Expression of Voltage-Gated Potassium Channels in Human and Rhesus Pancreatic Islets

To establish the profile of Kv channel subtypes in islets, we performed RT-PCR amplification on RNA extracted from human islets using a subtype-specific primer pair for each of the 26 members of the Kv1–Kv11 families.

To obtain efficient and specific primer pairs, we used the Vector NTI program (Informax, Frederick, MD) to select sequences. Each primer sequence then was submitted to a basic local alignment search tool search against GenBank to ensure specificity of the selected sequence. Specific primer pairs were then used to amplify Kv channel subtypes from human fetal brain cDNA. Finally, the most effective primer pairs for each subtype were used to study the expression of Kv channels in human islets (Table 1).

A rabbit polyclonal antibody for Kv1.6 was raised against the peptide RRSSYLPTPHRAYAEKRM, corresponding to residue 509–526 of the rat Kv1.6 (34). Human Kv1.6 shares 17 of 18 amino acid residues with the rat channel in this region. Rabbit polyclonal antibodies against Kv2.1 and Kv3.2 proteins were purchased from Alomone Labs (Jerusalem, Israel). The Kv2.1 antibody was raised against the peptide HMLPGGGAHGSTRDQSI, corresponding to residue 837–853 of rat Kv2.1. Human Kv2.1 shares 15 of 17 amino acids with this region of the rat channel. The Kv3.2 antibody was raised against the peptide DLGGKRLGIEDAAGLGGPDGK(C), corresponding to residues 184–204 of rat Kv3.2. The human and rat Kv3.2 have 19 identical amino acids in this peptide sequence. Antibodies for Kv2.1 and Kv3.2 detected only a single band in Western blots of the cognate channels expressed in HEK 293 cells.

Human pancreatic islets were obtained from the University of Alberta (Edmonton, AB, Canada). The islets were purified based on staining with the β-cell-specific dye diphenylthiocarbazone (Sigma), as previously described (35). The total RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s instruction. Because most of the human Kv channel genes are intronless, we treated the total RNA with DNaseI (Ambion, Austin, TX) to eliminate traces of genomic DNA. A control PCR reaction was performed with β-actin primers (forward: 5′-GCCCTTTCTCACTGGTTCTC-3′; reverse: 5′-CTTTACACCAGCCTCATGGC-3′) located on an intron region to verify the absence of genomic DNA.

The DNaseI-digested RNA was transcribed into cDNA using SensiScript Reverse Transcriptase from Qiagen (Valencia, CA), per the manufacturer’s instruction. Human fetal brain poly A+ RNA was purchased from BD Biosciences Clontech (Palo Alto, CA). The RNA was transcribed into cDNA with the Omniscript Reverse Transcriptase Kit from Qiagen following the manufacturer’s instructions. The cDNA was used as templates for the amplification of individual channel subtypes. Approximately 100 ng of total RNA was used in each reaction. A blank reaction was used as a control. The reaction conditions were as follows: start with 95°C for 15 min to activate the HotStarTaq DNA Polymerase (Qiagen), then denature at 94°C for 30 s, annealing at 56°C for 30 s, followed by an extension at 72°C for 1 min. The total number of cycles was 35. The amplification was followed by a 10-min extension at 72°C. The primers for each Kv channel subtype are listed in Table 1. The sequences for the insulin primers are the following: sense primer 5′-CCAGCCGCAGCCTTTGTGA-3′, antisense primer 5′-GCTGGTAGAGGGAGCAGAT-3′. The sequences for the trypsin II primers are the following: sense primer 5′-GCCCCCTTTGATGATGATG-3′, antisense primer 5′-ACACGCGGGAATTGATGAC-3′. The sequences for the K_ir6.2 primers are the following: sense primer 5′-AAGAAGTGAAGTGGGACC-3′, antisense primer 5′-GTTGCCTTTCTTGGACAC-3′.

