Brain slice and dissociated cell culture preparations were used to investigate the role of KATP channels in controlling the excitability of cholinergic neurones from the medial septum, diagonal band and substantia innominata of 12- to 14-day-old rats. Animals were anaesthetised by chloroform inhalation before being killed according to Home Office guidelines.
In the absence of ATP in the pipette filling solution cell excitability fell markedly within the first few minutes of establishing whole-cell conditions. Typically, cell membrane potential hyperpolarised by 13.4 ± 1.6 mV (n = 24), whilst resting membrane conductance increased by 13.2 ± 1.7 nS (n = 24). This resulted in a 5- to 10-fold increase in the stimulating current required to evoke action potential discharge. These effects could be reversed by exposure to the sulphonylurea compounds glibenclamide (10 nM) or tolbutamide (100 µM). However, inclusion of MgATP (0.1-4 mM), in the pipette solution failed to prevent activation of the underlying conductance.
Under voltage clamp, the sulphonylurea-sensitive current had an almost linear I/V relationship between -120 and -40 mV, exhibited no time dependence and reversed close to EK. Changes in [K+]o shifted the reversal potential by 59.6 mV for a 10-fold increase in [K+]o (n = 5).
Simultaneous whole-cell and cell-attached patch recording with 118 mM K+-containing solution in both electrodes and 3 mM K+ in the bath solution, revealed that the current was associated with the opening of channels of conductance of 60-74.5 pS (mean ± S.E.M. 66.2 ± 1.9 pS, n = 7). Both the current and channel activity were inhibited reversibly by tolbutamide (100 µM) and irreversibly by glibenclamide (10 nM). In inside-out patches recorded in symmetrical 140 mM K+-containing solution (potassium acetate 140 mM, Hepes 20 mM, MgCl2 1 mM, EGTA 1 mM; pH 7.3) the KATP channels exhibited weak inward rectification at positive membrane potentials. Channel opening was reversibly inhibited by 10 µM MgATP. In the presence of 100 µM MgATP, ADP and diazoxide but not pinacidil (200 µM) increased channel opening.
These data indicate that KATP channels exert a profound modulatory influence upon the excitability of cholinergic basal forebrain neurones. Single-channel conductance and pharmacological data are consistent with them being composed of SUR1 and Kir 6.2 subunits. Furthermore, whilst they show a high affinity for inhibition by ATP in excised patches, in the intact cell their sensitivity to ATP is more than two orders of magnitude lower, indicating that the channels are under tonic modulation by factors such as PIP2 (Baukrowitz et al. 1998) that are washed out or lost following excision.
This work is supported by the MRC.