifying the complete cDNA in a single RT–PCR from mouse cerebellar total RNA. The mouse orthologue possesses 70% identity to the human γ7 at the nucleic acid level and, remarkably, 100% identity at the protein level. BLASTn searches and Genscan analysis of human chromosome 17 BAC AC005988 identified another related gene encoding a 275 amino acid protein. This gene also possessed five exons and predicted a protein with 70.5% amino acid identity compared with the human γ7 and 27% identity compared with human γ2. It exhibited 100% identity to the previously described human γ5 subunit (Burgess et al., 1999) over its first 190 amino acids but, like the γ7 subunit, the additional fifth exon encodes an alternative C-terminus. Unlike the previously predicted four-exon γ5 and γ7, the C-termini of the five-exon γ5 and γ7 share considerable identity (80%, Figure 1A). We therefore named this subunit γ5 (gene CACNG5) and subsequently identified the mouse orthologue on mouse chromosome 16 BAC AC079424. The mouse orthologue (cacng5) exhibited 89.5% identity at the nucleotide level and 97% identity at the protein level to the human sequence. Hydropathy plots predict that, like all the stargazin-related proteins, γ7 and γ5 have four transmembrane-spanning α-helices with predicted intracellular N- and C-termini (Figure 1C). The final transmembrane-spanning α-helices of γ7 and γ5 are predicted to be six and eight amino acids longer, respectively, than their equivalents in the previously predicted four-exon γ7 or γ5, and the full-length γ7 has a much more substantial cytosolic C-terminus than in the predicted four-exon γ7, in which only four intracellular amino acids are predicted after the final transmembrane segment. The sequences have been deposited in the DDBJ/EMBL/GenBank database with the following accession Nos: human γ7 (AF458897), human γ5 (AF458898), mouse γ7 (AF458899) and mouse γ5 (AF458900). Tissue distribution The tissue distribution of the novel γ7 mRNA was analysed by northern blot. Figure 2 shows a human multiple tissue northern blot (Figure 2A) and two brain region blots (Figure 2B and C) hybridized with a probe corresponding to nucleotides 576–763 of the γ7 ORF. This region was chosen because it contains the least identity when compared with other γ-subunits (53% to human γ2), and is unique to γ7. Thus, this probe will not detect the predicted four-exon γ7, should this be expressed. This γ7-specific probe reveals two transcripts of ∼2.4 and 3.0 kb, both of which are expressed only in brain. Both of these transcripts are expressed in all brain regions probed, although the shorter transcript is expressed at greater levels in several areas including cerebellum, amygdala, hippocampus and thalamus. These blots were stripped and re-probed using the same probe as that designed to detect the four-exon γ7 (Burgess et al., 2001). This probe, corresponding to nucleotides 80–482 of the γ7 ORF, would detect both the predicted four-exon γ7 and full-length γ7 transcripts. No additional transcripts were seen in any of the blots with this probe. Indeed, precisely the same expression profile was seen as with the full-length γ7-specific probe, including the same differential expression of the short and long transcripts seen in cerebellum, amygdala, hippocampus and thalamus (data not shown). Figure 2.The expression profile of the human γ7 subunit. (A) Multiple tissue northern blots probed specifically for the γ7 subunit show two mRNA species of ∼2.4 and 3.0 kb that are localized specifically to human brain. Multiple brain region blots (B and C) show that the γ7 subunit is expressed in all the individual brain regions probed. The bottom section of each panel displays the mRNA detected by the control β-actin probe for each blot. Download figure Download PowerPoint Influence of the γ7 subunit on heterologous expression of VDCCs and KV3.1b Having investigated the tissue distribution of the γ7 subunit in human brain, we next examined the effect of expression of this protein on Ba2+ currents recorded from neuronal VDCCs expressed in