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CACN4, the major α1 subunit isoform of voltage-dependent calcium channels in pancreatic β-cells: a minireview of current progress
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Diabetes Research and Clinical Practice 28 Suppl. (1995) S99-S103
CACN4, the major Q'1 subunit isoform of voltage-dependent calcium channels in pancreatic ,a-cells: a minireview of current progress Susumu Seino* Division of Molecular Medicine, Center for Biomedical Science, Chiba Uiversity School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260, Japan
Abstract Calcium influx through L-type voltage-dependent calcium channels (VDCCs) triggers insulin secretion. Until recently, the structure of VDCCs in pancreatic f3-cells and their regulation in altered metabolic states were not known. Study of the VDCC protein in skeletal muscle has shown that the a I subunit is functionally the most important subunit among the five subuints (ai, a2, 13, 'Y and 8), acting as a voltage sensor and an ion-conducting pore. Molecular cloning of a novel a I subunit (f3-celljneuroendocrine type, CACN4) of VDCCs from pancreatic islets and insulinoma have made it possible to study the electrophysiological and pharmacogical properties, regulation, and genetics of the VDCCs expressed in f3-cells. The CACN4 is structurally related to other members of the VDCC o l subunit family, including skeletal muscle, cardiac, and brain types. In situ hybridization experiments reveal that CACN4 mRNAs are expressed in f3-cells in the islets. Heterologous expession studies show that CACN4 in the presence of the 13 subunit elicits L-type VDCC currents, although expression of CACN4 alone is not sufficient for VDCC acitivity. Studies of animal models with chronic hyperglycemia and starvation have indicated that the reduced CACN4 mRNA levels in pancreatic islets are associated with impaired insulin responses to stimuli in both hyperglycemic and fasting states. These studies demonstrate that CACN4 is the major component of VDCCs in pancreatic f3-cells and suggest that it plays a crucial role in the regulation of insulin secretion in normal and altered metabolic states, Keywords: Ion channels; Calcium; Insulin; Non-insulin-dependent diabetes mellitus
1. Introduction
Intracellular calcium ([Ca2 +]i) is the principal signal for insulin secretion [1]. [Ca2+]i is regu-
* Corresponding author, TeL: +81432262187; Fax: +8143 2217803,
lated by calcium influx from an extracellular source and calcium mobilization from intracellular stores. The calcium influx across the plasma membrane through voltage-dependent calcium channels (VOCCs) is the major factor contributing to a rise in [Ca2+]i in pancreatic ,B-cells [1]. VOCC currents have been classified into four types, namely L, T, N, and P-types, according to
0168-8227/95/$09,50 © 1995 Elsevier Science Ireland Ltd. All rights reserved. SSDIOI68-8227(95)01085-R
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5. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103
their electrophysiological and pharmacological properties [2,3]. Since L-type VDCC blockers inhibit the rise in [Ca 2+]i in l3-cells and insulin seceretion, an L-type VDCC plays a crucial role in insulin secretion [4]. The VDCC is a multisubunit protein and, in skeletal muscle, the complex has been shown to consist of five distinct subunits, a 1, 0'2, 13, y and 0 [2,3]. Expession studies have shown that the a 1 subunit is the most important subunit for generating VDCC activity [2,3]. However, until recently, the molecular basis of the VDCCs in l3-cells and the regulation of their expession in altered metabolic states are not known. Since the identification by molecular cloning of a novel VDCC a l-subunit (13cell/neuroendocrine type, CACN4) as well as a previously reported cardiac type a l-subunit expressed in pancreatic islets [5], knowledge of the VDCCs in pancreatic l3-cells has been accumulating. This review focuses on the structure, electrophysiological properties, and regulation of CACN4 1• 2. Structure of the p-cell / neuroendocrine type VDCC o I subunit (CACN4) Overlapping cDNA fragments encoding the CACN4 expressed in human pancreatic islets and insulinoma were isolated by a combination of reverse-transcriptase polymerase chain reaction (RT-PCR)-based strategy and screening of a eDNA library constructed from human insulinoma. Human CACN4 is a protein of 2181 amino acids [5]. The sequence of human CACN4 shows that it has 68%, 64%, and 41% overall amino acid identity with the sequences of rabbit heart, skeletal muscle, and brain type VDCC a 1 subunits, respectively. Computer analysis of human CACN4 predicts that it has a structure similar to that originally proposed for the skeletal muscle type a 1 subunit. The four intramolecular homologous repeated units (I - IV), with each repeat having six putative membrane spanning regions (51-S6), are highly conserved, especially the fouth transmem-
I The CACN4 has ken classified into aID. according to the latest naming of voltage-dependent calcium channels [20].
