(Received for publication, July 18, 1995; and in revised form, August 24, 1995)
From the
Insulin secretion is associated with changes in pancreatic
-cell K
permeability. A degenerate polymerase
chain reaction strategy based on the conserved features of known
inwardly rectifying K
(K
) channel genes
was used to identify members of this family expressed in human
pancreatic islets and insulinoma. Three related human K
transcript sequences were found: CIR (also known as cardiac
KATP-1), GIRK1, and GIRK2 (KATP-2). The pancreatic islet CIR and GIRK2
full-length cDNAs were cloned, and their genes were localized to human
chromosomes 11q23-ter and 21, respectively. Northern blot analysis
detected CIR mRNA at similar levels in human islets and exocrine
pancreas, while the abundance of GIRK2 mRNA in the two tissues was
insufficient for detection by this method. Using competitive
reverse-transcription polymerase chain reaction, CIR was found to be
present at higher levels than GIRK2 mRNA in native purified
-cells. Xenopus oocytes injected with M2 muscarinic
receptor (M2) plus either GIRK2 or CIR cRNA expressed only very small
carbachol-induced currents, while co-injection of CIR plus GIRK2 along
with M2 resulted in expression of carbachol-activated strong inwardly
rectifying currents. Activators of K
channels failed to
elicit currents in the presence or absence of co-expressed sulfonylurea
receptor. These results show that two components of islet cell K
channels, CIR and GIRK2, may interact to form heteromeric
G-protein-activated inwardly rectifying K
channels
that do not possess the typical properties of K
channels.
The permeability of K ions plays a crucial role
in the control of pancreatic islet
-cell excitability and insulin
secretion(1, 2) . Electrophysiological studies have
revealed at least four classes of functionally distinct
K
currents in
-cells: 1) ATP-sensitive
K
channels that close in response to increased
intracellular ATP/ADP ratios generated by increased metabolic flux, 2)
voltage-gated K
channels activated by depolarization,
3) large and small conductance calcium-activated K
channels, and 4) ligand-gated K
channels that respond
to physiological agonists acting through G-protein-coupled
receptors(1, 3, 4, 5) . Because
pancreatic islet K
channel genes are only beginning to
be identified, the molecular basis for most of these currents remains
unknown(6, 7, 8) . Furthermore, their precise
contribution to insulin secretory activity is largely not understood.
The characterization of K
channel proteins synthesized
in islet cells is of great practical interest, as it will contribute to
understanding
-cell electrophysiology and potentially enhance the
development of more effective and specific drugs to manipulate insulin
secretory function. Furthermore, because defective insulin release is
central to the pathogenesis of non-insulin-dependent diabetes
mellitus(9) , these molecules provide a valuable source of
candidate genes to study the inherited basis of this disorder.
A
novel superfamily of genes encoding inward rectifying K (K
) channels has been recently
identified(10, 11, 12, 13) . Unlike
voltage-activated K
channels of the Shaker gene family
which are opened by membrane depolarization(14) ,
K
channels are open at hyperpolarized potentials. These
channels share an underlying conserved structure, with two predicted
membrane spanning domains, homologous to the fifth (S5) and sixth (S6)
transmembrane domains of voltage-gated channels, encompassing a region
homologous to the pore-forming portion of voltage-activated
channels(14) . K
channels, however, lack a portion
homologous to the amino-terminal region of voltage-gated channels
(S1-S4).
Islet -cells contain K
channels
which have gating properties similar to members of the K
family of channels, including ATP-sensitive channels (K
)
and G-protein-activated K
channels that do not possess
features of K
channels(2, 4) . These
channels may be involved in the regulation of insulin secretion by
glucose and/or neurotransmitters acting through G-protein-coupled
receptors(1, 4, 15, 16) . Because
islet K
proteins are likely to share homology to other
K
molecules, we have employed a degenerate polymerase
chain reaction strategy based on the conserved features of known
K
genes to identify and clone members of this family
expressed in pancreatic islets. We demonstrate here the presence of
three related K
transcript sequences, CIR, GIRK1, and
GIRK2, in human pancreatic islet cells. In contrast to previous work
based on studies with cultured tumor
-cell lines which disclosed
the presence of GIRK2, but not CIR(15, 17) , CIR was
found to be more abundant than GIRK2 in native purified pancreatic
-cells. Cloned islet cell CIR and GIRK2 cDNAs are shown to express
heteromultimeric G-protein-activated K
channels that do
not possess characteristic features of K
channels in the
presence or absence of co-expressed sulfonylurea receptor (18) .
