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The phosphatidylinositol 3-kinase inhibitor LY294002 potently blocks K_V currents via a direct mechanism¹

WASIM EL-KHOLY^*, PATRICK E. MACDONALD^*, JIA-HUI LIN^*, JING WANG^*, JOCELYN MANNING FOX, PETER E. LIGHT, QINGHUA WANG^*,, ROBERT G. TSUSHIMA^* and MICHAEL B. WHEELER^*,2

^* Departments of Medicine and Physiology, University of Toronto, Ontario, Canada;
Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada; and
Division of Endocrinology and Metabolism, St. Michael’s Hospital, Toronto, Canada

²Correspondence: University of Toronto, Department of Physiology, 1 King’s College Circle, RM 3352, Toronto, ON, M5S 1A8, Canada. E-mail: michael.wheeler{at}utoronto.ca

SPECIFIC AIMS

A potential role for modulation of voltage-gated potassium (Kv) channels by phosphatidylinositol 3-kinase (PI3K) in pancreatic ß cells was examined using the structurally distinct PI3K inhibitors wortmannin and LY294002. It was found that LY294002, but not wortmannin, blocked Kv currents in a strikingly potent manner. The acute effects of LY294002 in excitable cells may be due to an inhibition of Kv currents rather than through a PI3K-dependent mechanism, and LY294002 represents a compound that can be used as a precursor for development of potassium channel blockers.

PRINCIPAL FINDINGS

1. Structure and activity of PI3K pathway inhibitors
Both 100 nM wortmannin and 50 µM LY294002 inhibited basal- and insulin-stimulated PKB/Akt phosphorylation in the MIN6 ß cell line when applied for 10 min. At 50 µM, LY303511, a compound structurally related to LY294002, did not inhibit insulin-stimulated PKB phosphorylation. These data confirm the ability of LY294002 and wortmannin to inhibit PI3K activity and show that LY303511 does not inhibit PI3K activity at the concentrations used in the present study.

2. Effects of LY294002, LY303511, and wortmannin on Kv channels in MIN6 cells
Using MIN6 ß cells, Kv channel function was monitored by whole-cell voltage-clamp measurements at room temperature (Fig. 1 ). At 50 µM LY294002, a concentration known to inhibit PI3K activity, sustained Kv currents were reversibly blocked within 60–90 s of application to the bath solution; similar results were observed with 50 µM LY303511, although the effect took 3–4 times longer to occur. In contrast, 100 nM wortmannin had no significant effect on Kv currents when applied for > 10 min (Fig. 1A ). At +70 mV depolarizations, 50 µM LY294002, 100 µM LY303511, and 15 mM of the general potassium blocker tetraethylammonium (TEA) inhibited peak currents by 66.9 ± 2.5% (n=7, P<0.05), 60.2 ± 4.8% (n=6, P<0.001), and 40.6 ± 10.4% (n=4, P<0.05), respectively (Fig. 1B ). Sustained Kv channel currents were blocked by 93.2 ± 1.6% (n=6, P<0.01), 67.2 ± 4.1% (n=6, P<0.001), and 72.8 ± 4.7% (n=5, P<0.05), respectively. Wortmannin (100 nM) did not significantly alter Kv currents at any voltage. LY294002 was considerably more potent than TEA at inhibiting Kv currents.

View larger version (48K): [in this window] [in a new window]   Figure 1. The effect of LY294002, wortmannin, and LY303511 on KV currents in MIN6 cells. A) 50 µM, LY294002 blocked sustained currents primarily whereas LY303511 inhibited both sustained and peak currents equally at this concentration. 100 nM wortmannin (WORT) did not have an appreciable effect on currents after 10 min of incubation in the bath solution. The general K+ channel blocker tetraethylammonium (TEA) (15 mM) was used as a positive control. B) Measurements of current-voltage relationships for the above experiments in MIN6 cells. Addition of 50 µM LY294002 or 100 µM LY303511 inhibited sustained Kv currents, followed by a near-complete restoration of outward currents on rewashing of control solution into the bath. Wortmannin (100 nM) (WORT) had no effect on Kv currents (**P<0.01, ***P<0.001). C) Preincubation of MIN6 cells with 100 nM wortmannin [WORT (1)] for 10 min had no effect on current amplitude, but subsequent application of LY294002 blocked sustained currents, which returned rapidly after rewashing the cells with the 100 nM wortmannin solution [WORT (2)].

