Growing evidence suggests that the endocrine hormones insulin and leptin are important modulators of neuronal function, with actions not directly associated with energy homeostasis. For example, leptin inhibits hippocampal neurons by phosphoinositide 3-kinase (PI3K)-driven activation of large conductance calcium-activated K⁺ (BK) channels (Shanley et al., 2002). In this study we have used human embryonic kidney (HEK 293) cells stably transfected with the α (hSlo) subunit of the BK channel to examine the actions of insulin.

Cell-attached and inside-out patch clamp recordings were used to study single channel responses as described previously (Shanley et al. 2002). In all cell-attached recordings, a large conductance K⁺ channel was evident (n = 164). In symmetrical 140 mM K⁺ inside-out recordings, the channel displayed a linear I-V relationship (± 60 mV) with a slope of conductance 210 ± 8.7 pS (mean ± S.E.M., n = 6) and reversal potential close to 0 mV. Channel open probability (P_o) increased with membrane potential depolarisation or increasing [Ca²⁺]_i, such that the V_0.5 values obtained in 1 µM and 10 µM Ca²⁺ were 37.29 ± 7.1 mV (n = 6) and 12.29 ± 4.9 mV (n = 5), respectively. Pipette application of insulin (1-50 nM) during cell attached recordings caused a rapid increase in channel activity (n = 31/35) with mean channel activity (NfP_o) increasing from 0.09 ± 0.02 (2-4 min) to 0.31 ± 0.09 (n = 35, P < 0.05, ANOVA) after 15 min. In contrast, control recordings displayed no change in channel activity with time (n = 17). As insulin and insulin-like growth factor-1 (IGF-1) receptors have a high sequence homology, a comparison of potencies of insulin and IGF-1 was used to determine the receptor identity. Thus, in the presence of insulin (10 nM), channel activity at 15 min was 2.87 ± 0.6 (n = 8), while NfP_o 15 min after application of IGF-1 (10 nM, n = 5) was 2.50 ± 0.41 (means normalised to control at 2-4 min; P > 0.05), suggesting the involvement of the IGF-1 receptors. It is well established that PI3K is a key enzyme activated downstream of IGF-1 receptors. However, incubation with LY294002 (10 µM) or wortmannin (10-50 nM) failed to prevent insulin activation of BK channels. Thus, the normalised NfP_o values obtained 15 min after insulin addition following exposure to LY294002 or wortmannin were 3.45 ± 0.8 (n = 7) and 2.6 ± 0.66 (n = 7), respectively compared to a control value of 3.81 ± 0.75 (n = 35).

These data indicate that insulin acting on IGF-1 receptors stimulates BK channels via a PI3K-independent pathway. This process may be a novel mechanism for regulating excitability.

This work is supported by the Wellcome Trust and Tenovus Scotland.