Single Nucleotide Polymorphisms in K_ATP Channels: Muscular Impact on Type 2 Diabetes

Type 2 diabetes is a challenge to modern medicine. Although defects in glucose homeostasis of the disease have been recognized for decades, molecular mechanisms underlying the defects remain poorly understood. Recent discovery of a single nucleotide polymorphism (SNP) in the Kcnj11 gene provides an important genetic link to type 2 diabetes (1,2,3). This SNP results in a point mutation of Glu23 to lysine (E23K) in the pore-forming Kir6.2 subunit of the ATP-sensitive K⁺ channels (K_ATP channels). Genotypic frequencies average 42% for E/E (homozygous at residue 23), 47% for E/K (heterozygous), and 11% for K/K (4). Risk and frequency estimates have shown that ∼15% of type 2 diabetic cases in Caucasians are attributable to the K/K and E/K genotypes (4). Similar allelic frequencies of E23K (34%) have been reported in Japanese populations (5).

The K_ATP channels are composed of four pore-forming Kir6 subunits (Kir6.1 or Kir6.2) and four regulatory subunits of sulfonylurea receptor (SUR1, SUR2A, or SUR2B). The channels are normally inhibited by physiological concentrations of ATP and activated when the ATP level drops, allowing them to couple the cellular metabolism with membrane excitability (6,7). Experimental evidence suggests that the E23K mutation augments pancreatic K_ATP channels and reduces insulin secretion from β-cells (8,9). In addition to β-cells, the Kir6.2 subunit is expressed in cardiac and skeletal muscles (6,7). The muscular isoform of K_ATP channels consisting of Kir6.2 and SUR2A subunits regulates muscular excitability and plays a role in glucose uptake (10,11,12,13). Therefore, genetic variations in the muscular K_ATP channels may contribute to abnormalities of systemic glucose homeostasis. Since proton is a potent regulator of the K_ATP channels (14,15,16), and since intracellular pH levels change widely in the skeletal muscles during the exercise (14), alterations in the pH sensitivity of the E23K variant may affect K_ATP channel activity, membrane excitability, and muscular glucose uptake, contributing a risk factor for type 2 diabetes and obesity. To test this hypothesis, we studied the pH sensitivity and the effect of pH on the ATP sensitivity of the wild-type and E23K variant Kir6.2 expressed with SUR2A using inside-out patches with symmetric concentrations of K⁺ (145 mmol/l) applied to both sides of patch membranes.

When the internal surface of patch membranes was exposed to a perfusate with various pH levels in the presence of 0.3 mmol/l ATP, the wild-type Kir6.2/SUR2A currents increased with moderate acidification, reached a maximum activation at pH 6.2, and were inhibited at extremely acidic pH, consistent with the Kir6.2/SUR1 currents shown in our previous studies (16,17) (Fig. 1A, online appendix [available at http://diabetes.diabetesjournals.org]). Such a biphasic response was seen in the presence of 1.0 mmol/l ATP, although the peak activation took place at pH 5.9 (Fig. 1B, online appendix). Since the inhibition or channel rundown was not seen in whole-cell recording (16), further studies focused on pH-dependent channel activation. The pH-current relationship was described using the Hill equation. In the presence of 0.3 mmol/l ATP, the pH level for 50% current activation (pK_a) was 6.77 ± 0.02 (n = 10) with the Hill coefficient (h) 3.5 (Fig. 1C). The pH-current relationship shifted toward lower pH levels when 1.0 mmol/l ATP was part of the internal solution (pK_a = 6.43 ± 0.01, h = 3.4, n = 7).

Exposures of the internal patch membranes to ATP produced a concentration-dependent inhibition of the Kir6.2/SUR2A currents (Fig. 1C and D, online appendix). At pH 7.4, the ATP concentration for 50% current inhibition (IC₅₀) was 32 ± 2.0 μmol/l (n = 10) (Fig. 2C). The ATP sensitivity, however, was markedly reduced when the internal solution became acidic. At pH 6.8, the currents were half inhibited by 75 ± 4.0 μmol/l ATP (n = 6) (Fig. 2C).