brane segments of each repeat. This S4 segment has positively charged amino acid residues, arginine or lysine, at every third position and is thought to act as a voltage sensor [2,3]. By contrast, the sequences of the Nand C termini and the intracellular loop connecting repeat I and II and repeat II and III are divergent among different a 1 subunit isoforms, suggesting that these regions may contribute to the isoform-specific functional properties. We have also isolated cDNAs for two isoforms of rat CACN4 (rCACN4A and rCACN4B) from a rat insulinoma RINm5F cDNA library. rCACN4A is a protein of 2203 amino acids and is the rat homolog of human CACN4, while rCACN4B lacks 535 amino acids in the C-terminus [6]. Other groups have also reported the same a 1 subunit isoform cloned from rat brain (RBal) [7], human brain (am) [8], and the hamster insulin-secreting cell line HIT (HCa3A) [9]. Both RBal and HCa3A also are C-terminal truncated forms. But, since rCACN4B, RBal, and HCa3A are truncated at different sites, CACN4 has several structural variations in the C-terminus, probably generated by a tissuespecific alternative splicing. Although the functional significance of these variations is not clear at present, the regulation of each truncated form by intracellular signals may be different because of the many potential cAMP-dependent and protein kinase C-dependent phosphorylation sites in the C-terminal region of CACN4. 3. Expression of CACN4 mRNA in pancreatic p-cells Tissue distribution of CACN4 mRNA was examined by northern blot analysis. A single ll-kb mRNA is expressed at moderate to high levels in rat pancreatic islets and brain as well as in RINm5F. However, CACN4A mRNA is not present in skeletal muscle, heart, kidney, spleen, liver, jejunum, and colon. In situ hybridization of rat pancreas using rat CACN4, cardiac type 0'1 subunit, and insulin antisense RNA probes indicates that CACN4 mRNAs are expressed in 13cells in the islets, while the cardiac type 0'1 subunit mRNA is expressed at low levels in ex-
S. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103 Table 1 Characteristics of f3-cell/neuroencorinc type VDCC a 1 subunit (CACN4) CACN4 protein and its variants
2181 amino acids (human) Several variations in the C-terminal region are present probably due to alternative splicing.
Sequence identity with other isoforms
68%,64%, and 41% identity with rabbit cardiac. skeletal muscle, and brain (81-2) isoforms
Expression
Brain, pancreatic f3-cells
Electrophysiological Voltage-dependent, properties Ltype. Functional expression requires f3 subunit. Regulation
CACN4gene
CACN4 mRNA levels in the islets are reduced by both glucose infusion and fasting. but not yet determined in diabetes. The human CACN4 gene spans more than 12U kbp on the short arm of chromosome :I (band p14.:I) and comprises 49 exons.
