Volume 263, number 1, 121-126 FEBS 08305 April 1990
Expression of voltage-gated IS+ channels in insulin-producing cells
Analysis by polymerase chain reaction
Christer BetshoW, Arnd Baumann2, Susan Kenna3, Frances M. Ashcroft?, Stephen J.H. Ashcroft3, Per-Olof Berggrens, Andrew Grupe6, Olaf Pongs6, Patrik Rorsman’, John Sandblom’ and Michael Welsh*
‘Department of Pathology, University Hospital, S-751 85 Uppsala, Sweden, 2Zentrum fiir Molekulare Neurobiologie, Hamburg, D- 2000 Hamburg 20, FRG, 3Nu&?eld Department of Clinical Biochemistry, John Radcli@e Hospital, Headington. Oxford OX3 SDU, UK, 4University Laboratory of Physiology, Parks Rd., Oxford OX12 3PT, UK, SDepartment of Endocrinology, Karolinska Institute,
K&+olinska Hospital, Box 60500, S-104 01 Stockholm, Sweden, 6Ruhr-Universita”t Bochum, ~hrst~l~r Biochemie, D-4630, Bochum, FRG, ‘Dep~tment of medics Physics, Gothenb~g University, Box 33031, S-400 33 Gothen~rg, Sweden and
sDepartment of Medical Cell Biology, Box 571, Biomedical Centre* S-751 23 Vppsola, Sweden
Received 13 Febuary 1990
We have used the polymerase chain reaction (PCR) with primers against the SS and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two ins~in-pr~ucing cell lines HIT T15 and RINmSF. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which
distribute differently to a number of tissues and cell lines including insulin-producing cells.
Insulin-producing cell; Potassium channel; Polymerase chain reaction; Northern blot analysis
1. INTRODUCTION
Pancreatic P-cells possess a variety of potassium channels including both ligand- and voltage-gated K+
channels [1,2]. The best characterized of the ligand- gated K” channels in the ,&cell is the ATP-sensitive K+
channel (G channel) [2]. This channel is directly in- hibited by intracellular ATP ([ATP]i) and changes in [ATP]i are believed to couple metabolic events to regulation of channel activity. It is also inhibited by sulfo~ylur~ that bind to the channel with high affini- ty [3-51, The voltage-gated K+ channels in &cells in- clude two kinds of delayed rectifier channels [6], a transient (A-current) K+ channel [7] and a Ca2+-activated K+ channel which is gated by both voltage and intra~ellul~ Ca2’ [8]. The role of the delayed rectifier K+ channels is clear and, as in other tissues, activation of these channels produces repolarization of the action potential [9]. Whereas the Ca’+-activated K+ channel is believed also to con- tribute to action potential repolarization [lo], the physiologic function of the A-current is not fully understood.
Recent molecular biological studies [IO-121 have identified a family of voltage-gated K+ channels known Correspondence address: M. Welsh, Department of Medical Cell Biology, Box 571, Biomedical Centre, S-751 23 Uppsala, Sweden
as RCK (rat cortex) or MBK (mouse brain) which func- tionally resemble the delayed rectifier K+ channels. The protein encoded by RCKl displays some characteristics similar to those of the larger of the two delayed rectifier K+ channels found in the ,&cell. Notably, the channels display the same conductance (10 pS) under a similar ionic concentration gradient, inactivate slowly during a maintained depolarisation and have a similar sensitivity to TEA [6,1,0]. We have investigated the presence of mRNA sequences similar to the RCK family in insulin- producing cells using the polymerase chain reaction [13] and oligonucleotide primers directed against con- served regions of the RCK family. A similar approach has previously been successfully used to identify se- quences coding for K+ channels in genomic DNA [14].
Using cDNA derived from isolated islets of the obese hyperglycemic (&lob) mice fl5] and from the insulin secreting cell lines HIT T15 [16] and RINm5F [17], we have identified 8 different cDNA sequences which show close homology to the RCK and MBK family. Three of these sequences have not been described previously.