For in situ hybridization (ISH), oligonucleotide probes specific for human Kv2.1, Kv2.2, Kv3.1, Kv6.1, Kv6.2, and Kv9.3 (Table 2) were end labeled with biotin-16-dUTP (Roche Molecular Biochemicals, Indianapolis, IN). The digoxigenin oligonucleotide tailing kit (Roche Molecular Biochemicals) was used according to the manufacturer’s protocol, except for replacement of digoxigenin-dUTP with biotin-16-dUTP. For Kv3.3 and Kv9.2, riboprobes instead of oligo probes were used for ISH. A 298-bp fragment from the 3′-untranslated region of the human Kv3.3 cDNA insert was prepared by RT-PCR amplification from human fetal brain cDNA, using a Kv3.3-specific primer pair (Table 1). Similarly, a 543-bp fragment from the 3′-untranslated region of the human Kv9.2 cDNA was amplified, using a Kv9.2-specific primer pair (Table 1). The reaction conditions were the same as described above for subtype RT-PCR. The PCR fragments were subcloned into the plasmid vector pCRII-TOPO (Invitrogen, Carlsbad, CA) and sequenced to verify their identity. To prepare riboprobes for ISH, the Kv3.3 and Kv9.2 containing vectors were linearized with the restriction enzymes SpeI and NotI, respectively, to create template DNA. Biotinylated sense and antisense riboprobes were then generated by in vitro transcription using Biotin RNA Labeling Mix (Roche Molecular Biochemicals).

Cryostat sections (8 μm) of human pancreas (the National Disease Research Interchange) and rhesus pancreas tissue, obtained under the approval of the Merck Research Laboratories Institutional Animal Care and Use Committee, were thaw mounted on SuperFrost plus slides (Fisher Scientific) and fixed with 4% paraformaldehyde. To improve the signal strength, a cocktail mixture of two labeled oligonucleotide probes (listed in Table 2) specific for a particular Kv channel subunit was used at a final concentration of 2 pmol/ml each. The ISH conditions were the same as those previously described (36). Bound probes were detected using the TSA direct red FISH tyramide amplification kit (PerkinElmer Life Sciences, Boston, MA) according to the manufacturer’s instructions. ISH using mRNA probes, immunohistochemistry (IHC) using antibodies, and combined double-label ISH/IHC were performed as previously described (37). ISH and IHC expression experiments were carried out on human and Rhesus pancreatic sections. The expression of Kv2.1, 2.2, 3.1, 3.2, 3.3, 6.1, 6.2, 9.2, and 9.3 subunits were examined in sections from both species, and in all cases, expression patterns in Rhesus and human were consistent. Double-staining cell identification experiments were carried out on the specimens that gave the best signal. Kv1.6 and Kv4.1 were only monitored in Rhesus.

Cell identification markers were antibodies specific for glucagon (Dako, Carpinteria, CA), insulin (Zymed Laboratories, South San Francisco, CA), and somatostatin (Dako). Matched preimmune sera or nonimmune control sera were used as negative controls for IHC. All antibodies were used at the manufacturer’s specified dilutions and incubated on the sections for 2 h at room temperature following the ISH procedures. Bound antibodies were detected using fluorescein isothiocyanate-conjugated donkey (MultipleLabel) IgG (Jackson Immunoresearch, West Grove, PA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR). Digital acquisition and image reassembly were carried out using a MicroMax CCD camera (Princeton Instruments, Princeton, NJ) and Metamorph imaging software (Universal Imaging, West Chester, PA).

The level of effectiveness of the Kv channel subtype-specific primers was tested by PCR using human fetal brain cDNA as templates. In all cases, a PCR product was visible on a 1% agarose gel (Fig. 1A). The expression profile for Kv channels was determined in at least two different human islet cDNA preparations. PCR fragments for Kv1.3, 1.6, 1.7, 2.1, 2.2, 3.1, 3.2, 3.3, 4.1, 4.4, 6.1, 6.2, 9.1 9.2, 9.3, 10.1, and 11.1 are identified as being present in the pancreatic islet preparation (Fig. 1B and Table 3). Probes for Kir6.2, representative of islet RNA, and trypsin, representative of acinar tissue RNA, indicate that there is some nonislet RNA present in the human islet preparation used for Fig. 1.

Kv3.4, Kv4.4, and Kv9.1 were only found in one of three human islet preparations. Kv4.4 and Kv9.1 appear as faint bands compared with brain and may be due to nonislet tissue in the one preparation. However, Kv3.4 was also found in only one of three human islet cDNA preparations, but as a more prominent band. This channel is usually responsible for an A-type potassium current and has been reported to be in β- and δ-cells from rat (21). This expression profile could be a natural variation within the human population or a marker for a possible disease state.

The expression of Kv1.7 was found in all three human preparations, but the band was weaker than the signal in brain and the signal of the other Kv channels in islets. These results were confirmed in a separate experiment where Kv1.7 and Kv1.6 were tested in a tissue panel including islets, brain, and skeletal muscle among others, where Kv1.7 was most prominent in skeletal muscle, confirming a previous report (38). While RT-PCR is not quantitative, these experiments suggest that the expression of Kv1.7 is lower in islets than for other Kv channels observed in this tissue.