ocrine panreas as well as in the islets [5,10]. These results suggest that CACN4 is the major isoform of the VOCC o l subunit in {3-cells [10]. 4. Electrophysiological properties of CACN4
To characterize the electrophysiological properties of CACN4, we have used two heterologous expression systems: Xenopus oocytes and Chinese hamster ovary (CHO) cells. Since coexpression of the {3 subunit with the skeletal muscle or cardiac type o l subunit in Xenopus oocytes has been shown to increase VOCC currents, the {3 subunit is important in modulating the VOCC activity [2,3]. Accordingly, we have also isolated a full-length cONA for the {31 subunit, one of the four {3 subunit isoforms, from a rat brain cONA library. The amino acid sequence of the rat {31 subunit which we have cloned is identical to that reported previously [11]. Full length cONAs for rCACN4A and the rat {31 subunit were subcloned into a plasmid vector, pGEM. In vitro synthesized cRNAs for rCACN4A alone or cRNAs for rCACN4A together with cRNAs for rat
S101
{31 subunit were injected into Xenopus oocytes. In addition, we have established CRO cells stably expressing rCACN4A alone or coexpressing rCACN4A and the rat {31 subunit, using the mammalian expression vector, pCMV. Electrophysiological recordings were performed using the voltage-clamp method [12]. In both Xenopus 00cytes and CRO cells, coexpression of rCACN4A and the rat {31 subunit elicited L-type VOCC currents, while expression of rCACN4A alone did not. This property is different from that found in skeletal muscle and cardiac L-type VOCCs in which the o l subunit is sufficient for functional expression of L-type VOCe. Although the subunit structure of the VOCC protein in pancretic {3-cells is not yet known, these findings suggest that the {3 subunit is necessary for CACN4-directed VOCC activity. 5. Regulation of CACN4 mRNA expression in altered metabolic states It is possible that an alteration of expression or activity of VOCCs in {3-cells is associated with impaired insulin secretion. As a first step to understand how CACN4 is regulated in normal and altered metabolic states, the effects of chronic hyperglycemia and fasting on CACN4 mRNA levels have been examined in rats [10,13]. For quantitative information on mRNA levels, a competitive RT-PCR procedure was used. The levels of CACN4 mRNA in rats made hyperglycemic by infusion of high glucose are reduced to 20-25% of those in control rats infused by saline, suggesting that chronic hyperglycemia results in downregulation of CACN4. Furthermore, it has been found that the reduced mRNA levels in hyperglycemic rats are associated with enhanced basal insulin secretion but reduced insulin secertory responses to an L-type VOCC agonist, Bay K8644. On the other hand, a 72-h fast also induces a 3-fold decrease in CACN4 mRNA levels, compared to those in fed and refed rats. Fasting results in a dramatic decrease in insulin secretory responses to Bay K8644 as well. Interestingly, after a 24-h refeeding, insulin secretory responses to glucose and Bay K 8644 are significantly im-
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S. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) S99-S103
proved, with a normalization of CACN4 mRNA levels. These findings suggest that a decreased expression level of CACN4 may be one of the factors contributing to impaired insulin secretory responses to various stimuli in both chronic hyperglycemia and fasting. 6. Molecular genetics of CACN4 Because an alteration of the structure or expression of the CACN4 gene could contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM), we have attempted a determination of the structure of the human CACN4 gene. Using fluorescence in situ hybridization to metaphase chromosomes, the human CACN4 gene was shown to be localized to chromosome 3, band p14.3 [14]. To determine the structure of the human CACN4 gene, we have isolated A clones from human genomic libraries using 32 P-Iabelled human CACN4 cDNA frgments as probes. The human CACN4 gene spans more than 120 kbp and has 49 exons [15]. Determination of the exon-intron organization of the human CACN4 gene indicates that each of the 24 putative transmembrane domains in the CACN4 protein tends to be encoded by a single exon, suggesting the correspondence between the exon and the functional domain of the protein. It would be interesting to screen genetic variations of the CACN4 gene in NIDDM patients, as has been reported in a mutation of the skeletal muscle a 1 subunit in hypokalemic periodic paralysis [16] and mutations of the sodium channel a 1 subuint in hyperkalemic periodic paralysis [17,18] and paramyotonia congenita [19]. 7. Conclusion Understanding the molecular mechanisms of insulin secretion as well as insulin action should reveal causes of NIDDM and facilitate the development of novel hypoglycemic drugs. The cloning and functional characterization of the VDCC a 1 subunit CACN4 provide a perspective to clarify the molecular basis for the regulation of the