COS-7 cells. For comparison with immunocytochemistry data (see below), we transfected an N-terminal green fluorescent protein-tagged CaV2.2 construct (GFP–CaV2.2), previously shown to have no significant differences in its biophysical properties compared with the non-tagged channel (Raghib et al., 2001). These experiments revealed an almost total abolition of whole-cell GFP–CaV2.2 Ba2+ current (IBa) in cells co-transfected with γ7 (Figure 3A, upper trace). In 1 mM Ba2+, mean current density of cells expressing GFP–CaV2.2/β1b/α2δ2 was −13.5 ± 4.3 pA/pF at 0 mV (n = 20) (Figure 3B) but, even when extracellular [Ba2+] was increased to 10 mM, currents from GFP–CaV2.2/β1b/α2δ2/γ7-transfected COS-7 cells remained extremely small (−0.23 ± 0.08 pA/pF at 0 mV, n = 20, P <0.01) (Figure 3A, lower trace, and B). In a recent report, it was stated that inhibition of CaV2.2 currents by the γ2 subunit was dependent upon co-expression of an α2δ subunit (Kang et al., 2001). To investigate if the same is true of the much more robust suppressive effect of γ7, recordings were made from cells in the absence of co-transfected α2δ2 subunit. The histograms in Figure 3B show that the influence of the γ7 subunit on CaV2.2/β1b currents was independent of co-expression of an α2δ subunit (CaV2.2/β1b, −14.9 ± 2.7 pA/pF at 0 mV in 1 mM Ba2+, n = 16; CaV2.2/β1b/γ7, −0.02 ± 0.03 pA/pF at 0 mV in 10 mM Ba2+; n = 18, P <0.01). Figure 3.GFP–CaV2.2/β1b cDNAs were transiently transfected into COS-7 cells with or without α2δ2 and γ7 subunits. (A) Example traces elicited by a 200 ms step depolarization to +10 mV from a holding potential of −80 mV in the presence of 1 mM Ba2+ (upper panel). In the presence of γ7, extracellular Ba2+ solution was also increased to 10 mM (lower panel). (B) Histogram of mean current densities measured at +10 mV in 1 mM Ba2+ for controls and 10 mM Ba2+ in the presence of γ7. Co-expression of γ7 abolished currents in both the presence and absence of the α2δ2 subunit. The number of experiments (n) for each condition is given in parentheses above the columns, and data from all cells tested are included (**P <0.01, Student's t-test). (C) Peak I–V relationships and individual representative traces for CaV2.2 (solid diamonds, n = 26) and CaV2.2 + γ7 (open diamonds, n = 24), expressed in Xenopus oocytes with the β1b auxiliary subunit were determined by measuring peak Ba2+ current amplitudes recorded during 100 ms test pulses between −70 and +40 mV (holding potential −100 mV; +10 mV increments; [Ba2+] in extracellular medium: 5 mM). Download figure Download PowerPoint To examine whether these effects were peculiar to transfection in mammalian expression systems, we next looked at the effect of γ7 on CaV2.2 currents expressed in Xenopus oocytes, where there cannot be any question as to whether all cDNAs are present in each cell. Figure 3C shows recordings made in 5 mM Ba2+ from Xenopus oocytes expressing CaV2.2/β1b either with or without co-expression of γ7. The maximum conductance (Gmax), determined from the current–voltage (I–V) plots, was dramatically and significantly reduced when the human γ7 subunit was co-expressed compared with oocytes where it was not, with a corresponding 92.6% reduction in peak current amplitude at 0 mV (Figure 3C and Table I). The half-maximal value for the voltage dependence of activation (V50) was also shifted 3.4 mV more depolarized upon co-expression of γ7. An almost identical 94% inhibition of CaV2.2/β1b/α2δ2 currents was observed by γ7 in this system (data not shown). To examine whether the residual current represents the current induced by auxiliary subunits in Xenopus oocytes (Lacerda et al., 1994), we examined currents from oocytes transfected with only the auxiliary subunit β1b. However, we observed that these currents were also reduced from −28.1 ± 9.6 nA (n = 10) to −8.3 ± 5.3 nA (n = 9) upon co-expression of γ7. We next investigated the effects of γ7 on other CaVα1 subtypes. The γ7 subunit also significantly reduced the Gmax of CaV2.1/β1b and CaV1.2/β1b VDCCs by 48 and 52%, respectively (Figure 