2. MATERIALS AND METHODS 2.1. Polymerase chain reactions
Total cellular RNA was prepared [18] from the hamster and rat in- sulinoma cell lines HIT T15 [16] and RINmSF [17], the human epidermal carcinoma cell line A431 [19] and from ob/ob mouse pan- creatic B-cells [15]. isolated as previously described [ZO]. Poly(A+)
Published by Elsevier Science Publishers B. V. (Biomedical Division)
RNA was selected using an oligo(dT) cellulose column (Pharmacia, Uppsala) and converted to single-stranded cDNA using oligo(dT) primers as previously described [21]. Approximately 50 ng cDNA was used as a template for enzymatic amplification with a ther- mostable Thermus aquaticus (Taq) DNA polymerase (Perkin Elmer Cetus, Norwalk, CT) and a DNA thermal cycler from the same com- pany. Alternatively, DNA prepared from a hgtll cDNA library generated from HIT T15 cells was used as template for the amplifica- tion reaction. The first two amplification cycles had the profile 94”C, 1 min; 37OC, 3 min; 72”C, 15 s and the following 28 cycles had the profile 94”C, 1 min; WC, 2 mitt; 72”C, 15 s with an extra 5 s at 72°C added in each cycle. The following two degenerate oligonucleotide primers were employed in the reactions:
5’ATTGGATCCAT-(C/A)TT(C/T)TTCCTCTTCAT (sense
primer) and ATTGAATTCAC(A/C)CC(A/C/T)GC-
(A/G)AT(G/T)GCACA (antisense primer). The amplification pro- ducts were analysed on agarose Tris-acetate buffer gels and DNA fragments of the expected size were excised and purified using a Geneclean kit (Bio 101, La Jolla, CA). DNA fragments were then digested with EcoRI and BumHI (to facilitate subcloning, the 5’
parts of the two primers were designed to contain an EcoRI and a BumHI site respectively), cloned into EcoRI/BumHI cut Ml3mpl8/19 or Bluescript vectors (Stratagene, USA) and sequenced using the Sequenase dideoxy nucleotide sequencing [22] system (United States Biochemical Corp., Cleveland, OH, USA).
FIGURE la
s5 region
2.2. Northern (RNA) blot analysis
DNA fragments corresponding to each K+ channel cDNA were subcloned in pUC vectors. Plasmids were then grown, linearized and labeled with ‘*P using random hexanucleotide priming [23]. Total cellular RNA was prepared from various rat tissues and rat and human cell lines using the LiCl/urea method [16], poly(A+) RNA was selected, size fractionated on 0.8% agarose/formaldehyde slab gels and transferred to nitrocellulose filters (Schleicher and Schiill, Dassel, FRG) using standard procedures [24]. Filters were dried and baked under vacuum, prehybridized and hybridized in 50% for- mamide at 42°C against the 32 P- labeled DNA probes as previously described [24]. Following washings under high stringency conditions (3 x 30 min at 55°C in 0.1 x SSC, 0.1% SDS), filters were exposed to Hyperfilm (Amersham, UK) using intensifying screens for 2-5 days at -70°C.
