ELSEVIER Diabetes Research and Clinical Practice 28 Suppl. (1995) S99-S103

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CACN4, the major Q'1 subunit isoform ofvoltage-dependent calcium channels in pancreatic ,a-cells:

a minireview of current progress

Susumu Seino*

Division ofMolecular Medicine, Center for Biomedical Science, Chiba Uiversity School ofMedicine, 1-8-1, Inohana,Chuo-ku, Chiba 260, Japan

Abstract

Calcium influx through L-type voltage-dependent calcium channels (VDCCs) triggers insulin secretion. Untilrecently, the structure of VDCCs in pancreatic f3-cells and their regulation in altered metabolic states were notknown. Study of the VDCC protein in skeletal muscle has shown that the a I subunit is functionally the mostimportant subunit among the five subuints (ai, a2, 13, 'Y and 8), acting as a voltage sensor and an ion-conductingpore. Molecular cloning of a novel a I subunit (f3-celljneuroendocrine type, CACN4) of VDCCs from pancreaticislets 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 ofthe VDCC o l subunit family, including skeletal muscle, cardiac, and brain types. In situ hybridization experimentsreveal that CACN4 mRNAs are expressed in f3-cells in the islets. Heterologous expession studies show that CACN4in the presence of the 13 subunit elicits L-type VDCC currents, although expression of CACN4 alone is not sufficientfor VDCC acitivity. Studies of animal models with chronic hyperglycemia and starvation have indicated that thereduced CACN4 mRNA levels in pancreatic islets are associated with impaired insulin responses to stimuli in bothhyperglycemic and fasting states. These studies demonstrate that CACN4 is the major component of VDCCs inpancreatic f3-cells and suggest that it plays a crucial role in the regulation of insulin secretion in normal and alteredmetabolic states,

Keywords: Ion channels; Calcium; Insulin; Non-insulin-dependent diabetes mellitus

1. Introduction

Intracellular calcium ([Ca2 +]i) is the principalsignal for insulin secretion [1]. [Ca2+]i is regu-

*Corresponding author, TeL: +81432262187; Fax: +81432217803,

lated by calcium influx from an extracellularsource and calcium mobilization from intracellu­lar stores. The calcium influx across the plasmamembrane through voltage-dependent calciumchannels (VOCCs) is the major factor contribut­ing to a rise in [Ca2+]i in pancreatic ,B-cells [1].VOCC currents have been classified into fourtypes, namely L, T, N, and P-types, according to

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Sloo 5. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103

their electrophysiological and pharmacologicalproperties [2,3]. Since L-type VDCC blockers in­hibit the rise in [Ca2+]i in l3-cells and insulinseceretion, an L-type VDCC plays a crucial rolein insulin secretion [4]. The VDCC is a multisub­unit protein and, in skeletal muscle, the complexhas been shown to consist of five distinct subu­nits, a 1, 0'2, 13, y and 0 [2,3]. Expession studieshave shown that the a 1 subunit is the mostimportant subunit for generating VDCC activity[2,3]. However, until recently, the molecular basisof the VDCCs in l3-cells and the regulation oftheir expession in altered metabolic states are notknown. Since the identification by molecularcloning of a novel VDCC a l-subunit (13­cell/neuroendocrine type, CACN4) as well as apreviously reported cardiac type a l-subunit ex­pressed in pancreatic islets [5], knowledge of theVDCCs in pancreatic l3-cells has been accumulat­ing. This review focuses on the structure, electro­physiological properties, and regulation ofCACN41

•

2. Structure of the p-cell / neuroendocrine typeVDCC o I subunit (CACN4)

Overlapping cDNA fragments encoding theCACN4 expressed in human pancreatic islets andinsulinoma were isolated by a combination ofreverse-transcriptase polymerase chain reaction(RT-PCR)-based strategy and screening of aeDNA library constructed from human insuli­noma. Human CACN4 is a protein of 2181 aminoacids [5]. The sequence of human CACN4 showsthat it has 68%, 64%, and 41% overall amino acididentity with the sequences of rabbit heart, skele­tal muscle, and brain type VDCC a 1 subunits,respectively. Computer analysis of human CACN4predicts that it has a structure similar to thatoriginally proposed for the skeletal muscle typea 1 subunit. The four intramolecular homologousrepeated units (I - IV), with each repeat having sixputative membrane spanning regions (51-S6), arehighly conserved, especially the fouth transmem-