Like the wild-type channel, the E23K variant displayed a biphasic response to acidic pH with peak activation at pH 6.5 and 6.2 in the presence of 0.3 and 1.0 mmol/l ATP, respectively (Fig. 1A and B). As a result, its pH-current relationship clearly differed from that of the wild-type channel. With 0.3 mmol/l ATP, the E23K variant showed a pK_a of 6.92 ± 0.03 (n = 8) and an h of 3.7 (Fig. 1C). Such a change was even more obvious in the presence of 1.0 mmol/l ATP (pK_a 6.62 ± 0.03, h = 3.4, n = 8).

We have previously shown pH sensitivity of the truncated Kir6.2 channel (Kir6.2ΔC36) expressed without SUR (16,17). For a comparison purpose, we studied the Kir6.2ΔC36-E23K in the absence of ATP and fitted the pH-current relationship using a sum of two Hill equations as described previously (16,17). The E23K mutation enhanced the pH sensitivity by 0.34 pH units (Fig. 2A–C, online appendix). Such pH sensitivity was also seen in the wild type and E23K coexpressed with SUR2A, although the pH-current relationship shifted toward more alkaline pH (Fig. 2C, online appendix).

The ATP sensitivity of the E23K polymorphic Kir6.2/SUR2A decreased modestly at pH 7.4 compared with the wild-type channel (Fig. 2A). The ATP-current relationship plot showed an IC₅₀ of 68 ± 4.0 μmol/l (n = 8) for the E23K variant (Fig. 2C). The ATP sensitivity dropped greatly at pH 6.8, with an IC₅₀ of 225 ± 5.0 μmol/l (n = 7), three times as high as that for the wild-type channel at the same pH level (Fig. 2B and C). Consistent with these observations, a reduction in the ATP sensitivity and augmentation of the pH sensitivity were also found in the E23K mutant of rodent Kir6.2/SUR2A channels (Fig. 3A and B, online appendix).

In addition to ATP and protons, K_ATP channels are modulated by ADP. The channels are activated with micromolar concentrations of ADP in the presence of Mg²⁺ and inhibited with millimolar concentrations (18). Thereby, the ATP-current relationship was studied with 0.3 mmol/l ADP and 1.0 mmol/l free Mg²⁺ in the internal solution. Under this condition, the E23K currents continued to be activated by ADP and inhibited by ATP. However, the ATP-current relationship curves shifted in parallel toward higher ATP levels in the E23K variant, with an IC₅₀ of 150 ± 9.0 μmol/l (n = 6) at pH 7.4 and 400 ± 29.0 μmol/l (n = 7) at pH 6.8 (Fig. 3A and E), indicating that the ATP sensitivity of the E23K variant is further reduced in the presence of ADP.

Another polymorphism, I337V in the Kcnj11 gene, was studied as a control. Both ATP and pH sensitivities remained close to the wild-type channel (Fig. 3C–F), indicating that the changes in ATP and proton sensitivities of the E23K polymorphism are specific.

Single-channel studies showed that conductance of the E23K (74 ± 8.4 pS, n = 27) remained comparable with that of the wild-type channels (72 ± 6.0 pS, n = 23; P > 0.05). The channel open-state probability (P_open) was measured in inside-out patches at −80 mV membrane potential. To have a pseudo-physiologic condition, 0.3 mmol/l ADP and 1.0 mmol/l ATP were added to the internal solution. At pH 7.4, the P_open was low (0.029 ± 0.006, n = 22) in the wild-type channel. Although it remained low, the P_open (0.079 ± 0.014, n = 18) was doubled in the E23K variant over the wild type (P < 0.001). At pH 6.8, the P_open increased in E23K (0.191 ± 0.015, n = 26) compared with wild type (0.112 ± 0.013, n = 17; P < 0.001). The P_open of the E23K at pH 6.8 was approximately sevenfold as high as the wild-type channel at pH 7.4 (Fig. 4). Similar results were obtained in the absence of ATP (Fig. 2D, online appendix).