calcium signal in pancreatic f3-cells in normal and altered metabolic states and they also may lead to novel strategies to stimulate insulin secretion. Acknowledgements S.S. thanks Y. Fujii, N. Inagaki, and T. Gonoi at the Chiba University, Y. Ihara and Y. Yamada at the Kyoto University, and Y. Iwashima at the Asahikawa Medical College for contributing to the study and providing the unpublished data. This study was supported by Scientic Research Grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan and by a Grant from the Juvenile Diabetes Foundation International. An initial part of the study was done at the Howard Hughes Medical Institute at the University of Chicago. References D]
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[5]
[6]
[7]
[8]
[9]
Wollheim, c.a and Sharp, G.w.G. (1981) Regulation of insulin release by calcium. Physiol. Rev. 61, 914-973 Catterall, W.A (1988) Structure and function of voltage-sensitive ion channels. Science 242, 50-61. Miller, R.J. (1992) Voltage-sensitive Ca 2 + channels. J. BioI. Chern. 267, 1403-1406. Aschcroft, F.M. and Rorsman, P. (1989) Electrophysiology of the pancreatic ,B-cell. Prog. Biopys. Mol. BioI. 54,87-143. Seino, S., Chen, L., Seino, M., Blondel, D., Takeda, J., Johnson, J.H. and Bell, G.I. (1992) Cloning of the al subunit of a voltage-dependent calcium channel expressed in pancreatic ,B-cell. Proc. Natl. Acad. Sci. USA 89, 584-588. Ihara, Y, Yamada, Y, Fujii, Y. et al. (1995) Molecular diversity and functional characterization of voltage-dependent calcium channels (CACN4) expressed in pancreatic ,B-cells. Mol. Endocrinol. 9, 121-130. Hui, A, Ellinor, P.T., Krikanova, 0., Wang, J., Diebold, R.J. and Schwartz, A (1991) Molecular cloning of multiple subtypes of a novel rat brain isoform of a a 1 subunit of the voltage-dependent calcium channel. Neuron 7, 35-44. Williams, M.E., Feldman, D.H., McCue, AF., Brenner, R., Velicelebi, G., Ellis, S.B. and Harpold, M.M. (1992) Structure and functional expression of rat brain type ai, 2, and ,B subunits of a novel human neuronal calcium channel subtype. Neuron 8, 71-84. Yaney, G.c., Wheeler, M.B., Wei, X., Perez-Reyes, E., Birnbaumer, L., Boyd III, AE. and Moss, L.G. (1992)
S Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103
[10]
[11]
[12]
[13]
[14]
[15]
Cloning of a novel a I-subunit of the voltage-dependent calcium channel from the f3-cell. Mol. Endocrinol. 6, 2143-2152. Iwashima, Y, Pugh, W., Depaoli, AM., Takeda, J., Seino, S., Bell, G.1. and Polonsky, KS. (1993) Expression of calcium channel mRNAs in rat pancreatic islets and downregulation after glucose infusion. Diabetes 42, 948-955. Pragnell, M., Sakamoto, J., Jay, S.D. and Campbell, KP. (1991) Cloning and tissue-specific expression of the brain calcium channel f3-subunit. FEBS Lett. 291, 253-258. Gonoi, T. and Hasegawa, S. (1998) Postnatal disappearance of transient calcium channels in mouse skeletal muscle: effect of denervation and culture. J. Physiol. (Lond) 401, 617-637. Iwashima, Y, Kondo, A, Seino, S., Takeda, J., Eto, M., Plolonsky, KS. and Makino, 1. (1994) Reduced levels of messenger ribonucleic acid for calcium, glucose transporter-2 and glucokinase are associated with alterations in insulin secretion in fasted rats. Endocrinololgy 135,1010-1017. Seino, S., Yamada, Y, Espinosa III, R., Le Beau, M.M. and Bell, G.I. (1992) Assignment of the gene encoding the £1'1 subunit of the neuroendocrine/brain-type calcium channel (CACNLA2) to human chromosome 3, band pI4.3. Genomics 13, 1375-1377. Yamada, Y, Masuda, K, Ki, Q. et al. (1995) The struc-
[16]
[17]
[18]
[19]
[20]
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tures of the human calcium channel aJ subunit (CACNLlA2) and f3 subunit (CACNLB) genes. Genomics 27, 321-319. ROll, KJ., Horn, F.L., Elbaz, A, Heine, R., Gregg, RG., Hogan, K, Plwera, P.A, Laple, P., Vale-Santos, J.E., Wessenbach, J. and Fontaine, B. (1994) A calcium channel mutation causing hypokalemic periodic paralysis. Hum. Mol. Genet. 3, 1415-1419. Rojas, C.V., Wang, J., Schwartz, L.S., Hoffman, E.P., Powell, RR. and Brown, RH. (1991) A Met-to-Val mutation in the skeletal muscle Na + channel a-subunit in hyperkalaemic periodic paralysis. Nature 354, 387-389. Ptacek, LJ., George, AL., Griggs, R.C., Tawaii, R, Kallen, RG., Barchi, R.L., Robertson, M. and Leppert, M. (1992) Identification of a mutation in the gene causing hyperkalemic periodic paralysis. Cell 67, 1021-1027. Ptacek, L.J., George, AL., Barchi, RL., Griggs, R.C., Riggs, J.E., Robertson, M. and Leppert, M. (1992) Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita. Neuron 8,891-897. Birnbaumer, L., Campbell, KP., Catterall, W.A., Harpold, M.M., Hofmann, F., Horne, W.A, Mori, Y., Schwartz, A, Snutch, T.P., Tanabe, T. and Tsien, R.W. (1994) The naming of voltage-gated calcium channels. Neuron 13, 505-506.