I The CACN4 has ken classified into aID. according to thelatest naming of voltage-dependent calcium channels [20].

brane segments of each repeat. This S4 segmenthas positively charged amino acid residues, argi­nine or lysine, at every third position and isthought to act as a voltage sensor [2,3]. By con­trast, the sequences of the Nand C termini andthe intracellular loop connecting repeat I and IIand repeat II and III are divergent among dif­ferent a 1 subunit isoforms, suggesting that theseregions may contribute to the isoform-specificfunctional properties. We have also isolated cD­NAs for two isoforms of rat CACN4 (rCACN4Aand rCACN4B) from a rat insulinoma RINm5FcDNA library. rCACN4A is a protein of 2203amino acids and is the rat homolog of humanCACN4, while rCACN4B lacks 535 amino acidsin the C-terminus [6]. Other groups have alsoreported the same a 1 subunit isoform clonedfrom rat brain (RBal) [7], human brain (am) [8],and the hamster insulin-secreting cell line HIT(HCa3A) [9]. Both RBal and HCa3A also areC-terminal truncated forms. But, since rCACN4B,RBal, and HCa3A are truncated at differentsites, CACN4 has several structural variations inthe C-terminus, probably generated by a tissue­specific alternative splicing. Although the functio­nal significance of these variations is not clear atpresent, the regulation of each truncated form byintracellular signals may be different because ofthe many potential cAMP-dependent and proteinkinase C-dependent phosphorylation sites in theC-terminal region of CACN4.

3. Expression of CACN4 mRNA in pancreaticp-cells

Tissue distribution of CACN4 mRNA was ex­amined by northern blot analysis. A single ll-kbmRNA is expressed at moderate to high levels inrat pancreatic islets and brain as well as inRINm5F. However, CACN4A mRNA is not pre­sent in skeletal muscle, heart, kidney, spleen,liver, jejunum, and colon. In situ hybridization ofrat pancreas using rat CACN4, cardiac type 0'1subunit, and insulin antisense RNA probes indi­cates that CACN4 mRNAs are expressed in 13­cells in the islets, while the cardiac type 0'1subunit mRNA is expressed at low levels in ex-

S. Seino / Diabetes Research and Clinical Practice 28 Suppl. (1995) 599-5103 S101

Table 1Characteristics of f3-cell/neuroencorinc type VDCC a 1 sub­unit (CACN4)

CACN4 proteinand its variants

Sequence identitywith other isoforms

Expression

Electrophysiologicalproperties

Regulation

CACN4gene

2181 amino acids (human)Several variations in the C-terminalregion are present probably due toalternative splicing.

68%,64%, and 41% identity with rabbitcardiac. skeletal muscle, and brain(81-2) isoforms

Brain, pancreatic f3-cells

Voltage-dependent,Ltype.Functional expression requires f3 sub­unit.

CACN4 mRNA levels in the islets arereduced by both glucose infusion andfasting. but not yet determined in dia­betes.The human CACN4 gene spansmore than 12U kbp on the short arm ofchromosome :I (band p14.:I) andcomprises 49 exons.

{31 subunit were injected into Xenopus oocytes. Inaddition, we have established CRO cells stablyexpressing rCACN4A alone or coexpressingrCACN4A and the rat {31 subunit, using themammalian expression vector, pCMV. Electro­physiological recordings were performed using thevoltage-clamp method [12]. In both Xenopus 00­

cytes and CRO cells, coexpression of rCACN4Aand the rat {31 subunit elicited L-type VOCCcurrents, while expression of rCACN4A alone didnot. This property is different from that found inskeletal muscle and cardiac L-type VOCCs inwhich the o l subunit is sufficient for functionalexpression of L-type VOCe. Although the subu­nit structure of the VOCC protein in pancretic{3-cells is not yet known, these findings suggestthat the {3 subunit is necessary for CACN4-di­rected VOCC activity.