4A and B, Table I). There were no significant effects of γ7 on their voltage dependence of activation (Table I). Thus, the effect of γ7 on these channels is not as striking as that seen with CaV2.2. Figure 4.Effect of heterologous expression of γ7 with other channels. (A and B) Mean I–V relationships and individual representative traces for (A) CaV2.1 (solid squares, n = 17) and CaV2.1 + γ7 (open squares, n = 19); (B) CaV1.2 (solid triangles, n = 10) and CaV1.2 + γ7 (open triangles, n = 15), expressed in Xenopus oocytes with β1b. Peak Ba2+ current amplitudes were recorded during 100 ms test pulses between −70 and +40 mV (holding potential −100 mV; +10 mV increments; [Ba2+] 10 mM). (C) Peak I–V relationship for KV3.1b (solid squares, n = 10), and KV3.1 + γ7 (open squares, n = 10), and current traces for KV3.1 (top left panel) and KV3.1 + γ7 (bottom left panel) expressed in Xenopus oocytes. Holding potential was −100 mV, and steps were between −30 and +100 mV for 100 ms. The scale bar refers to both panels. Download figure Download PowerPoint Table 1. Influence of the γ7 subunit on the activation properties of VDCCs expressed in Xenopus oocytes Channel Activation n V50 (mV) k Gmax (μS) Peak IBa (μA) CaV2.2 −7.34 ± 0.79 4.34 ± 0.24 12.7 ± 2.29 −0.50 ± 0.10 26 CaV2.2 + γ7 −3.88 ± 0.92** 4.66 ± 0.40 1.50 ± 0.27*** −0.04 ± 0.01*** 24 CaV2.1 −0.63 ± 0.73 5.03 ± 0.24 5.76 ± 0.95 −0.17 ± 0.02 17 CaV2.1 + γ7 0.92 ± 0.68 4.71 ± 0.41 2.98 ± 0.46** −0.09 ± 0.02** 19 CaV1.2 −0.68 ± 2.08 6.80 ± 0.31 5.88 ± 1.40 −0.17 ± 0.04 10 CaV1.2 + γ7 1.75 ± 0.70 5.68 ± 0.59 2.84 ± 0.57* −0.10 ± 0.02 14 Data are expressed as mean ± SEM of the number of replicates, n. *P <0.05, **P <0.01, ***P <0.001 according to an unpaired Student's t-test. The peak IBa was at +10 mV for CaV2.1 and CaV1.2 and at 0 mV for CaV2.2. Together with the data generated in COS-7 cells, the near complete abolition of CaV2.2 current seen in Xenopus oocytes upon co-expression of the γ7 subunit suggests that it may be affecting the expression of these channels rather than altering their biophysical properties. However, since the effect of γ7 on the Ba2+ currents through CaV2.2 VDCCs expressed in COS-7 cells or Xenopus oocytes was so striking, we investigated the possibility that it down-regulates other heterologously expressed ion channels by co-expressing it with the Shaw-like voltage-dependent potassium channel, KV3.1b. Figure 4C shows representative traces and the peak I–V relationship of KV3.1b currents expressed in Xenopus oocytes alone and co-expressed with the γ7 subunit, which indicate that the γ7 subunit had no effect on the peak current amplitude of heterologously expressed KV3.1b channels. Influence of the γ7 subunit on endogenous Ca2+ currents recorded from cultured sympathetic neurones We investigated the electrophysiological consequence of the acute expression of γ7 upon the endogenous VDCC currents from sympathetic neurones of the rat superior cervical ganglion (SCG). These neurones express 80–90% N-type and 10% L-type current (Plummer et al., 1989; Delmas et al., 1998) and provided a vehicle in which we could determine if the γ7 subunit could exert its influence upon pre-existing Ca2+ channels. Whole-cell patch-clamp recordings, performed 36–48 h post-isolation and transfection from control sympathetic neurones transfected with the GFP marker, gave a mean IBa current density at 0 mV of −13.6 ± 2.6 pA/pF (n = 6). However, this was not significantly altered in neurones transfected with GFP and the γ7 subunit (−15.2 ± 3.5 pA/pF, n = 6) (Figure 5A). Expression of γ7 protein in γ7-transfected neurones was verified by immunocytochemistry (Figure 5B), which also demonstrated the presence of only a very low level of immunoreactivity for endogenous γ7 in neurones transfected with GFP alone (Figure 5B). Although the morphology of sympathetic neurones cultured for 36–48 h is quite variable, in a preliminary observation, we also noted an alteration in neurite morphology in sympathetic neurones transfected with γ7 (Figure 5B), but this will require further