The 3` rapid
amplification of human islet KATP-1/CIR cDNA was performed as follows:
reverse transcription of RNA was primed with T(A/G/C)N,
where N is A, G, C, and T. PCR amplification was performed for 35
cycles at 94, 48, and 72 °C for 1 min each step with primer
5`-GGGCATTATACTCCTCTTGG-3`, derived from the insulinoma CIR degenerate
PCR fragment sequence, and T
(A/G/C)N. A unique 2.4-kb band
was purified and partially sequenced directly.
Northern blots were
prepared with selected human poly(A) RNA-enriched
samples, hybridized with
P-labeled probes, and washed at a
final stringency of 0.1
SSC, 0.1% SDS, 65 °C before
exposure to x-ray film for 48 h.
Figure 1:
Nucleic acid sequence comparison of the
three human islet-cell K cDNAs identified by degenerate
PCR. Bases that match a consensus, defined as at least 2 conserved
bases, are shaded in black. Primer sequences used to
amplify these fragments have been omitted and are noted in the
text.
Figure 2: Alignment of the deduced amino acid sequence of human islet GIRK2 (hi-GIRK2), mouse brain GIRK2 (mb-GIRK2), human cardiac KATP-1/CIR (hc-KATP-1/CIR), and rat islet CIR (ri CIR). Sequence alignments were created with Megalign (DNASTAR) and visual modification. Amino acid identities are indicated by background shading. Predicted M1 and M2 transmembrane domains and the P pore region are highlighted by a line above the sequence lineup. Conserved consensus serine/threonine phosphorylation sites are boxed. Two possible N-glycosylation sites are marked, one presumed extracellular site present only in the CIR sequences (*), the other of uncertain topology but conserved in the four proteins (**).
The
expression of hi-GIRK2 in human tissues was evaluated by Northern blot
and RT-PCR analysis. A distinct band of approximately 5.7 kb, and a
more diffuse signal of approximately 2.4-2.8 kb, were observed by
Northern analysis in poly(A)-enriched RNA from human
insulinoma (Fig. 3), while the abundance in purified human
islets was insufficient to detect a similar signal. To further define
the tissue distribution of hi-GIRK2 mRNA, RT-PCR analysis of a serial
dilution of cDNAs was performed under reduced cycling conditions that
allowed relative semiquantitation among tissues (Fig. 3). Using
primers specific for hi-GIRK2, a unique PCR product of the expected
size was observed to be most abundant in insulinoma and cerebellum RNA,
while lower levels of expression were detected in all other tissues
examined, including human islet and pancreatic exocrine tissue (Fig. 3). The fact that RT-PCR product signals were only
slightly enhanced in islets relative to exocrine samples could reflect
significant cross-contamination of the exocrine and islet preparations
and/or the existence of GIRK2 mRNA at lower levels in exocrine tissue.
Figure 3:
Distribution of GIRK2 and CIR mRNA in
adult human tissues. A, Northern blot analysis.
Poly(A) RNA from human islets (HI) (2 and 1.5
µg), insulinoma surgical specimen (INS) (2 µg), and
pancreatic exocrine (EXO) (1.5 µg) was blotted and
hybridized with
P-labeled hi-GIRK2 cDNA first, then
stripped and rehybridized with a
P-labeled human islet CIR
0.7-kb PCR product. B, reverse transcription-PCR analysis.
Total RNA from human tissues was treated with RNase-free DNase,
reverse-transcribed, and cDNA corresponding to 80, 20, 5, and 1.25 ng
of RNA was amplified for 25 or 28 cycles using primers specific for
human islet GIRK2 and CIR, respectively. INS, insulinoma; HI, pancreatic islets; EXO, exocrine; LIV,
liver; CER, cerebellum; MUS, muscle; VEN,
left ventricle; and DUO, duodenum.
