Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes
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ATP-sensitive potassium (KATP) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. KATP channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the KATP channel to inhibition by MgATP, and enhanced whole-cell KATP currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the KATP current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.
KeywordsATP-sensitive potassium channel Neonatal diabetes Kir6.2 Insulin secretion Sulphonylureas SUR1
This issue of Pflugers Archiv celebrates the 25th anniversary of a milestone in electrophysiology—the development of giga-seal patch clamp recording . This technique has not only confirmed that ion channels are the molecular entities responsible for the electrical activity of excitable cells; it has elucidated, in detail, how ion channels work . Equally importantly, it has increased our understanding of the role of ion channels in physiological functions of cells as diverse as neurones, lymphocytes, and pancreatic beta cells [2, 3] and revealed that mutations in ion channel genes underlie a wide spectrum of human diseases [3, 4]. About 20 years ago, the patch clamp technique was used to demonstrate that ATP-sensitive potassium (KATP) channels play a key role in insulin secretion from pancreatic beta cells . In this paper, we use patch clamp methods to show how mutations in the KATP channel give rise to impaired insulin secretion and neonatal diabetes.
Neonatal diabetes is usually diagnosed within the first 6 months of life . In some patients, the diabetes is transient, although it may frequently relapse, whereas in other cases it is permanent and requires insulin treatment for life. The commonest cause of permanent neonatal diabetes (PNDM) are heterozygous activating mutations in the KCNJ11, the gene encoding Kir6.2, which constitutes the pore-forming subunit of the KATP channel in the pancreatic beta cell [7, 8, 9]. KCNJ11 mutations account for around 50% of cases of PNDM [7, 8, 9, 10, 11, 12, 13]. Some of these mutations lead to a more severe syndrome in which developmental delay and epilepsy accompany neonatal diabetes, a condition known as DEND syndrome . Mutations in KCNJ11 can also result in transient neonatal diabetes mellitus (TNDM) , although in most patients TNDM is caused by an abnormality of the imprinted region on chromosome 6q24 . Kir6.2 associates with the sulphonylurea receptor SUR1 (ABCC8) to form the KATP channel of the pancreatic beta cell [16, 17] and mutations in ABCC8 can also cause neonatal diabetes .
The beta cell KATP channel is a hetero-octameric complex of Kir6.2 and SUR1 . Four Kir6.2 subunits form a central tetrameric pore, and they are surrounded by four regulatory SUR1 subunits , which modulate the opening and closing (gating) of the pore. The most important modulators of KATP channel gating are adenine nucleotides: in particular, ATP closes the channel by binding to Kir6.2, whereas interaction of Mg-nucleotides (MgATP, MgADP) with the nucleotide-binding domains (NBDs) of SUR1 stimulates channel activity and reverses channel inhibition by ATP [21, 22, 23]. Nucleotide modulation enables KATP channels to couple changes in plasma glucose levels to changes in insulin secretion [24, 25].
Mutations in KATP channel subunits that reduce channel function lead to congenital hyperinsulinism, whereas those that enhance channel function cause diabetes [8, 9]. To date, mutations in Kir6.2 associated with TNDM and PNDM have been shown to enhance KATP channel activity by decreasing ATP inhibition at Kir6.2 [7, 8, 9, 13, 14, 26, 27, 28, 29, 30, 31]. In addition, they may also increase channel activation by Mg-nucleotides, by enhancing the stimulatory action of SUR1 on Kir6.2 [28, 29].
KATP channels are also the target for sulphonylurea drugs, which are widely used to treat type 2 diabetes . These drugs stimulate insulin secretion by binding to SUR1, and closing KATP channels directly, thus bypassing beta cell metabolism. They have proved effective in treating neonatal diabetes that results from gain-of-function mutations in Kir6.2 [9, 10, 33].
In this paper, we examine the functional effects of six novel missense mutations recently reported to be associated with neonatal diabetes in the Spanish population: H46Y, N48D, E227K, E229K, V252A, and E292G. These mutations are associated with TNDM (N48D, E227K, E229K), PNDM (H46Y, E292G) or both (V252A).
Materials and methods
Human Kir6.2 (Genbank NM000525 with E23 and I337) and rat SUR1 (Genbank L40624) were used in this study. Site-directed mutagenesis of Kir6.2, preparation of mRNA, and isolation of Xenopuslaevis oocytes was performed as described previously [29, 34]. Oocytes were injected with 0.8 ng wild-type or mutant Kir6.2 mRNA and ∼4 ng of SUR1 mRNA (giving a 1:5 ratio). To simulate the heterozygous state, SUR1 was coexpressed with a 1:1 mixture of wild type and mutant Kir6.2 (28). Currents were recorded 1–3 days after injection.