3. RESULTS
We designed our PCR primers against the two best conserved membrane-spanning domains SS (sense primer) and S6 (antisense primer) of the RCK family of voltage-gated brain K+ channels [ 11,121. Sequencing of
RCK-1 GGC GTC ATA CTG TTT TCT AGT GCA GTG TAC TTT GCG GAG GCG CAA GAA GCT GAG TCG CAC TTC TCC
HK-1 G C
RCK-2 A C C C C C T C C A ATCT C T CT
IN-2 A c c C C T C C A TTTT C T CT
RCK-3 G C T C C C T A C CC TCT GGT T AA
KK-3 G C T C C c T A C CC TCT GGT T AA
RCK-4 G c c C C T T A A T C ACC A C T CAA
HK-4 G T C C C C T T A T cc ACT A C T CAA
RCK-5 G c c c T C T A A T T G CGA T C G C
HK-5 G c c c T C T A A T T G AGA T C G C
IN-6 CCC C c c C T AT CCGG TG CAC T A
HaK-6 CCC C c c c T AT CCGG TG CAC T A
RK-6 T GGC C c c c c c A TT C CGG TG CTC T AT HaK-7 ____________________--- C A C AAC CAG G C
RK-6 c c T T T A T C ACT A C T CAA
RCK-1 AGT ATC CCC CAT GCT TTC TGG TGG GCG GTG GTG TCC ATG ACC ACT GTG GGA TAC GGT GAC ATG TAC KKK-1
RCK-2 m-2 RCK-3 MK-3 RCK-4 xX-4 RCK-5 MK-5 MK-6 HaK-6 RK-6 HaK-7 RK-8
C A C T
C A C
G C
G C
C T A G T
C T A G T
C C G C
C G
c G G C T
C G GTC T
C T CTC
A c
c A A T
T TAA
T TAA
C AA
A A AA
T AA
T CA A
A C C
A C
A CA
A CA
G A CA
A A CA
T A
A A T T G G A T T G
A C T T T C
A T T T T C
A c G AC
A C T G AC
A A C T A GTT
A A C T A GTT
C G T C T G GCA
G T C T G GCA
T A T C T A GCA
CACTGG AGG
A C T G AC
Fig. 1. Nucleotide (a) and predicted amino acid (b) sequences of amplified K+ channel DNA fragments. Sequences are compared with those of RCK-1 [ll], RCK-2 (0. Pongs, unpublished sequence) and RCK-3,4,5 [12]. Diverging positions are indicated. Identical positions are left out.
(- - -) Sequence not revealed.
Volume 263, number 1 FEBS LETTERS April 1990 fragments amplified from ob/ob mouse islets revealed
6 different, but closely homologous, sequences related to this family. Five of these appear to be the mouse islet counterparts (MKl-5) of previously identified members of the rat brain RCK family (RCKl-5) (Fig. 1). MKl is
almost identical to MBKI which is derived from mouse brain [lo]. A sixth mouse islet sequence (MK6) is, however, novel and may derive from a hitherto uniden- tified K+ channel gene (Fig. 1). We repeated the same procedure on two insulin-producing tumour cell lines,
Fig. I continued. FIGURE: la (continued)
Sxi region --
BCK-1 CCT GTG ACA ATT GGA GGC AAG ATC GTG GGC ICC TTG TGT m-1
RCK-2 C A GGG T AC
HZ-2 CA GGA G A AC
RCK-3 A C A T T TCT
log-3 A c A T TCT
RCR-4 C ATC G G A T G C
MK-4 C ATC G C G A T G C RCK-5 A ACT C G A A T TC
MK-5 A ACT C C G A T TC
HK-6 c c CGG T TC
l&K-6 C C CGG T TC
la-6 c c TGG T ----_-___
f&K-? CAC TGG T GC
RX-8 CAC TGA G T CG C
FIGURE lb
RCK-1 m-1 RCK-2 u-2 RCK-3 HK-3 RCK-4 nK-4 RCK-5 XK-5 KK-6 HaK-6 RK-6 SIaK-7
RCK-1 m-1 RCK-2 HK-2 RCK-3 IN-3 RCK-4 HK-4 RCK-5 HICK-5 XK-6 HaK-6 RK-6 RaK-7
55 region --
GVILPSSAVYFAEAEBAESHFSSIPDAFWWAVVSMTTVGYGDHY w
DDVD L P DDVD L P DDPS G If DDPS G B D PTT DDPTT
D RD Q P D RD Q P
V SDRVDT T
V VDRVDT T
V VDRVDS T
________ DRQG
S6 region PVTIGGKIVGSLC
x v
R v
I v I v T T
V V V I v
T T
T H
T H
T I(
AT K
V V
E TI A
ES T A
S T A
T G R
A a b d e f g
28S- 18S-
28S- 18S-
Fig. 2. Northern blot analysis using MK-1 in (A) and MK-2 in (B) as probes. Samples of 10 fig poly(A)+ RNA each from RINm5F cells (lane a), HIT ‘IX cells (lane b), rat brain (lane c), rat liver (lane d), rat spleen (lane e), rat heart (lane f), rat skeletal muscle (lane g) and A431 cells (lane h) were electrophoresed, transferred to nitrocellulose filters and hybridized with the probes. Note that lanes a, b, c and h have been overexposed relative to the other lanes to visualize the mRNAs that hybridized. The arrow indicates a faint 6-7 kb band in brain RNA that
hybridized with MK-2.