Segregation analysis of a panel of human-Chinese hamster ovary/mouse somatic cell hybrids with specific oligonucleotides that amplified a 130-bp fragment from the 3`-untranslated region of the hi-GIRK2 gene allowed unequivocal localization of this gene to chromosome 21 (data not shown). This confirms data reported by others during revision of this manuscript, which indicated that this gene maps to chromosome 21q22(28) .
When these
studies were initiated, only the rat CIR nucleic acid sequence was
known. To identify the human CIR sequence, an oligonucleotide was
synthesized based on the human -cell tumor PCR product
(hi-CIR.pcr) sequence and 3` rapid amplification of cDNA was performed
to obtain further 3` human sequence from human islet mRNA (data not
shown). Partial sequence analysis, which eventually proved to be
identical to that entered in public nucleic acid data bases with the
designation of human cardiac KATP-1, allowed us to design
oligonucleotide primers for chromosomal localization studies. The
116-bp PCR product that was thus generated was shown to segregate with
chromosome 11 in human-rodent somatic cell hybrids. A panel of
chromosome 11 deletion hybrids was utilized for further physical
mapping of the human CIR gene to chromosome 11q23-ter (Fig. 4).
Given the role of islet cell K
channels in the
regulation insulin secretion, CIR may be regarded as a candidate gene
for any form of diabetes mapping to this chromosomal region.
Interestingly, a 45,X male with a translocation (Y;11)(q11.2;q24) has
been reported, and the phenotype includes hypoglycemia along with
multiple dysmorphic features(29) .
Figure 4: Localization of human CIR gene to 11q23-ter. Top panel, ethidium bromide-stained agarose gel showing the segregation of human CIR 116-bp PCR products in a panel of human chromosome 11 deletion somatic cell hybrids (hamster-human). Bottom panel, schematic of human chromosome 11 content of the different samples. Hu, human genomic DNA; J1, chromosome 11; J1-11, 11p only; J1-44, 11 (deletion q11-q14); J1-46, 11 (deletion q11-q13); R29-4D, 11q14-qter only; C, no DNA control; LSH, translocation retaining 11pter-11q23ter; R229-3A, translocation retaining 11q23-qter only.
Northern blot analysis, using a radiolabeled 0.7-kb human islet CIR PCR fragment as a probe, disclosed the presence of three major transcripts of 6.8, 5.4, and 2.4 kb, which appeared to be abundantly expressed in both islet and exocrine pancreas RNA, but very weakly expressed in insulinoma (Fig. 3). Hybridization of a multiple human tissue Northern blot (Clontech, Palo Alto, CA) with the same probe disclosed the same pattern of transcript sizes in total pancreas and, with lower intensity, in heart. No clear signal was detected in the remaining tissues (data not shown). Using RT-PCR analysis, a unique band of the expected size was seen in all human tissues examined, although human pancreatic islet and pancreatic exocrine samples showed the highest intensity (Fig. 3). Interestingly, the relative intensity of hybridization and RT-PCR signals was not indicative of specific expression in either endocrine or exocrine pancreatic cells, suggesting that CIR mRNA is present in both fractions.
