Modulation of ASIC channels in rat cerebellar Purkinje neurons by ischaemia-related signals

Abstract

Acid-sensing ion channels (ASICs), activated by a decrease of extracellular pH, are found in neurons throughout the nervous system. They have an amino acid sequence similar to that of ion channels activated by membrane stretch, and have been implicated in touch sensation. Here we characterize the pH-dependent activation of ASICs in cerebellar Purkinje cells and investigate how they are modulated by factors released in ischaemia. Lowering the external pH from 7.4 activated an inward current at -66 mV, carried largely by Na+ ions, which was half-maximal for a step to pH 6.4 and was blocked by amiloride and gadolinium. The H+-gated current desensitized within a few seconds, but approximately 30 % of cells showed a sustained inward current (11 % of the peak current) in response to the maintained presence of pH 6 solution. The peak H+-evoked current was potentiated by membrane stretch (which occurs in ischaemia when [K+]o rises) and by arachidonic acid (which is released when [Ca2+]i rises in ischaemia). Arachidonic acid increased to 77 % the fraction of cells showing a sustained current evoked by acid pH. The ASIC currents were also potentiated by lactate (which is released when metabolism becomes anaerobic in ischaemia) and by FMRFamide (which may mimic the action of related mammalian RFamide transmitters). These data reinforce suggestions of a mechanosensory aspect to ASIC channel function, and show that the activation of ASICs reflects the integration of multiple signals which are present during ischaemia.

Figure 1. ASIC currents in cerebellar Purkinje neurons

Membrane potential was −66 mV and internal solution contained Cs⁺ as the main cation, except where stated. A, response of an isolated Purkinje cell to pH 6.0 solution in control conditions, in the presence of 100 μM amiloride, and after washout of amiloride. B, increase of sustained component of response of an isolated Purkinje cell to pH 6.0 solution during deterioration of cell condition (shown by increase of holding current: distance below dashed line). Slashes on dashed line denote time intervals of ≈2 min between the responses shown. Middle trace shows that amiloride does not block the sustained current. C, response of a Purkinje cell in a cerebellar slice to pH 6.0 solution (solutions contained 10 μM NBQX, 40 μM bicuculline and 1 μM TTX, to block AMPA and GABA_A receptors and Na⁺ action potentials). D, specimen I-V relations for the peak ASIC current evoked by pH 6.0 solution in three isolated cells whole-cell clamped with internal solutions in which the main cation was Na⁺ (•), Cs⁺ (▪) or K⁺ (▴); data were normalized to the inward current recorded at the most negative voltage tested. E, responses of an isolated cell to solutions of pH 6.5, 6.0 and 5.0. F, mean current-pH curve from 20 isolated cells. Smooth curve has the form [H⁺]_o³/([H⁺]_o³ + H_0.5³) with pH_0.5 = -log₁₀H_0.5 = 6.4.

Figure 4. Modulation by lactate and neurotransmitters of ASIC currents in isolated Purkinje cells

A, response of an isolated cell to pH 6.7 solution in control conditions, in 15 mm lactate, and after washing out the lactate. B, mean peak ASIC current (evoked by pH 6.0 solution) in the presence of 100 μM glutamate (GLU) + 10 μM NBQX (n = 5 cells), 100 μM GABA + 40 μM bicuculline (BIC, n = 5), 100 μM ATP (n = 5), and 100 μM adenosine (ADO, n = 3). C, response of a Purkinje cell to pH 5.0 solution in control conditions, in the presence of 100 μM FMRFamide, and after washout of the neuropeptide. D, mean potentiation by FMRFamide of the peak and sustained components of the ASIC current evoked by pH 5.0 solution, in 5 and 3 cells respectively. E, alignment of sequences of FMRFamide and met-enkephalin-arginine-phenylalanine (M-enk-RF); vertical lines show identical amino acids. F, M-enk-RF (30 μM) did not potentiate the ASIC current evoked by pH 6.0 solution. Cells were clamped at −66 mV with internal solution containing Cs⁺. Dashed lines show mean amplitude of control responses bracketing responses in lactate, FMRFamide and M-enk-RF.

Figure 2. Modulation of ASIC currents by gadolinium and cell swelling

A, response of an isolated Purkinje cell to pH 6.0 solution in control conditions, after 1 min in the presence of 100 μM gadolinium, and 1 min after washout of the 2 min exposure to gadolinium. Solutions were phosphate-free (see Methods). B, response of an isolated Purkinje cell to pH 6.0 solution in normal external solution, after 1 min in 25 % hypotonic solution, and again in normal solution 1 min after the end of a 2 min exposure to hypotonic solution. Dashed line shows mean amplitude of control responses before and after hypotonic solution. Cells were clamped at −66 mV, with internal solution containing Cs⁺.

Figure 3. Modulation of ASIC currents in isolated Purkinje cells by arachidonic acid

A, response of a Purkinje cell to pH 6.7 solution in the absence of arachidonic acid, in the presence of 10 μM arachidonic acid, and after washout of the arachidonic acid. B, potentiation of the peak current response to pH 6.0 solution as a function of arachidonic acid concentration (data from 6, 4, 4, 5 and 20 cells for 1, 4, 5, 7 and 10 μM arachidonic acid). C, potentiation by 5 μM arachidonic acid of the peak response to solutions of different pH (5, 3, 2 and 4 cells for pH 6.7, 6.5, 6.3 and 6.0). D, induction by 5 μM arachidonic acid of a sustained component to the ASIC response to pH 6.7 solution. Top traces show entire responses; bottom traces are at higher amplification to show the sustained component. E, percentage of 44 cells lacking a sustained component in control solution in which arachidonic acid induced (I) a sustained component, percentage of 13 cells showing a sustained component in control solution which arachidonic acid enhanced (E), and percentage of all 57 cells in which arachidonic acid induced or enhanced a sustained component (total, T). F, lysophosphatidylinositol (LPI, 2 μM) does not alter the Purkinje cell response to pH 6.0 solution (data typical of 7 cells). Cells were clamped at −66 mV, with internal solution containing Cs⁺. Dashed lines show mean amplitude of control responses bracketing responses in arachidonic acid and LPI.