lli]ililM.ill.:CIEI
Aff.D
'ClIINil'!J\J1 IPMmm
ELSEVIER
Diabetes Research and Clinical Practice 28 Suppl. (1995) S99-S103
CACN4, the major Q'1 subunit isoform of voltage-dependent calcium channels in pancreatic ,a-cells: a minireview of current progress Susumu Seino* Division of Molecular Medicine, Center for Biomedical Science, Chiba Uiversity School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260, Japan
Abstract Calcium influx through L-type voltage-dependent calcium channels (VDCCs) triggers insulin secretion. Until recently, the structure of VDCCs in pancreatic f3-cells and their regulation in altered metabolic states were not known. Study of the VDCC protein in skeletal muscle has shown that the a I subunit is functionally the most important subunit among the five subuints (ai, a2, 13, 'Y and 8), acting as a voltage sensor and an ion-conducting pore. Molecular cloning of a novel a I subunit (f3-celljneuroendocrine type, CACN4) of VDCCs from pancreatic islets and insulinoma have made it possible to study the electrophysiological and pharmacogical properties, regulation, and genetics of the VDCCs expressed in f3-cells. The CACN4 is structurally related to other members of the VDCC o l subunit family, including skeletal muscle, cardiac, and brain types. In situ hybridization experiments reveal that CACN4 mRNAs are expressed in f3-cells in the islets. Heterologous expession studies show that CACN4 in the presence of the 13 subunit elicits L-type VDCC currents, although expression of CACN4 alone is not sufficient for VDCC acitivity. Studies of animal models with chronic hyperglycemia and starvation have indicated that the reduced CACN4 mRNA levels in pancreatic islets are associated with impaired insulin responses to stimuli in both hyperglycemic and fasting states. These studies demonstrate that CACN4 is the major component of VDCCs in pancreatic f3-cells and suggest that it plays a crucial role in the regulation of insulin secretion in normal and altered metabolic states, Keywords: Ion channels; Calcium; Insulin; Non-insulin-dependent diabetes mellitus
1. Introduction
Intracellular calcium ([Ca2 +]i) is the principal signal for insulin secretion [1]. [Ca2+]i is regu-
* Corresponding author, TeL: +81432262187; Fax: +8143 2217803,
lated by calcium influx from an extracellular source and calcium mobilization from intracellular stores. The calcium influx across the plasma membrane through voltage-dependent calcium channels (VOCCs) is the major factor contributing to a rise in [Ca2+]i in pancreatic ,B-cells [1]. VOCC currents have been classified into four types, namely L, T, N, and P-types, according to
0168-8227/95/$09,50 © 1995 Elsevier Science Ireland Ltd. All rights reserved. SSDIOI68-8227(95)01085-R
Sloo
5. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103
their electrophysiological and pharmacological properties [2,3]. Since L-type VDCC blockers inhibit the rise in [Ca 2+]i in l3-cells and insulin seceretion, an L-type VDCC plays a crucial role in insulin secretion [4]. The VDCC is a multisubunit protein and, in skeletal muscle, the complex has been shown to consist of five distinct subunits, a 1, 0'2, 13, y and 0 [2,3]. Expession studies have shown that the a 1 subunit is the most important subunit for generating VDCC activity [2,3]. However, until recently, the molecular basis of the VDCCs in l3-cells and the regulation of their expession in altered metabolic states are not known. Since the identification by molecular cloning of a novel VDCC a l-subunit (13cell/neuroendocrine type, CACN4) as well as a previously reported cardiac type a l-subunit expressed in pancreatic islets [5], knowledge of the VDCCs in pancreatic l3-cells has been accumulating. This review focuses on the structure, electrophysiological properties, and regulation of CACN4 1• 2. Structure of the p-cell / neuroendocrine type VDCC o I subunit (CACN4) Overlapping cDNA fragments encoding the CACN4 expressed in human pancreatic islets and insulinoma were isolated by a combination of reverse-transcriptase polymerase chain reaction (RT-PCR)-based strategy and screening of a eDNA library constructed from human insulinoma. Human CACN4 is a protein of 2181 amino acids [5]. The sequence of human CACN4 shows that it has 68%, 64%, and 41% overall amino acid identity with the sequences of rabbit heart, skeletal muscle, and brain type VDCC a 1 subunits, respectively. Computer analysis of human CACN4 predicts that it has a structure similar to that originally proposed for the skeletal muscle type a 1 subunit. The four intramolecular homologous repeated units (I - IV), with each repeat having six putative membrane spanning regions (51-S6), are highly conserved, especially the fouth transmem-
I The CACN4 has ken classified into aID. according to the latest naming of voltage-dependent calcium channels [20].