3. Dose-response effect of LY294002 and LY303511 on Kv currents in MIN6 cells
Dose-response curves for LY294002 and LY303511 on Kv channel function in MIN6 cells were generated. LY294002 maximally inhibited sustained Kv currents by 90% at a concentration of 50 µM, with an IC₅₀ of 9.0 ± 0.7 µM (n5 cells at each concentration). LY303511 blocked currents with an IC₅₀ of 64.6 ± 9.1 µM, with a maximal inhibition of 90% at 500 µM (n5 cells at each concentration). This suggests that the oxygen-nitrogen substitution on LY303511 plays a functional role in its ability to directly inhibit Kv currents, although the near-complete inhibition observed at higher concentrations implies that both LY294002 and LY303511 block the same population of Kv channel subtypes.

4. LY294002 inhibits Kv2.1 and Kv1.4 expressed in HEK293 cells
Western blotting has revealed that Kv2.1 and Kv1.4 are highly expressed in ß cells. The ability of these compounds to inhibit specific Kv channel currents in a recombinant system was therefore tested. Using HEK293 cells transfected with the rat Kv2.1 -subunit, the previous experiments in MIN6 cells were replicated. Kv2.1 was inhibited by 50 µM LY294002, with a maximal inhibition on sustained currents of 98.8 ± 0.3% (n=10, P<0.001) Addition of 100 µM LY303511 reduced Kv2.1 currents by 41.1 ± 5.8% (n=9, P<0.05). Wortmannin (100 nM) had no significant effect on the same currents, but 30 mM TEA reduced currents by 61.7 ± 7.6% (n=7, P<0.05). The rate of inactivation of Kv2.1 increased with increasing concentrations of LY294002, implying an open-channel mechanism of block. These data also implicate LY294002 as a more potent Kv channel inhibitor than TEA, suggesting a possible difference in their mechanisms of channel block.

LY294002 reduced the inactivation time constant (TAU2) of Kv1.4 from 73.4 ± 8.3 ms to 18.2 ± 2.9 ms (n=13, P<0.0001). No significant effect on the time constant of activation (TAU1) for these channels was observed. Wortmannin had no significant effect on the peak currents, TAU1 or TAU2.

5. Kv currents in rat ß cells at near-physiological temperature are inhibited by LY294002
Rat pancreatic ß cells were voltage-clamped in the whole-cell configuration at 32–35°C to assess the effect of LY294002 on native Kv channels at near-physiological temperature. Voltage-dependent outward K⁺ currents were similar to those we have described at 22°C, but inactivated to a greater degree over 500 ms (50%). In cells treated with 25 µM LY294002 for 5 min, sustained Kv currents were inhibited by 92.3 ± 1.1% (n=6, P<0.001) compared with controls whereas 100 nM wortmannin had no effect. Since LY294002 is expected to block channels by an open-channel mechanism, the remaining inactivating currents likely result from activation of both Kv1.4 and 2.1 channels.

6. The effect of LY294002 on other important ß cell channels
The ß cell is known to express both ATP-sensitive potassium (K_ATP) channels and voltage-dependent calcium channels (VDCCs). Given the extent to which LY294002 was able to block Kv channels, we examined whether it could also directly affect these other channels. This compound slightly but significantly enhanced K_ATP currents from tsA201 cells overexpressing Kir6.2/SUR1 in the inside-out excised patch configuration by 31.4 ± 10.1% (n=13, P<0.05). TSA cells transfected with the ₁c and ß_2A L-type VDCC subunits were examined to assess whether LY294002 could affect L-type calcium channel function. LY294002 (50 µM) did not significantly alter peak currents at +10 mV whereas 10 µM nifedipine, a calcium channel blocker, reduced peak currents by 88.8 ± 1.1% (n=4, P<0.05). These findings indicate that LY294002 does not exert its insulinotropic effects by blocking K_ATP channels or via a calcium channel-dependent mechanism.