5. Regulation of CACN4 mRNA expression inaltered metabolic states

ocrine panreas as well as in the islets [5,10]. Theseresults suggest that CACN4 is the major isoformof the VOCC o l subunit in {3-cells [10].

4. Electrophysiological properties of CACN4

To characterize the electrophysiologicalproperties of CACN4, we have used two heterolo­gous expression systems: Xenopus oocytes andChinese hamster ovary (CHO) cells. Since coex­pression of the {3 subunit with the skeletal muscleor cardiac type o l subunit in Xenopus oocyteshas been shown to increase VOCC currents, the{3 subunit is important in modulating the VOCCactivity [2,3]. Accordingly, we have also isolated afull-length cONA for the {31 subunit, one of thefour {3 subunit isoforms, from a rat brain cONAlibrary. The amino acid sequence of the rat {31subunit which we have cloned is identical to thatreported previously [11]. Full length cONAs forrCACN4A and the rat {31 subunit were sub­cloned into a plasmid vector, pGEM. In vitrosynthesized cRNAs for rCACN4A alone or cR­NAs for rCACN4A together with cRNAs for rat

It is possible that an alteration of expression oractivity of VOCCs in {3-cells is associated withimpaired insulin secretion. As a first step to un­derstand how CACN4 is regulated in normal andaltered metabolic states, the effects of chronichyperglycemia 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 ofCACN4 mRNA in rats made hyperglycemic byinfusion 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 beenfound that the reduced mRNA levels in hyper­glycemic rats are associated with enhanced basalinsulin secretion but reduced insulin secertoryresponses to an L-type VOCC agonist, Bay K8644.On the other hand, a 72-h fast also induces a3-fold decrease in CACN4 mRNA levels, com­pared to those in fed and refed rats. Fastingresults in a dramatic decrease in insulin secretoryresponses to Bay K8644 as well. Interestingly,after a 24-h refeeding, insulin secretory responsesto 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 mRNAlevels. These findings suggest that a decreasedexpression level of CACN4 may be one of thefactors contributing to impaired insulin secretoryresponses 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 tothe development of non-insulin-dependent dia­betes mellitus (NIDDM), we have attempted adetermination of the structure of the humanCACN4 gene. Using fluorescence in situ hy­bridization to metaphase chromosomes, the hu­man CACN4 gene was shown to be localized tochromosome 3, band p14.3 [14]. To determine thestructure of the human CACN4 gene, we haveisolated A clones from human genomic librariesusing 32 P-Iabelled human CACN4 cDNA frg­ments as probes. The human CACN4 gene spansmore than 120 kbp and has 49 exons [15]. De­termination of the exon-intron organization ofthe human CACN4 gene indicates that each ofthe 24 putative transmembrane domains in theCACN4 protein tends to be encoded by a singleexon, suggesting the correspondence between theexon and the functional domain of the protein. Itwould be interesting to screen genetic variationsof the CACN4 gene in NIDDM patients, as hasbeen reported in a mutation of the skeletal mus­cle a 1 subunit in hypokalemic periodic paralysis[16] and mutations of the sodium channel a 1subuint in hyperkalemic periodic paralysis [17,18]and paramyotonia congenita [19].

7. Conclusion

Understanding the molecular mechanisms ofinsulin secretion as well as insulin action shouldreveal causes of NIDDM and facilitate the devel­opment of novel hypoglycemic drugs. The cloningand functional characterization of the VDCC a 1subunit CACN4 provide a perspective to clarifythe molecular basis for the regulation of the

calcium signal in pancreatic f3-cells in normal andaltered metabolic states and they also may lead tonovel strategies to stimulate insulin secretion.

Acknowledgements

S.S. thanks Y. Fujii, N. Inagaki, and T. Gonoiat the Chiba University, Y. Ihara and Y. Yamadaat the Kyoto University, and Y. Iwashima at theAsahikawa Medical College for contributing tothe study and providing the unpublished data.This study was supported by Scientic ResearchGrants from the Ministry of Education, Scienceand Culture, and the Ministry of Health andWelfare, Japan and by a Grant from the JuvenileDiabetes Foundation International. An initial partof the study was done at the Howard HughesMedical Institute at the University of Chicago.

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