Whole-cell currents were recorded using the two-electrode voltage-clamp method in response to voltage steps of ±20 mV from a holding potential of −10 mV . They were filtered at 1 kHz and digitized at 4 kHz. Oocytes were constantly perfused at 20–22°C with a solution containing (in mM): 90 KCl, 1 MgCl2, 1.8 CaCl2, and 5 HEPES (pH 7.4 with KOH). Metabolic inhibition was produced by 3 mM Na-azide and the sulphonylurea tolbutamide (0.5 mM) was used to confirm currents flowed through KATP channels.
Macroscopic currents were recorded from giant excised inside-out patches using the patch-clamp technique in response to 3-s voltage ramps from −110 to +100 mV (holding potential, 0 mV) at 20–22°C. Currents were filtered at 0.15 kHz and digitised at 0.5 kHz. The pipette solution contained (mM): 140 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES (pH 7.4 with KOH). The internal (bath) solution contained (mM): 107 KCl, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES (pH 7.2 with KOH) and MgATP as indicated. For single-channel recordings, we used the same external solution and the internal solution contained (in mM): 107 KCl, 1 K2SO4, 10 EGTA, 10 HEPES (pH 7.2 with KOH).
ATP sensitivity of wild-type and mutant heterozygous Kir6.2/SUR1 channels
% I, 1 mM MgATP
% I, 3 mM MgATP
All data were analyzed with pCLAMP8 (Axon Instruments, CA, USA), Origin 6.02 (Microcal Software, Northampton, MA, USA) and Igor (Wavemetrics, Lake Oswego, OR, USA) software, and are given as mean±SEM. Statistical significance was evaluated using an unpaired two-tailed Student t test. A probability value of P<0.05 was taken as the criteria for a significant difference.
Location of mutations
Residue E292 is predicted to form an inter-subunit ion pair with R301, which makes a backbone interaction with the adenine ring of ATP (Fig. 2c). E292 also lies within one of the two gating loops that link the ATP-binding site to the slide helix, and which may, therefore, be involved in coupling ATP binding to channel opening [35, 40]. Thus, disruption of the putative E292-R301 ion pair can be hypothesized to either alter the mechanism by which ATP binding is linked to channel gating, or to destabilize ATP binding indirectly, by altering the conformation of the ATP-binding site.
Finally, the model predicts that E227 and E229 in one Kir6.2 subunit form putative ion pairs with residues in the adjacent subunit (R192 and R314, respectively). Thus, E227 and E229 may be involved in electrostatic interactions between subunits (Fig. 2d). Their mutation to a positively charged residue would be expected to break these interactions and thereby might influence channel gating. Indeed, functional studies have previously shown that E229 makes an ion pair with R314 that is critical for maintaining channel activity .
Effects on whole-cell currents
Because all patients are heterozygotes, their pancreatic beta cells will contain a mixture of wild-type and mutant Kir6.2. We, therefore, explored the functional effects of Kir6.2 mutations on KATP channel function in the simulated heterozygous state, by coinjecting a 1:1 mixture of mutant and wild-type Kir6.2 together with SUR1 . This will produce a mixed population of homomeric wild-type channels, homomeric mutant channels and heteromeric channels containing between 1 and 3 mutant subunits. We refer to this channel population as heterozygous channels.
Effects on KATP channel ATP sensitivity
We next explored the effect of the mutations on the sensitivity of heterozygous channels to inhibition by MgATP. Experiments were carried out in the presence of 2 mM Mg2+ to most closely approximate the physiological condition.
In the case of the E227K and E229K mutations, the heterozygous currents ran down rapidly after patch excision into nucleotide-free solution. However, they increased substantially after application of high concentrations of MgATP (>1 mM), as previously reported for homomeric channels with mutations at E229 . The ATP sensitivity was measured following activation with high MgATP concentrations.
Effects of the mutations on the intrinsic open probability
Previous studies have shown that mutations in Kir6.2 can reduce ATP inhibition of the KATP channel by several mechanisms. For example, they may disrupt ATP binding or impair the mechanism by which ATP binding is translated into closure of the channel pore [26, 29, 30]. They may also affect ATP sensitivity indirectly by stabilizing the intrinsic (unliganded) open state of the KATP channel [26, 27]. This shifts the gating equilibrium in the presence of ATP towards the open state, and, therefore, reduces its inhibitory effect [42, 43].
Despite the increased Po(0), the homomeric E227K and E229K currents ran down faster than wild type (Fig. 5b). The rate of rundown could be well fit with a single exponential with a time constant of 41±10 s (n=5) for E227K and 29±9 s (n=5) for E229K, compared with 135±67 s (n=6) for wild-type channels. These values are approximately five- to tenfold slower than those reported for mutation of E229 to lysine, alanine or cysteine .