brane segments of each repeat. This S4 segment has positively charged amino acid residues, arginine or lysine, at every third position and is thought to act as a voltage sensor [2,3]. By contrast, the sequences of the Nand C termini and the intracellular loop connecting repeat I and II and repeat II and III are divergent among different a 1 subunit isoforms, suggesting that these regions may contribute to the isoform-specific functional properties. We have also isolated cDNAs for two isoforms of rat CACN4 (rCACN4A and rCACN4B) from a rat insulinoma RINm5F cDNA library. rCACN4A is a protein of 2203 amino acids and is the rat homolog of human CACN4, while rCACN4B lacks 535 amino acids in the C-terminus [6]. Other groups have also reported the same a 1 subunit isoform cloned from rat brain (RBal) [7], human brain (am) [8], and the hamster insulin-secreting cell line HIT (HCa3A) [9]. Both RBal and HCa3A also are C-terminal truncated forms. But, since rCACN4B, RBal, and HCa3A are truncated at different sites, CACN4 has several structural variations in the C-terminus, probably generated by a tissuespecific alternative splicing. Although the functional significance of these variations is not clear at present, the regulation of each truncated form by intracellular signals may be different because of the many potential cAMP-dependent and protein kinase C-dependent phosphorylation sites in the C-terminal region of CACN4. 3. Expression of CACN4 mRNA in pancreatic p-cells Tissue distribution of CACN4 mRNA was examined by northern blot analysis. A single ll-kb mRNA is expressed at moderate to high levels in rat pancreatic islets and brain as well as in RINm5F. However, CACN4A mRNA is not present in skeletal muscle, heart, kidney, spleen, liver, jejunum, and colon. In situ hybridization of rat pancreas using rat CACN4, cardiac type 0'1 subunit, and insulin antisense RNA probes indicates that CACN4 mRNAs are expressed in 13cells in the islets, while the cardiac type 0'1 subunit mRNA is expressed at low levels in ex-
S. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103 Table 1 Characteristics of f3-cell/neuroencorinc type VDCC a 1 subunit (CACN4) CACN4 protein and its variants
2181 amino acids (human) Several variations in the C-terminal region are present probably due to alternative splicing.
Sequence identity with other isoforms
68%,64%, and 41% identity with rabbit cardiac. skeletal muscle, and brain (81-2) isoforms
Expression
Brain, pancreatic f3-cells
Electrophysiological Voltage-dependent, properties Ltype. Functional expression requires f3 subunit. Regulation
CACN4gene
CACN4 mRNA levels in the islets are reduced by both glucose infusion and fasting. but not yet determined in diabetes. The human CACN4 gene spans more than 12U kbp on the short arm of chromosome :I (band p14.:I) and comprises 49 exons.