The E23K has been shown to be associated with type 2 diabetes, both alone and in combination with I337V (1,2). Previous studies suggest that the homozygous K/K is much more likely to predispose the carrier to type 2 diabetes than the heterozygous E/K (1,2,3). The channel activity is increased by 1.4- and 2.2-fold in the E23K heterozygous and K23K homozygous models, respectively (8). The E23K polymorphism enhances baseline open-state probability and reduces ATP sensitivity of the K_ATP channels in pancreatic β-cells. The overactivation of β-cell K_ATP channels prevents the intracellular Ca²⁺ rise necessary for insulin secretion and thus inhibits insulin release (8,9).

In addition to the β-cells, several lines of evidence suggest that K_ATP channels in peripheral tissues are also involved in glucose homeostasis. The effect of the E23K genotype on β-cell function has been inconsistent and even contradictory, as indicated by different research groups (8,9,19,20). The β-cell hypothesis cannot totally explain the disruption of glucose homeostasis in type 2 diabetes, as glucose uptake is problematic in type 2 diabetes (21). As a subunit of muscular K_ATP channels, Kir6.2 is expressed in both skeletal and cardiac muscles, which constitute the predominant tissues for glucose utilization in the human body (6,7). K_ATP channel openers suppress glucose uptake in human skeletal muscles, and such an effect can be blocked by K_ATP channel inhibitors (22). The K_ATP channel openers also reduce force development leading to muscle fatigue, whereas they have no effect on action potentials and fatigue kinetics of skeletal muscles in Kir6.2^−/− mice (10). Genetical disruption of K_ATP channels lowers muscular excitability and enhances glucose uptake in skeletal muscles (12,13). In knockout mice lacking both Kcnj11 and insulin receptor substrate-1 genes, K_ATP channel–mediated glucose uptake is found to be independent of insulin receptor substrate-1 and phosphatidylinositol-3 kinase signaling pathway (11).

The E23K variant has been previously shown to reduce ATP sensitivity when expressed with SUR1; at pH 7.15, the IC₅₀ for the E23K is about twice as high as the wild type (8), consistent with our observation in the muscular isoform at pH 7.4. It is worth noting that the IC₅₀ level of the wild-type channels varies in a similar range depending on experimental conditions (8,17,23). Such a variation might have prevented some previous studies from showing different ATP sensitivity between the wild-type and E23K variant (24).

To our surprise, we found that the E23K defect in muscular K_ATP channels was more related to pH than ATP. Intracellular acidification not only activates the E23K channel more readily than the wild type but also relieves the channel inhibition by ATP, leading to a vast activation of the muscular K_ATP channels. The activation of K_ATP channels can cause hyperpolarization of sarcolemmal membranes, muscle fatigue, and a reduction in glucose uptake, as shown in previous studies in genetically disrupted K_ATP channels (10,11,12,13). Therefore, it is likely that the enhanced pH sensitivity together with the reduced ATP sensitivity in the E23K variant leads to overactivation of the muscular K_ATP channels, impairs glucose uptake during exercise, and contributes to the development of type 2 diabetes.

Our results also suggest a link of the genetic factor to the environmental factors, such as sedentary lifestyle and obesity, as the easy activation of polymorphic muscular K_ATP channels tends to hyperpolarize muscular cells, leading to muscle fatigue, reduction in glucose uptake, and fat deposition. Then, why is such an adverse genetic variant widely reserved in human populations? We believe that the E23K variant, which potentially results in slow glucose utilization, muscle fatigue, and obesity, may facilitate population survival of the early humans, as their food supplies may be seasonal and discontinuous.