The role of N-, Q- and R-type Ca2+ channels in feedback inhibition of ACh release from rat basal forebrain neurones
T G Allen, T G Allen
Abstract
1. The Ca2+ channel subtypes controlling ACh release from basal forebrain neurones and the ionic basis underlying muscarinic receptor-mediated autoinhibition were studied using skeletal myoballs to detect ACh release from individual rat basal forebrain neurones in culture. 2. Somatic Ca2+ currents evoked using a simulated action potential waveform revealed that Ca2+ entry was primarily through N-, Q- and to a lesser extent R-, T- and L-type Ca2+ channels. 3. Muscarine (10 microM) inhibited N- and Q- but not R-, T- or L-type somatic Ca2+ channels. Agonist inhibition was totally blocked by pre-treatment with pertussis toxin (500 ng ml-1). 4. ACh release from discrete sites along basal forebrain neurites (1. 2 mM extracellular Ca2+) could be largely abolished by blocking Ca2+ entry through either N-type or Q-type Ca2+ channels. Inhibition of Ca2+ entry through L- or T-type channels had no effect upon release. Following inhibition of either N- or Q-type Ca2+ channels, release could be restored to near control levels by raising [Ca2+]o. After selectively blocking N-, Q-, L- and T-type channels, low levels of release could still be evoked as a result of Ca2+ entry through R-type Ca2+ channels. 5. Muscarinic receptor activation reversibly inhibited ACh release due to Ca2+ entry through N-, Q- and R-type Ca2+ channels. In contrast, inhibition of inwardly rectifying K+ channels using Ba2+ (3-10 microM) or substance P (0.03-0.1 microM), or block of SK or BK Ca2+-activated K+ channels with apamin (100 nM) or charbydotoxin (100 nM) respectively, had no effect upon either ACh release or its modulation by muscarinic agonists. 6. These results show that ACh release from individual release sites on basal forebrain neurones is controlled by multiple Ca2+ channel subtypes with overlapping Ca2+ microdomains and that autoinhibition of release results from M2 muscarinic receptor-mediated inhibition of these presynaptic Ca2+ channels rather than as a consequence of K+ channel activation.
Figures
A, log-log plot of mean peak agonist-induced current versus ACh concentration for a typical excised myoball membrane patch. The curve was constructed according to a binding scheme of the form I/Imax = 1/(1 +K/A)nH, where A is the agonist concentration, nH is the Hill coefficient, K is the equilibrium constant and I is the response amplitude. The specific values of Imax, K and nH used to construct the curve were mean values obtained from 11 patches reported by Allen & Brown (1996) with Imax, K and nH being 640 pA, 5.3 μM and 2.1, respectively. From the form of this curve, it can be seen that provided I < = 1/6 Imax (i.e. it lies within the roughly linear region of the curve), Δ[ACh]∝ΔI/10nH - 1. In the case of a myoball, this means that provided the amplitude of the detector response from a given site does not exceed 1/6 of maximum, then assuming the binding constants of the receptors in the excised patches and whole myoball are the same, the same linear relationship can be applied to relate changes in detector current to changes in ACh concentration. B, the response to detection of ACh release from a discrete site on a basal forebrain neurone in response to a single action potential (i) under control conditions, 1.2 mM extracellular Ca2+/Mg2+, and (ii) in elevated (5 mM) Ca2+ and 0 mM Mg2+. From this it can be seen that under the control conditions used in the present study, the peak response meets the criterion of being 1/6 Imax. Note, the individual records show the mean and s.d. values for 5-7 repetitions of single action potential-evoked release.
A, plot of the time course of the block of the Ca2+ current by ω-Aga IVA, ω-CgTX and nifedipine in a typical basal forebrain neurone. B, data from the same cell as in A, showing somatic Ca2+ currents evoked by stepping from a VH of -80 mV to 0 mV for 100 ms (2.5 mM Cao2+) under control conditions and in the presence of various selective Ca2+ channel blockers: low (20 nM) and high (200 nM) concentrations of ω-Aga IVA to block P-type and P/Q-type channels, respectively; ω-CgTX (100 nM) to block N-type channels; and nifedipine (3 μM) to block L-type Ca2+ channels. In each case, currents have been corrected for leak, by subtracting the current recorded in the presence of 200 μM Cd2+. C, cumulative data showing the relative contributions played by the different Ca2+ channels to the peak somatic Ca2+ current. All values are means ±s.e.m. for the number of cells indicated above the individual histogram bars.
A, Ca2+ current I-V relationship generated using a voltage ramp from -80 to +60 mV (ramp rate, 1.4 V s−1) recorded under control conditions and in the presence of ACh (30 μM) or Cd2+ (200 μM). B, effect of ACh (30 μM) upon the R-type high threshold Ca2+ current after pre-incubation for 40 min prior to patching the cell, and continued application during the recording, of ω-Aga IVA (200 nM), ω-CgTX (200 nM) and nifedipine (3 μM). C, effect of ACh (30 μM) upon the Bay K 8644-enhanced (3 μM) L-type Ca2+ current evoked after inhibition of P-, Q- and N-type channels by pre-incubation of the cell for 45 min prior to patching the cell, and continued application during the recording, of ω-Aga IVA (200 nM) and ω-CgTX (100 nM). D, inhibition of the high threshold Ca2+ current by ACh (30 μM) after blocking Q-type channels by pre-incubating with ω-Aga IVA (200 nM) for 11 min. E, inhibition of the high threshold Ca2+ current by ACh (30 μM) after blocking N-type channels by pre-incubation with ω-CgTX (200 nM) for 7 min. F, lack of effect of ACh upon the T-type low threshold Ca2+ current evoked by stepping from -80 to -30 mV. Note, [Ca2+]o was 2.5 mM throughout except in B where it was 5.0 mM.