detailed examination in a future study. It would therefore appear that the γ7 subunit is unable to acutely influence the properties of pre-existing N-type VDCCs in these neurones, and that γ7 does not co-exist endogenously in SCG neurones with N-type channels. Figure 5.Effect of heterologous expression of γ7 in cultured sympathetic neurones. (A) Left: example traces from sympathetic neurones transiently transfected with GFP or GFP and the γ7 subunit, elicited by a 100 ms step depolarization to +10 mV from a holding potential of −80 mV in medium containing 10 mM Ba2+. Right: histogram of mean current densities measured from sympathetic neurones at +10 mV. The number of experiments (n) for each condition is given in parentheses above the columns. (B) Upper row: a GFP-transfected sympathetic neurone (left panel), showing lack of immunostaining for endogenous γ7 (right panel). Lower row: a GFP- plus γ7-transfected sympathetic neurone (left panel), showing immunostaining for γ7 (middle panel). Data are representative of three transfections. Scale bars = 15 μm. Download figure Download PowerPoint Immunocytochemical analysis of the effects of the γ7 subunit The subcellular distribution of the expressed γ7 subunit and its effects upon the expression of CaV2.2 subunits were determined using immunocytochemistry and confocal laser scanning microscopy. When the γ7 subunit alone was transiently transfected into COS-7 cells (Figure 6A), expression of γ7 protein was observed throughout the cytoplasm and not specifically associated with the plasma membrane (delineated by Oregon Green–phalloidin). Figure 6B shows the typical fluorescence pattern of a cell transiently transfected with GFP–CaV2.2/β1b. Figure 6C shows two cells where the γ7 subunit has also been co-transfected. A striking reduction in the fluorescence of GFP–CaV2.2 was observed upon co-expression of the γ7 subunit, whilst the γ7 levels remained comparable with those in cells transfected with the γ7 subunit alone. Figure 6.Co-expression of γ7 with GFP–CaV2.2 almost abolishes GFP fluorescence in COS-7 cells. Cells were transfected with (A) the γ7 subunit alone, (B) GFP–CaV2.2/β1b, (C) GFP–CaV2.2/β1b/γ7, (D) γ7-myc/his, (E) KV3.1b or (F) KV3.1b/γ7-myc/his. The panels in column 1 show staining with Oregon Green–phalloidin, GFP fluorescence or FITC-labelled myc Ab as stated; the panels in column 2 show Texas red staining for the γ7 subunit or KV3.1b subunit as stated; the panels in column 3 display the blue DAPI staining of the nucleus; and the panels in column 4 show the merged images. The scale bar in (A–C) represents 10 μm and in (D–F), 20 μm. Download figure Download PowerPoint Immunocytochemical methods also confirmed the earlier electrophysiological findings that the γ7 subunit does not adversely affect the expression of the KV3.1b subunit. Figure 6D displays the expression of a γ7-myc/his fusion protein expressed alone in a COS-7 cell. The expression pattern of this construct is similar to that of the untagged γ7 (Figure 6A). Shown in Figure 6E is the expression of KV3.1b when transfected alone. The distribution and the expression level of KV3.1b were unaltered by the co-expression of the γ7-myc/his fusion protein (Figure 6F). In transient transfections, co-transfected subunits are not always co-expressed in every cell. We therefore sought to quantify the extent of the reduction in observed GFP–CaV2.2 fluorescence caused by γ7 subunit co-expression. This was measured by counting the number of GFP-positive cells in both the GFP–CaV2.2/β1b- and GFP–CaV2.2/β1b/γ7-transfected cultures. The mean percentage of the total cells per 16 mm2 field of view with GFP–CaV2.2 fluorescence, which entirely co-localizes with β1b expression, was reduced from 16.4 ± 3.4% in GFP–CaV2.2/β1b-transfected cells to 3.3 ± 0.7% in cells additionally transfected with the γ7 subunit (Figure 7A). The same histogram also shows that almost all cells that exhibited GFP–CaV2.2 fluorescence in the CaV2.2/β1b/γ7 transfections were also labelled f
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