Pancreatic islets are composed primarily of three cell types (i.e., α-, β-, and δ-cells); however, vascular endothelial cells (39) and interneurons (40) are found in islets as well. To identify the presence of channel subtypes in islet cells and to obviate the possibility of contamination from surrounding tissues, each of the most prominent islet Kv channel subtypes identified by PCR were tested by either or both ISH and IHC and compared with cell markers in sections of either Rhesus or human pancreatic tissue. Because Kv2.1 and Kv3.2 have been reported to exist in β-cells and in insulin-secreting cell lines (16,28), the cellular presence of these two channel subtypes was determined first. Pancreatic sections were probed for Kv2.1 mRNA with labeled antisense oligonucleotides by ISH (Table 2) and with an insulin-specific antibody by IHC in a double-staining experiment. Kv2.1 mRNA-positive cells located within an islet (Fig. 2A) exclusively colocalize with the insulin-containing cells, which confirms the expression of Kv2.1 in β-cells (Fig. 2B and C). Consistent with this finding, there is no colocalization of Kv2.1 and the glucagon signals (Fig. 2D). The presence of Kv2.1 protein in islets, but not in surrounding pancreatic tissue, was confirmed by immunostaining (Fig. 2E). Taken together, our results are consistent with the expression of Kv2.1 in pancreatic β-cells.

Expression of Kv3.2 was studied with the same double-labeling protocol used for Kv2.1. Kv3.2 antisense oligonucleotides (Fig. 3G) and insulin antibody (Fig. 3H) labeled the same population of islet cells (Fig. 3I), suggesting that Kv3.2 is expressed in β-cells.

Because both Kv2.1 and Kv3.2 are expressed in β-cells, we tested for their colocalization in pancreatic sections. Accordingly, we probed with a Kv3.2 antibody (Fig. 3J) and with Kv2.1 antisense oligonucleotides (Fig. 3K). As expected, both positive cell populations overlap completely, further confirming that Kv2.1 and Kv3.2 are coexpressed in β-cells, based on colocalization with insulin and each other (Fig. 3L).

Pancreatic tissue was probed for Kv2.2 mRNA and insulin, using the same protocol as for Kv2.1 (Fig. 2F and G). In marked contrast to Kv2.1, colocalization of Kv2.2 with insulin was not observed (Fig. 2H). Subsequently, a section was probed with Kv2.2 antisense oligonucleotides and a somatostatin antibody. Both probes labeled the same cell population (Fig. 2I–K), indicating that Kv2.2 is expressed in the δ-cells of the islet.

The double staining of Kv3.1 mRNA (Fig. 3A) and insulin (Fig. 3B) did not overlap, suggesting that Kv3.1 is not expressed in β-cells (Fig. 3C). Subsequently, a section was probed with Kv3.1 antisense oligonucleotides (Fig. 3D) and a glucagon antibody (Fig. 3E). Both probes labeled the same cell population (Fig. 3F), indicating that Kv3.1, in contrast to Kv3.2, is expressed in pancreatic α-cells.

Several silent subunits were found to be present in human islet cDNA by RT-PCR (see above). Human pancreatic sections were probed for Kv6.1 and Kv6.2 channels with labeled antisense oligonucleotides (Table 2) and with cell marker-specific antibodies in a double-staining experiment. The results shown in Fig. 4A–H demonstrate that Kv6.1 is colocalized with glucagon, but not with insulin. In Figs. 4I–L, Kv6.2 appears to colocalize with all of the insulin-containing cells. However, in a number of islets, Kv6.2 labels only a major fraction of the insulin-containing cells (data not shown). This distribution could indicate the existence of a subset of β-cells within some islets.

To determine whether Kv9.2 is expressed in pancreatic β-cells, we first performed an ISH experiment using Kv9.2-specific oligonucleotide probes, but could not detect a significant signal over background levels (data not shown). We then used a Kv9.2 riboprobe for ISH and the insulin antibody for IHC experiments. The results in Fig. 5A–C show that the Kv9.2 mRNA is expressed in islets, but does not colocalize with insulin.