7. 5,6-Dichlorobenzimidazole riboside (DRB) does not affect Kv currents in MIN6 cells
It has been reported that LY294002 nonspecifically inhibits the action of casein kinase II (CKII). In at least one instance, CKII has been shown to modulate Kv channel function, causing a shift in the voltage dependence of Kv3.1. To test whether inhibition of CKII was a potential mechanism for LY294002’s action on Kv channels, the CKII inhibitor DRB was applied to MIN6 cells while monitoring their whole-cell Kv currents. Application of 50 µM DRB had no significant effect on sustained currents. These data suggest that LY294002 does not inhibit Kv channels through CKII.

8. LY294002 potentiates glucose-stimulated insulin secretion from MIN6 cells
To provide a functional assessment of the properties of LY294002 and LY303511 as Kv channel blockers in ß cells, their ability to affect GSIS from MIN6 cells was examined (Fig. 2 ). It has previously been demonstrated that inhibition of Kv currents from insulinoma cells potentiates their ability to release insulin at elevated glucose concentrations only. Addition of 8 µM LY294002 and 50 µM LY303511, the approximate IC₅₀ values for their inhibition of Kv currents, to MIN6 cells with 10 mM glucose potentiated insulin secretion to 200.7 ± 29.7% (P<0.01) and 163.2 ± 1.7% (P<0.05) of control levels, respectively. Addition of 100 nM wortmannin potentiated secretion to only 130.4 ± 0.6% (P<0.05) of control. Insulin secretion from basal glucose levels was not significantly different between groups. These data indicate that LY294002 enhances insulin secretion at least in part through inhibition of Kv channels. It is likely that Kv channel inhibition serves as the primary insulinotropic mechanism of LY294002, as evidenced by the significant difference in capacity between LY294002 and wortmannin to enhance insulin secretion.

View larger version (18K): [in this window] [in a new window]   Figure 2. LY294002, LY303511, and wortmannin potentiate glucose stimulated insulin secretion. MIN6 cells were incubated for 1 h in KRB solution containing 10 mM glucose in the presence and absence of the three compounds. Insulin release from these cells was subsequently assayed and 8 µM LY294002, 50 µM LY 303511, or 100 nM wortmannin enhanced secretion by 200.7 ± 29.7%, 163.2 ± 1.7%, and 130.4 ± 0.6%, respectively. No significant increase in secretion was observed from cells incubated under 0 mM glucose conditions between control and treated groups (**P<0.01, *P<0.05).

CONCLUSION AND SIGNIFICANCE

We have identified the Kv channels as novel targets for the PI3K inhibitor LY294002 in the MIN6 insulinoma cell line, rat pancreatic ß cells, and HEK293 cells expressing recombinant Kv2.1 and Kv1.4. It has been demonstrated that LY294002 is acting through a PI3K-independent mechanism, since treatment of cells with wortmannin was unable to affect Kv channel function and the LY294002 negative control, LY303511, also blocked currents. Based on its rapid time course of action and its ability to increase the rate of channel inactivation dose dependently, it is likely that LY294002 is acting directly on the channel, with current inhibition usually starting to occur < 1 min after introduction to the bath solution (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Schematic diagram.

The ubiquitous nature of Kv channels and the observation that LY294002 appears to block their currents quite extensively, combined with the fact that numerous studies in excitable tissues have used this compound, further emphasize the importance of these findings. In the future, the molecular nature of the interaction between LY294002, LY303511, and other quercetin-derived compounds and Kv channels should be investigated. This may ultimately bring about the discovery of a novel, potent, and specific potassium channel blocker.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0802fje; to cite this article, use FASEB J. (February 5, 2003) 10.1096/fj.02-0802fje

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