The close sequence
homology shared by CIR and GIRK2 (Fig. 2) was employed to assess
their relative abundance in pancreatic islet cell subpopulations using
competitive RT-PCR. To circumvent the fact that minor differences in
the efficiency of PCR amplification of different sets of primers can
greatly affect the rate of accumulation of two different PCR products,
one pair of PCR primers expected to amplify both cDNAs was used. As
predicted from the sequences of GIRK2 and CIR mRNAs, RT-PCR from total
RNA derived from fluorescence-activated sorted purified pancreatic
islet -cells and non-
islet cells yielded a single 213-bp
band after 28 cycles (Fig. 5). After Sau3AI digestion,
which was expected to cleave CIR but not GIRK2, a major component of
the amplified product appeared to be cleaved, while a 213-bp band of
lower intensity remained (Fig. 5). Conversely, after selective
cleavage of GIRK2 sequence with HincII, a predominant
component of the 213-bp PCR product was left intact. Cleavage with both
enzymes resulted in complete digestion of the 213-bp band. An analogous
restriction enzyme pattern was observed using an independent pair of
primers and sequence-specific restriction enzyme digestion (data not
shown). The restriction pattern in all cases was consistent with that
expected if the major products of amplification were only CIR and
GIRK2, rather than additional closely related known K
genes such as GIRK1 and GIRK3(12, 27) . The
relative accumulation of GIRK2 and CIR PCR products was independent of
cycle number between 25 and 35 cycles, suggesting that both templates
were amplified with a similar efficiency. Thus, both CIR and GIRK2 mRNA
were expressed in
-cell and non-
-cell enriched preparations,
while in the the two primary islet cell preparations the relative
abundance of CIR mRNA clearly exceeded that of GIRK2. This finding is
in apparent contrast to the report of Ashford et
al.(15) , that CIR (KATP-1) did not hybridize to
poly(A)
RNA from a rat insulinoma cell line. However,
our Northern blot data also suggest that GIRK2 is more abundant than
CIR in a human insulinoma specimen, but not in native islets. One
likely explanation for this observation is that tumor
-cell lines
often either do not express the characteristic markers of the native
-cell phenotype or do so at abnormal levels (30) .
Figure 5:
GIRK2 and CIR mRNA expression in purified
islet cells. Total RNA from fluorescence-activated cell sorted
and non-
islet cells was reverse-transcribed, treated with DNase,
and amplified by PCR with one pair of primers expected to yield a
single 213-bp band from both CIR and GIRK2. Products were analyzed by
agarose gel electrophoresis after selective restriction digestion. Sau3AI (SAU3) is expected to cleave only CIR-derived
amplicon (61-, 75-, and 77-bp fragments), HincII (HINC
II) is expected to cleave only the GIRK2 amplicon (101- and 112-bp
fragments). Double digestion (SAU+HINC) completely
cleaves the 213-bp PCR product.
Figure 6:
Expression of carbachol (CCh)-activated K currents in oocytes
expressing GIRK subunits. A, two microelectrode currents
before (pre), during (CCh), and after (post)
exposure to 10 mM CCh from an oocyte that had been injected
with
15 ng each of M2, GIRK2, and CIR cDNAs. Experiment performed
at room temperature. The bath contained 98 mM KCl, pH 7.5. B, currents during exposure to carbachol with mean current
before and after subtracted from A. C, steady-state
current (I)-voltage (V) relationship from B. D, carbachol-activated current at -80 mV from oocytes
expressing M2 receptor plus GIRK2, CIR, or a mixture of both cDNAs
(each injected at
15 ng/oocyte). Bars show mean ±
S.E. (n indicated).
Although CIR was initially reported as
the cardiac ATP-sensitive K channel
(KATP-1)(15) , inhibition of ATP production failed to activate
K
currents in oocytes injected with CIR and/or GIRK2.
This was also true when the sulfonylurea receptor(18) , a
candidate site for endowment of ATP sensitivity to islet K
channels, was co-expressed. The inward rectification of the
expressed receptor-activated K
channels, which is
likely to be determined by the primary structure of the pore forming
subunit itself, is much stronger than that of native K
channels (31) (Fig. 6). The studies presented here thus
provide evidence that oocytes injected with GIRK2 do not express
K
channels and confirms data recently presented by others
that CIR expression in multiple cell systems does not result in
K
channels(26) . A role for these molecules in
K
channel complexes in association with as yet
unidentified subunits nevertheless remains possible.
The precise
relationships between the molecular components of human islet K channels identified in the present study and native
-cell
G-protein-activated K
currents remain to be defined.
In addition to K
channels, which are known to be gated
through G-proteins, small conductance channels that are activated
through G-protein-coupled receptors and that are insensitive to ATP and
sulfonylureas have been recorded(4) . A role for
G-protein-activated K
channels in islet
-cell
physiology can be anticipated given that multiple hormones and
neurotransmitters cause hyperpolarization and inhibit insulin secretion
via G-protein-coupled receptors and K
channels(2, 4, 16) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U24660[GenBank].