Our results demonstrate that the six Kir6.2 mutations associated with PNDM or TNDM that we have studied are gain-of-function mutations, which act by reducing the ability of MgATP to inhibit the KATP channel. This adds to accumulating evidence that KCNJ11 mutations that cause neonatal diabetes do so by affecting KATP channel ATP sensitivity, either directly or indirectly [8, 9, 13, 14, 26, 27, 28, 29, 30].
Four of the six mutations studied, H46Y, N48D, V252A, and E292G, are found close to the predicted ATP-binding site. Thus, it seems likely that mutation of these residues impairs ATP binding, probably by allosterically altering the conformation of the binding site itself. The lack of effect of the H46Y mutation on the single-channel kinetics is consistent with this idea. Two mutations, E227K and E229K, lie distant from the ATP-binding site at the interface between adjacent subunits. These mutations alter the channel ATP sensitivity indirectly by increasing the intrinsic open probability of the channel [42, 43]. The increase in Po(0) (to 0.68 and 0.72) is similar to that found for mutations at residue F35, which cause PNDM (∼0.75, (31)), but less than that observed for mutations associated with DEND syndrome (∼0.85, [26, 27]).
The data presented in this paper further emphasize that it is the magnitude of the increase in the KATP current in the presence of physiologically relevant concentrations of ATP that determines the severity of the clinical phenotype, and not the mechanism of action of the mutation. Thus, mutations that impair the gating of the channel can cause TNDM, as shown here, as well as PNDM  and DEND syndrome [26, 27]. When Po(0) is greater than about ∼0.8, very small changes in P(0) can have large effects on ATP sensitivity because of the steep relationship between Po(0) and the IC50 for ATP inhibition . Mutations that lie in the ATP-binding site can also produce different clinical phenotypes, depending on the extent to which they impair the channel ATP sensitivity .
Effects of reduced ATP sensitivity
The reduction in ATP sensitivity produced by the KCNJ11 mutations we studied is associated with a small increase in the resting KATP current when expressed in Xenopus oocytes. Although in some cases the increase in resting current was not significant, this may not necessarily be the case in pancreatic beta cells, where resting ATP concentrations appear to be lower and native wild-type KATP channels are open in the absence of glucose. An increase in KATP current may be expected to hyperpolarize the beta cell membrane and reduce or abolish the membrane depolarization evoked by glucose. This will prevent electrical activity, calcium influx and insulin secretion, and could thereby account for the diabetic phenotype of the patients.
Our results suggest neonatal diabetes was produced by rather small changes in the ability of MgATP to block the KATP channel: for example, a 2.7-fold reduction in the IC50 (V252A) and a fivefold increase in the current at 1 mM MgATP (E229K). This is consistent with previous reports that diabetes can be associated with small changes in nucleotide sensitivity in man [14, 26, 29, 30, 31] and mouse . Given that the beta cell membrane potential is largely determined by the activity of the KATP channel, it is not surprising that small changes in ATP sensitivity, which produce tiny changes in KATP current, can result in marked changes in electrical activity, and insulin secretion .
It is important to note, however, that with a single exception (N48D), the unblocked currents in the presence of 3 mM MgATP (7–10%) lay within the range of values previously reported for TNDM and PNDM mutations. Furthermore, they were much smaller than those found for Kir6.2 mutations that give rise to a DEND syndrome, where the unblocked current was between 28 and 40% (Fig. 6).
The reason why some mutations cause a relapsing remitting form of diabetes whereas others cause permanent diabetes is unclear. As previously suggested, this may be due to a reduced insulin requirement at the time of remission; to changes in beta cell turnover due to the Kir6.2 mutation; or to compensatory changes (at the level of the beta cell, pancreas, or whole body), which are able to overcome the effects of the channel defect.
The ability of tolbutamide to block the whole-cell currents in oocytes ranged from 82 to 96%, compared with 96% for wild-type channels (Fig. 3c). This suggests that it should be possible to control the diabetes of all the patients described here with sulphonylureas, as has been successfully achieved for Kir6.2 mutations, which cause a similar degree of reduction in ATP sensitivity [7, 9, 10, 33, 45].
We thank C. Luzuriaga, J.M. Gomez Vida, A. Aragones, C. Fernandez, R. Barrio, J.P. Lopez-Siguero, and J. Prieto for informing us of patients with neonatal diabetes, and Vicky Ball and Itziar Estalella for technical assistance. FMA thanks the Wellcome Trust, the Royal Society and the EU (EuroDia) for support. The Spanish group was partially supported by grants RGDM (G03/212) and RCMN (C03/08) from the Instituto de Salud Carlos III, Madrid, Spain. GPN is a FIS Research Scientist supported by the Spanish Ministry of Health (Fellowship no. 03/0064). FMA is a Royal Society Research Professor.