The double-staining protocol with insulin antibody was used to determine the cell distribution of Kv9.3 mRNA. Kv9.3-positive cells completely overlap with insulin-containing cells (Fig. 5D–F), but not with those cells containing glucagon (Fig. 5I). These data suggest that Kv9.3 mRNA is exclusively expressed in β-cells. Although Kv10.1 and Kv11.1 are found in human islets by RT-PCR (see above), we did not attempt to further characterize the cell distribution of these two silent subunits.

Other subunits, such as Kv1.6, Kv3.3, and Kv4.1, were identified by RT-PCR in human islets. In ISH/IHC protocols, the three subtypes appear to be located outside of the islet, and no colocalization with insulin was observed for any of them (Fig. 6A–I). Morphologically, the tubular configuration of the Kv1.6-expressing cells suggests that these cells could be acinar, Schwann, or nerve cells (41–43).

The expression of Kv channels in islet cell types other than α-, β-, or δ-cells might contribute to the observed PCR signals (Fig. 1). However, this contribution does not appear to be significant because, by ISH, Kv channels that coexpress with either insulin, somatostatin, or glucagon are only found in cells that contain that marker. If other cell types were responsible for the PCR signal, at least some cells would be expected to show the Kv signal separate from the marker.

The identification of the molecular components for the I_DR in human β-cells is critical for the development of an inhibitor of this channel that would function as a glucose-dependent insulin secretagogue for the treatment of type 2 diabetes. This study is an attempt to identify the Kv subunits that are present in human β-cells, with the ultimate goal of correlating their biophysical and pharmacological properties with the currents found in β-cells. The initial ISH experiments on both human and Rhesus islets demonstrate that expression of the Kv channels under investigation exhibit the same pattern in both species. Our results combining RT-PCR with ISH and IHC strongly suggest that Kv2.1 and Kv3.2 are the major subunits in β-cells. In addition, silent subunits Kv6.2 and 9.3 are also present in β-cells (Table 3).

In heterologous expression systems, Kv2.1 and Kv3.2 express delayed rectifier-type currents that resemble those present in β-cells (44–46). Therefore, either one or the other or both may contribute to I_DR in β-cells. Because the tetraethylammonium ion and hanatoxin sensitivities of individually heterologously expressed channels are quite different, it may be possible to use these tools to distinguish the relative contributions of these channel subtypes to the β-cell I_DR (44).

It is interesting that two silent subunits, Kv6.2 and Kv9.3, are also expressed in human β-cells. In heterologous expression systems, these subunits are known to coassemble with subunits from either Kv2 or Kv3 families and to modify their function (24,47–49). It remains to be determined if this also occurs in β-cells. In addition two other silent subunits, Kv10.1 and 11.1, previously reported to be in pancreas and also known to associate with subunits from Kv2 and Kv3 family (50), are found in islets. Their presence in β-cells and their significance will require further investigation.

It is curious that members from two particular families distribute to different cell types within the islet. For instance, Kv2.1 and Kv3.2 distribute to the β-cell, whereas Kv2.2 and Kv3.1 are present in δ- and α-cells, respectively. This differential distribution has significant implications for the development of inhibitors that specifically target channels present in β-cells.

There have been reports of Kv1 family channels in islets, β-cells, and insulin-secreting cell lines (16,51,52). In human islets, others (51) have reported that by RT-PCR Kv1.1, Kv1.2, and Kv1.4 are not found, while Kv1.5 and Kv1.6 were present. In our studies of human islets, only Kv1.3 and Kv1.6 were identified by RT-PCR, with a very weak indication for Kv1.7. Kv1.6 is external to the islet, in contrast to the recent report of its presence in rat β-cells (53). While Kv1.4 seems to be absent from human islets by RT-PCR, it appears to be present in rat β-cells by Western blot and PCR (51), but not in mouse by immunostaining (53). These data could indicate differences in channel composition between species and highlights the importance of the identification of the relevant subunits in human β-cells.

The identification of different channel types exclusive to each of the three major cell types found in the islet suggests that it may be possible to select for cell-type-dependent intervention through block of their respective I_DRs. All of the Kv subunits tested in islets have been found in other tissues, but, in general, the exact combination of subunits in these tissues is unknown. For the β-cell, determination of I_DR composition may aid significantly in the identification of a glucose-dependent insulin secretagogue applicable in type 2 diabetes, without the hypoglycemic liabilities found with K_ATP inhibitors.

We would like to thank Drs. Maria Garcia, Jim Herrington, and Owen B. McManus for critical discussion and constructive comments. We would also like to thank Dr. H.G. Knaus for generously supplying antibodies for Kv1.6 and Dr. Jonathan Lakey for supplying human islets.