A, plot of the time course of the block by amiloride (300 μM), ω-Aga IVA (20 and 200 nM), ω-CgTX (100 nM), nifedipine (3 μM) and CdCl2 (200 μM) of the peak whole-cell Ca2+ current. Individual currents were generated every 10 s using a simulated action potential waveform (2.5 mM Cao2+). B, data from the same cell as in A, showing individual currents under control conditions and after equilibration with each of the different blockers. C, cumulative data showing the relative contribution played by each of the different Ca2+ channel subtypes to total Ca2+ entry following an action potential. All values are the means ±s.e.m. for the number of cells indicated above the individual histogram bars. D, the contribution of amiloride- sensitive (300 μM) T-type channels to peak and total Ca2+ influx in a cell expressing a larger number of somatic T-type channels. All currents shown in B and D have been corrected for leak by subtracting the current recorded in the presence of 200 μM Cd2+.
A, upper panel, graph of peak myoball detector current generated in response to ACh release by single action potentials evoked every 60 s under control conditions and in the presence of ω-CgTX (100 nM). Middle and lower panels, original data from the same experiment. Under control conditions (1.2 mM Cao2+; left-hand middle panel), stimulation of ACh release evoked a large rapidly activating and inactivating inward current due to activation of the nAChRs in the detector myoball. After incubation with ω-CgTX (100 nM) for 7 min to block Ca2+ entry through N-type channels, release was largely abolished (centre panel). Subsequently elevation of [Ca2+]o to 5 mM in the continued presence of ω-CgTX restored release to near control levels. B, similarly, inhibition of Ca2+ entry through Q-type channels by incubation of the cell with ω-Aga IVA (200 nM) for 20 min greatly reduced ACh release. In the continued presence of ω-Aga IVA release could again be restored to near control levels by elevating [Ca2+]o to 5 mM. Note, each of the records shown is the mean and s.d. of several repetitions (4-6) of single action potential-evoked responses.
A, the effect of applying nifedipine (3 μM) to block L-type channels. B, the effect of inhibiting T-type channels with Ni2+ (10 μM). In each case, records show the release evoked in response to single action potentials in 1.2 mM Cao2+ before and after application of the blockers. Note, each of the records illustrated is the mean and s.d. of 4-8 individual responses evoked at 1 min intervals.
A, control ACh release in response to single action potentials in 1.2 mM Cao2+. B, release after 11 min incubation with ω-Aga IVA (200 nM) to block Q-type channels. C, raising [Ca2+]o to 5 mM in the continued presence of ω-Aga IVA restores release to near control levels. D, after an additional 9 min incubation in ω-Aga IVA (200 nM) plus ω-CgTX (100 nM) with 5 mM Cao2+ a small component of release could still be detected. E, after subsequent addition of nifedipine (3 μM) to block L-type channels and depolarization of the cell to -50 mV using elevated K+ to inactivate T-type channels, release could still be detected as the result of Ca2+ entry through residual ‘R-type’ Ca2+ channels (7.5 mM Cao2+). Note, each of the records shown is the mean and s.d. of 4-9 individual responses evoked at 1 min intervals.
Data shown are of the inhibition of single action potential-evoked ACh release produced by exogenous application of ω-CgTX (100 nM), ω-Aga IVA (200 nM), nifedipine (3 μM) or Ni2+ (30 μM) to naive cells in 1.2 mM Cao2+ in order to block Ca2+ entry through N-, Q-, L- and T-type Ca2+ channels, respectively. The histogram bars on the left of the graph show the percentage inhibition in terms of a change in measured myoball detector current, whilst those on the right show the same data expressed in terms of the change in ACh concentration after correcting for the non-linear concentration-response characteristics of the nAChRs (see Methods). All data are the means ±s.e.m. for the number of cells indicated above each bar.
A, single action potential-evoked release due to Ca2+ entry through N-type channels (5.0 mM Cao2+) recorded under control conditions (after 10 min pre-incubation in 200 nM ω-Aga IVA, 3 μM nifedipine and 30 μM Ni2+; left-hand panel) and after application of 30 μM muscarine (right-hand panel). B, ACh release due to Ca2+ entry through Q-type channels under control conditions (after 10 min pre-incubation with 100 nM ω-CgTX, 3 μM nifedipine and 30 μM Ni2+; left-hand panel) and after application of 30 μM muscarine. C, release arising from Ca2+ entry through R-type channels following 12 min pre-incubation with all of the above blockers (7.5 mM Cao2+; left-hand panel) and after application of 30 μM muscarine. Note, each of the records shown is the mean and s.d. of 4-9 individual responses evoked at 40 s intervals.
A, application of Ba2+ (10 μM) to selectively inhibit KIR channels in magnocellular basal forebrain neurones had no effect upon either control release (middle panel; 1.2 mM Cao2+) or its modulation by 10 μM muscarine (right-hand panel). B and C, similarly, incubation (5 min) with 100 nM apamin or charybdotoxin to inhibit selectively SK- and BK-type Ca2+-activated K+ channels, respectively, had no effect upon control release in response to individual action potentials (centre panels) or its modulation by exogenous application of 10 μM muscarine (right-hand panels). Note, all traces are the mean and s.d. of between 4 and 9 individual action potential-evoked release events generated every 40 s.
Source: PubMed