HIT Tl5 and RINmSF. Four different PCR amplifica- tion products were obtained from size-fractionated (> 4 kb) first-strand cDNA prepared from mRNA from RINmSF cells. Two of these (RKl and RK4) were iden- tical to RCK-1 and RCK-4 respectively over the amplified region whereas the third (RK6) probably
represents the rat counterpart to MK6 (Fig. 1). The fourth amplification product (RK8) was novel. The amino acid sequence encoded by the RK8 fragment was identical to that of RK4 despite several differences in the nucleotide sequence (Fig. 1).
Two RCK counterparts were identified in cDNA
Volume 263, number 1 FEBS LETTERS April 1990 prepared from HIT T15 mRNA. One of these, HaKl,
was closely similar to RCK-1 (not shown), but the other, HaK7, was novel (Fig. 1). When DNA prepared from a HIT T15 hgtll cDNA library was used as a source of DNA for PCR reaction, two sequences were found. One was identical to RCK-2 whereas the other (HaK6) was closely homologous to MK6 and RK6 (Fig. 1).
When the PCR ~pli~cation products were studied by Northern blot analysis, all probes tested (MKl-5) hybridized strongly to a large (approximately 8 kb) skeletal muscle mRNA (Fig. 2A, B). A fainter band of similar size was seen in rat liver mRNA, whereas no detectable hybridization occurred to spleen or HIT T15 mRNA (Fig. 2A, B). MKl hybridized weakly to an 8 kb rat brain mRNA (Fig. 2A). MK2 hybridized weak- ly to a smaller mRNA (6-7 kb) in rat brain (Fig. 2B).
No detectable hybridization occurred to rat brain mRNA using MK3-6, HaK7 or RK8 as probes (results not shown). Two faint bands of sizes 7-10 kb were detected in heart mRNA using MKl , MK3 and MK5 as a probe (Fig. 2B), whereas none of the other PCR amplification products (MK2, MK4, MK6, HaK7 and RK8) generated a detectable signal in heart mRNA (results not shown). Two bands of about 2 and 4 kb were seen in RINmSF and A431 mRNA when hybridiz- ing with MK2 (Fig. 2B), MK4, MK6, HaK7 and an only 4 kb band was detected in these RNAs using RK8 as a probe (results not shown). HaK7 hybridized weakly to 2 and 4 kb mRNAs in HIT T15 mRNA (result not shown).
4. DISCUSSION
Voltage-gated K+ channels are known to be present in pancreatic P-cells where they play an important physiological role in repolarization of the action poten- tial. We have now identified 8 distinct but closely homologous putative I<+ channel sequences in cDNA prepared from isolated pancreatic islets or insulin- producing cell lines. These PCR amplification products show close similarity to the SS-S6 region of the RCK family of voltage-gated I(+ channels. This suggests that members of the RCK family are also expressed in non- neuronal tissue and that the novel sequences (MK6, HaK7, RK8) may constitute further members of this family.
Without knowledge of the gene structures, the possibility that the S5-S6 region of the RCK family of proteins is contained within a single exon, and thus the risk that one or several of the amplified sequences originate from chromosomal DNA fragments con- taminating the poly(A)+ RNA has to be considered.
However, the differences in the spectrum of putative channel sequences obtained from the various mRNA sources (Table I) favour ~p~~cation from cDNA and not from genomic DNA, especially since no one single
source amplified all 8 channels. The largest diversity of channel sequences was seen in isolated pancreatic islets.