ocrine panreas as well as in the islets [5,10]. These results suggest that CACN4 is the major isoform of the VOCC o l subunit in {3-cells [10]. 4. Electrophysiological properties of CACN4
To characterize the electrophysiological properties of CACN4, we have used two heterologous expression systems: Xenopus oocytes and Chinese hamster ovary (CHO) cells. Since coexpression of the {3 subunit with the skeletal muscle or cardiac type o l subunit in Xenopus oocytes has been shown to increase VOCC currents, the {3 subunit is important in modulating the VOCC activity [2,3]. Accordingly, we have also isolated a full-length cONA for the {31 subunit, one of the four {3 subunit isoforms, from a rat brain cONA library. The amino acid sequence of the rat {31 subunit which we have cloned is identical to that reported previously [11]. Full length cONAs for rCACN4A and the rat {31 subunit were subcloned into a plasmid vector, pGEM. In vitro synthesized cRNAs for rCACN4A alone or cRNAs for rCACN4A together with cRNAs for rat
S101
{31 subunit were injected into Xenopus oocytes. In addition, we have established CRO cells stably expressing rCACN4A alone or coexpressing rCACN4A and the rat {31 subunit, using the mammalian expression vector, pCMV. Electrophysiological recordings were performed using the voltage-clamp method [12]. In both Xenopus 00cytes and CRO cells, coexpression of rCACN4A and the rat {31 subunit elicited L-type VOCC currents, while expression of rCACN4A alone did not. This property is different from that found in skeletal muscle and cardiac L-type VOCCs in which the o l subunit is sufficient for functional expression of L-type VOCe. Although the subunit structure of the VOCC protein in pancretic {3-cells is not yet known, these findings suggest that the {3 subunit is necessary for CACN4-directed VOCC activity. 5. Regulation of CACN4 mRNA expression in altered metabolic states It is possible that an alteration of expression or activity of VOCCs in {3-cells is associated with impaired insulin secretion. As a first step to understand how CACN4 is regulated in normal and altered metabolic states, the effects of chronic hyperglycemia and fasting on CACN4 mRNA levels have been examined in rats [10,13]. For quantitative information on mRNA levels, a competitive RT-PCR procedure was used. The levels of CACN4 mRNA in rats made hyperglycemic by infusion of high glucose are reduced to 20-25% of those in control rats infused by saline, suggesting that chronic hyperglycemia results in downregulation of CACN4. Furthermore, it has been found that the reduced mRNA levels in hyperglycemic rats are associated with enhanced basal insulin secretion but reduced insulin secertory responses to an L-type VOCC agonist, Bay K8644. On the other hand, a 72-h fast also induces a 3-fold decrease in CACN4 mRNA levels, compared to those in fed and refed rats. Fasting results in a dramatic decrease in insulin secretory responses to Bay K8644 as well. Interestingly, after a 24-h refeeding, insulin secretory responses to glucose and Bay K 8644 are significantly im-
S102
S. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) S99-S103
proved, with a normalization of CACN4 mRNA levels. These findings suggest that a decreased expression level of CACN4 may be one of the factors contributing to impaired insulin secretory responses to various stimuli in both chronic hyperglycemia and fasting. 6. Molecular genetics of CACN4 Because an alteration of the structure or expression of the CACN4 gene could contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM), we have attempted a determination of the structure of the human CACN4 gene. Using fluorescence in situ hybridization to metaphase chromosomes, the human CACN4 gene was shown to be localized to chromosome 3, band p14.3 [14]. To determine the structure of the human CACN4 gene, we have isolated A clones from human genomic libraries using 32 P-Iabelled human CACN4 cDNA frgments as probes. The human CACN4 gene spans more than 120 kbp and has 49 exons [15]. Determination of the exon-intron organization of the human CACN4 gene indicates that each of the 24 putative transmembrane domains in the CACN4 protein tends to be encoded by a single exon, suggesting the correspondence between the exon and the functional domain of the protein. It would be interesting to screen genetic variations of the CACN4 gene in NIDDM patients, as has been reported in a mutation of the skeletal muscle a 1 subunit in hypokalemic periodic paralysis [16] and mutations of the sodium channel a 1 subuint in hyperkalemic periodic paralysis [17,18] and paramyotonia congenita [19]. 7. Conclusion Understanding the molecular mechanisms of insulin secretion as well as insulin action should reveal causes of NIDDM and facilitate the development of novel hypoglycemic drugs. The cloning and functional characterization of the VDCC a 1 subunit CACN4 provide a perspective to clarify the molecular basis for the regulation of the
calcium signal in pancreatic f3-cells in normal and altered metabolic states and they also may lead to novel strategies to stimulate insulin secretion. Acknowledgements S.S. thanks Y. Fujii, N. Inagaki, and T. Gonoi at the Chiba University, Y. Ihara and Y. Yamada at the Kyoto University, and Y. Iwashima at the Asahikawa Medical College for contributing to the study and providing the unpublished data. This study was supported by Scientic Research Grants from the Ministry of Education, Science and Culture, and the Ministry of Health and Welfare, Japan and by a Grant from the Juvenile Diabetes Foundation International. An initial part of the study was done at the Howard Hughes Medical Institute at the University of Chicago. References D]
[2] [3] [4]
[5]
[6]
[7]
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