Ca2+ permeability of nicotinic acetylcholine receptors in rat hippocampal CA1 interneurones

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain where they are involved in a variety of physiological processes, including cognition and development. The nAChRs are ligand-gated cationic channels, and different subtypes are known to be differentially permeable to Ca2+; the alpha7-containing nAChRs are generally considered to be the most permeable. Ca2+ can activate and regulate a variety of signal transduction cascades, and the influx of Ca2+ through these receptors may have implications for synaptic plasticity. To determine the Ca2+ permeability of the nAChRs in rat hippocampal interneurones in the slice, which contain diverse subtypes of alpha7- and non-alpha7-containing nAChRs, we combined patch-clamp electrophysiology recordings with conventional fura-2 fluorescence imaging techniques. We estimated the relative Ca2+ permeability of the channels by determining the ratio of the increase in [Ca2+]i level (Delta[Ca2+]i) in the soma to the integrated transmembrane current (charge, Q) induced by the activation of the nAChRs, and compared this ratio to the highly Ca2+ permeable NMDA subtype of glutamate receptor channel. In all cells tested, the Delta[Ca2+]i/Q ratio was significantly larger (i.e. more than twice as big) for responses activated by NMDA than for alpha7-containing nAChRs in interneurones; the activation of the non-alpha7 nAChRs did not produce any significant increase in [Ca2+]i. Interestingly, the Ca2+ permeability of native alpha7 nAChRs in PC12 cells was significantly larger than in hippocampal interneurones, and not significantly different from NMDA receptors. Therefore, the alpha7-containing nAChRs in rat hippocampal interneurones are significantly less permeable to Ca2+ than not only NMDA receptors but also alpha7 nAChRs in PC12 cells.

Figure 1. Increased [Ca²⁺]_i level due to activation of α7, but not non-α7, nAChRs

A, brief (20–200 ms) duration pressure applications (arrows) of choline (‘Ch’; 10 mm) selectively activated α7-containing nAChRs in a neurone, inducing a rapidly activating and decaying inward current response (bottom trace), and a slower activating and decaying increase in fluorescence due to an increasing [Ca²⁺]_i level (top trace). B, in a different neurone, the activation of non-α7 nAChRs by longer (10 s) pressure applications of ACh (2 mm), along with the α7-selective antagonist MLA (10 nm), induced a more slowly activating inward current response (bottom trace) without any significant change in [Ca²⁺]_i level (top trace).

Figure 2. [Ca²⁺]_i changes due to activation of α7 nAChRs are smaller than during depolarization

The increase in [Ca²⁺]_i level due to a brief application of choline (10 mm; arrow; continuous line) is smaller than the increase in [Ca²⁺]_i level due to depolarization to +10 mV for a duration of either 100 ms (‘Depol-1’; black dashed line) or 1 s (‘Depol-2’; grey dashed line).

Figure 3. Comparison of [Ca²⁺]_i signals due to activation of α7 and NMDA receptors

A, the application of choline (10 mm; black traces) and NMDA (1 mm; grey traces) in the same neurone induced inward current responses (bottom traces) with different peak amplitudes and kinetics, as well as increases in [Ca²⁺]_i (top traces). Even though the amplitude of the NMDA current response was much smaller, the resulting increase in [Ca²⁺]_i was larger than for choline. B, the total amount of Ca²⁺ influx (as indicated by the increase in [Ca²⁺]_i level; top) is proportional to the integrated membrane current (i.e. charge, Q), the latter of which is estimated as the area under the current trace (bottom). The time for current integration and [Ca²⁺]_i amplitude measurement was 1 s after the beginning of agonist application (horizontal bar). C, the plot of the peak increase in [Ca²⁺]_i level versus the corresponding integrated current (Q) for choline (filled black squares) and NMDA (open grey triangles) responses yielded a near-linear relationship (linear regressions yielded R and P values of 0.52 and < 0.003 for choline, and 0.80 and < 0.0001 for NMDA). We have termed the slope of these lines the Permeability Index (PI).

Figure 4. [Ca²⁺]_i signals due to α7 nAChR activation are not due to Ca²⁺ intracellular store depletion, nor the activation of VGCCs

A, after inducing a choline response, slices were treated for 10 min with ryanodine and CPA (both at 20 μm) to prevent intracellular store depletion during α7 nAChR activation. Neither the peak amplitude of the [Ca²⁺]_i signal (top traces) nor the current response (bottom traces) was significantly changed by pretreatment with ryanodine and CPA. B, the application of cadmium (Cd²⁺; 200 μm) had no significant effect on the PI (3 cells; tested both in control and with Cd²⁺).

Figure 5. Ca²⁺ permeability of native α7 nAChRs in PC12 cells and NMDA receptors in pyramidal neurones

The PI values for the choline-induced activation of α7 nAChRs (left open bars) in interneurones (‘intern.’) was significantly smaller than in PC12 cells. For NMDA responses (right bars), there was no significant differences in the PI between interneurones and pyramidal (‘pyramid.’) cells.