When comparing the amplification products from islets and &cell lines, two were always detected (RK/MK/HaKl and RK/MK/HaK6), suggesting that these are expressed in the &cell. HaK-6 was also ob- tained from a hgtl 1 cDNA library, minimizing the risk that this product arose from amplification of genomic sequences. Finally, RK4 and RK8 were abundant amplification products in size-selected first strand cDNA, which also argues against genomic amplifica- tion, since then no bias in favour of any specific chan- nel should exist. The absence of sequences corresponding to HaK7 and RK8 in the islet amplifica- tion reaction suggests that these are spuriously express- ed in insulin-producing tumour cells. Both HIT T15 and RINmSF cells display altered characteristics of in- sulin secretion, however, electrophysiological studies have not indicated any differences in voltage-gated K+
channels between normal &cells and insulin producing tumour cell lines. The Northern blot analyses do not entirely resolve the matter of expression of specific channels in RINmSF and HIT T15 cells because of significant cross-reactivity between the different pro- bes. Two mRNAs of 2 and 4 kb present in the insulin- producing cell lines hybridized with several of the amplification products. Since the PCR reactions using HIT T15 and RINm5F cDNAs yielded more than two products, these two bands are either heterogeneous or other, less abundant, mRNAs coding for voltage-gated K+ channels are expressed, in these cells.
Functional characterization of the channels in Xenopus oocytes following microinjection of in vitro transcribed RCK-specific RNAs has shown them to be a family of voltage-gated I<+ channels [ 121. Despite the extensive homology at the level of derived amino acid sequence, the channels show considerable diversity in their voltage-dependent gating and town-binding pro- perties. The RCK family are also homologous to the
Table I
Frequency with which each K+ channel sequence occurred following PCR
cDNA source Number of clones identified corresponding to different RCKs
I 2 3 4 5 6 7 8
0Wob islets 5 2 10 4 7 1 0 0
HIT T15 70 000020
RINmSF 10 03 0 10 2
HIT T15 library 0.1 000 100
Amplified DNA fragments were processed as described in section 2
and subcloned Ml3 or Bluescript vectors. Numerous DNA sequences
without homology to the RCK-family were revealed. The table gives the number of MI3/Blu~pt clones ~n~n~g inserts identical to
or homologous with previously known RCK sequences
Shaker family of Drosophila voltage-gated K+ chan- nels. An additional mammalian K+ channel, drk-1, less homologous to the RCK family in the SS-S6 region than the novel sequences obtained in the present study, is also voltage-gated 1251. We therefore consider it like- ly that the new K+ channel-like sequences (MK6, HaK7 and RKS) identified here represent additional voltage- gated K+ channels. Since the primers that we used did not encompass the putative voltage-sensor region (S4) of the K+ channel, but rather an extracellular in- termembranous domain, there remains the possibility that they may include other types of K+ channels, e.g.
the ATP-sensitive K+ channel which is of relatively high abundance in the p-cell. Furthermore, diversity of K+ channels could be generated by heteropolymer subunit arrangement in which the ATP-sensitive K+
channel could have a subunit which does not share the overall protein structure characteristic for the RCK/MBK family. However, the nature of these chan- nels will be resolved first by the isolation and functional expression of full length cDNA clones.
Acknowledgements: The technical assistance of Viveka Svensson and S&run Svanholm is gratefully acknowledged. The work was sup- ported by the Juvenile Diabetes Foundation Int., The Swedish Medical Research Council (12x-8273, 12x-109, 19x-00034 and 4x-08641), The Bank of Sweden Tercentenary Foundation, The Swedish Diabetes Association, The Hoechst Foundation, The Swedish Cancer Research Fund, The Medical Research Council (UK), the British Diabetic Association, E.P. Abraham Fund and the European Molecular Biology Organization. F.M.A. is a Royal Socie- ty 1983 University Research Fellow.
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