Altered functional properties of KATP channel conferred by a novel splice variant of SUR1

Abstract

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming (Kir6.x) and regulatory sulphonylurea receptor (SURx) subunits. We have isolated a novel SUR variant (SUR1bDelta33) from a hypothalamic cDNA library. This variant lacked exon 33 and introduced a frameshift that produced a truncated protein lacking the second nucleotide binding domain (NBD2). It was expressed at low levels in hypothalamus, midbrain, heart and the insulin-secreting beta-cell line MIN6. 2. We examined the properties of KATP channels composed of Kir6.2 and SUR1bDelta33 by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing these subunits. We also investigated the effect of truncating SUR1 at either the start (SUR1bT1) or end (SUR1bT2) of exon 33 on KATP channel properties. 3. Kir6.2/SUR1bDelta33 showed an enhanced open probability (Po = 0.6 at -60 mV) and a reduced ATP sensitivity (Ki, 86 microM), when compared with wild-type channels (Po = 0.3; Ki, 22 microM). However, Kir6.2/SUR1bT1 and Kir6.2/SUR1bT2 resembled the wild-type channel in their Po and ATP sensitivity. 4. Neither MgADP, nor the K+ channel opener diazoxide, enhanced Kir6.2/SUR1bDelta33, Kir6.2/SUR1bT1 or Kir6.2/SUR1bT2 currents, consistent with the idea that these agents require an intact NBD2 for their action. Sulphonylureas blocked KATP channels containing any of the three SUR variants, but in excised patches the extent of block was less than that for the wild-type channel. In intact cells, the extent of sulphonylurea block of Kir6.2/SUR1bDelta33 was greater than that in excised patches and was comparable to that found for wild-type channels. 5. Our results demonstrate that NBD2 is not essential for functional expression or sulphonylurea block, but is required for KATP channel activation by K+ channel openers and nucleotides. Some of the unusual properties of Kir6.2/SUR1bDelta33 resemble those reported for the KATP channel of ventromedial hypothalamic (VMH) neurones, but the fact that this mRNA is expressed at low levels in many other tissues makes it less likely that SUR1bDelta33 serves as the SUR subunit for the VMH KATP channel.

Figure 1. SUR1bΔ33 lacks exon 33

A, putative membrane topology of SUR1b (after Tusnady et al. 1997) with the position of the amino acids that are altered in SUR1b indicated (•). NBD, nucleotide binding domain. The truncation that occurs in SUR1bΔ33 is indicated in the expanded view of NBD2 (inset, right). B and C, differences in the amino acid sequence (B) and nucleotide sequence (C) of SUR1b and SUR1bΔ33. The boxed area shows exon 33 and the shaded area shows the amino acids that are different in SUR1bΔ33. W_A, Walker A motif; W_B, Walker B motif. The asterisk indicates the termination of translation.

Figure 2. RT-PCR amplification of SUR1 and SUR1Δ33 variants from mouse tissue

Photograph of ethidium bromide-stained gel, with RT-PCR products separated by electrophoresis in parallel with a 100 bp ladder as molecular weight marker. PCR fragments were generated with mouse SUR1-specific primers spanning exon 33. The prominent band at 655 bp corresponds to SUR1 and that at 525 bp to SUR1Δ33. MB, midbrain. HYP, hypothalamus. MIN6, mouse β-cell line MIN6.

Figure 3. Effects of ATP on wild-type and mutant K_ATP channels

A, macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV from oocytes injected with mRNAs encoding Kir6.2 and either SUR1b (left) or SUR1bΔ33 (right). ATP was added to the intracellular solution as indicated by the horizontal bars. B, mean ATP dose-response relationships for Kir6.2/SUR1b (•, n = 6), Kir6.2/SUR1bΔ33 (○, n = 5), Kir6.2/SUR1bT1 (▵, n = 7) and Kir6.2/SUR1bT2 (▴, n = 8) currents. Test solutions were alternated with control solutions and the slope conductance (G) is expressed as a fraction of the mean (G_c) of that obtained in control solution before and after exposure to ATP. The lines are the best fits of the data to the Hill equation (eqn (1)) for SUR1b (continuous line, K_i = 22 ± 3 μm, h = 1.30± 0.16) and for SUR1bΔ33 (dotted line, K_i = 86 ± 4 μm, h = 1.20± 0.07).

Figure 4. Single-channel currents

Single-channel currents recorded at -60 mV from inside-out patches excised from oocytes injected with mRNAs encoding Kir6.2 plus SUR1, SUR1b, SUR1bΔ33, SUR1bT1 or SUR1bT2. The dotted lines indicate the closed level.

Figure 5. Single-channel currents

A, single-channel currents recorded at the indicated membrane potentials from an inside-out patch excised from an oocyte injected with mRNAs encoding Kir6.2 and SUR1bΔ33. The dotted lines indicate the closed level. B, mean single-channel current-voltage relationship recorded for Kir6.2/SUR1bΔ33 currents (n = 3).

Figure 6. Effects of ADP and diazoxide on wild-type and mutant K_ATP channels

A and B, macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV from oocytes injected with mRNAs encoding Kir6.2 and either SUR1b (A) or SUR1bΔ33 (B). ATP, diazoxide (DZ) or MgADP were added to the intracellular solution as indicated by the horizontal bars. C, mean macroscopic slope conductance recorded in the presence of MgADP, ATP or ATP plus diazoxide, expressed as a fraction of the slope conductance in control solution (no additions), for Kir6.2/SUR1b, Kir6.2/SUR1bΔ33, Kir6.2/SUR1bT1 and Kir6.2/SUR1bT2. The dashed line indicates the current level in control solution. The number of oocytes is given above the bars.

Figure 8. Interactions between MgADP and tolbutamide

A, macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV from oocytes injected with mRNAs encoding Kir6.2 and either SUR1b, SUR1bΔ33 or SUR1bT1. Tolbutamide (TB) and MgADP were added to the intracellular solution as indicated by the horizontal bars. B, mean macroscopic slope conductance recorded in the presence of ADP, tolbutamide or ADP plus tolbutamide, expressed as a fraction of the slope conductance in control solution (no additions), for Kir6.2/SUR1b, Kir6.2/SUR1bΔ33 and Kir6.2/SUR1bT1. Experiments were carried out in the presence of Mg²⁺ except where indicated. The dashed line indicates the current level in control solution. The number of oocytes is given above the bars. *P < 0.05, **P < 0.01, compared with Kir6.2/SUR1b. ††P < 0.01, compared with data obtained for the same type of channel in the presence of Mg²⁺. C, mean macroscopic slope conductance recorded in the presence of ADP, ADP plus tolbutamide, and ADP in Mg²⁺-free solution for Kir6.2/SUR1b, Kir6.2/SUR1bΔ33 and Kir6.2/SUR1bT1. Currents are expressed as a fraction of the slope conductance in the absence of ADP. Experiments were carried out in the presence of Mg²⁺ except where indicated. The number of oocytes is given above the bars.

Figure 7. Effect of sulphonylureas on Kir6.2/SUR1b and Kir6.2/SUR1bΔ33 currents

A, macroscopic currents recorded from inside-out patches in response to a series of voltage ramps from -110 to +100 mV from oocytes injected with mRNAs encoding Kir6.2 and either SUR1b (top) or SUR1bΔ33 (bottom). Tolbutamide (TB), glibenclamide (Glib) or ATP were added to the intracellular solution as indicated by the horizontal bars. B, mean relationships between tolbutamide concentration and the macroscopic K_ATP conductance, expressed as a fraction of its amplitude in the absence of the drug. •, continuous line: Kir6.2/SUR1b currents (n = 4). ○, dashed line: Kir6.2/SUR1bΔ33 currents (n = 7). The lines are the best fit of the data to eqn (2). Kir6.2/SUR1b currents: K_i1 = 2.1 μm, h₁ = 0.83, K_i2 = 1.2 mm, h₂ = 1.0, L = 26%. Kir6.2/SUR1bΔ33 currents: K_i1 = 1.2 μm, h₁ = 1.5, K_i2 = 1.4 mm, h₂ = 1.1, L = 72%.

Figure 9. Effects of metabolic inhibition

Mean whole-cell current amplitudes recorded in response to a 300 ms voltage step to -100 mV in control solution (□), 15 min after exposure to 3 mm azide (), and in the continued presence of azide plus either 0.01 mm tolbutamide (TB; ) or 0.1 mm tolbutamide (▪). Oocytes were coinjected with mRNAs encoding Kir6.2 and SUR1, SUR1b, SUR1bΔ33 or SUR1bT1, as indicated, or were injected with mRNA encoding Kir6.2ΔC26. The number of oocytes is given above the bars. *Data taken from Tucker et al. (1997). Inset, mean whole-cell current amplitudes recorded at -100 mV in control solution, in the presence of 3 mm azide, in the presence of 3 mm azide plus 0.01 mm tolbutamide, 0.1 mm tolbutamide or 0.1 μm glibenclamide (Glib), as indicated. Oocytes were coinjected with mRNAs encoding Kir6.2 and SUR1bΔ33. The number of oocytes is given above the bars.

Figure 10

Mean current amplitudes recorded at -100 mV in the excised patch or whole-cell (TEVC) configuration, expressed as a fraction of the mean current recorded for Kir6.2/SUR1. Whole-cell currents were recorded in the presence of 3 mm azide. Oocytes were coinjected with mRNAs encoding Kir6.2 and SUR1, SUR1b, SUR1bΔ33 or SUR1bT1, as indicated, or were injected with mRNA encoding Kir6.2ΔC26. *Data taken from Tucker et al. (1997). † Data from Trapp et al. (1998). The number of oocytes is given above the bars.