Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones

Abstract

Permeation of the endogenous cation Zn2+ through calcium-permeable AMPA/kainate receptor-gated (Ca-A/K) channels might subserve pathological and/or physiological signalling roles. Voltage-clamp recording was used to directly assess Zn2+ flux through these channels on cultured murine hippocampal neurones. Ca-A/K channels were present in large numbers only on a minority of neurones (Ca-A/K+ neurones), many of which were GABAergic. The presence of these channels was assessed in whole-cell or outside-out patch recording as the degree of inward rectification of kainate-activated currents, quantified via a rectification index (RI = G+40/G-60), which ranged from <0.4 (strongly inwardly rectifying) to >2 (outwardly rectifying). The specificity of a low RI as an indication of robust Ca-A/K channel expression was verified by two other techniques, kainate-stimulated cobalt-uptake labelling, and fluorescence imaging of kainate-induced increases in intracellular Ca2+. In addition, the degree of inward rectification of kainate-activated currents correlated strongly with the positive shift of the reversal potential (V(rev)) upon switching to a sodium-free, 10 mM Ca2+ buffer. With Zn2+ (3 mM) as the only permeant extracellular cation, kainate-induced inward currents were only observed in neurones that had previously been identified as Ca-A/K+. A comparison between the V(rev) observed with 3 mM Zn2+ and that observed with Ca2+ as the permeant cation revealed a P(Ca)/P(Zn) of approximately 1.8. Inward currents recorded in 3 mM Ca2+ were unaffected by the addition of 0.3 mM Zn2+, while microfluorimetrically detected increases in the intracellular concentration of Zn2+ in Ca-A/K+ neurones upon kainate exposure in the presence of 0.3 mM Zn2+ were only mildly attenuated by the addition of 1.8 mM Ca2+. These results provide direct evidence that Zn2+ can carry currents through Ca-A/K channels, and that there is little interference between Ca2+ and Zn2+ in permeating these channels.

Figure 2. Strong correlation between inward rectification of kainate-induced currents and other methodologies in the identification of putative Ca-A/K channel-expressing (Ca-A/K(+)) neurones

A, Ca-A/K(+) neurones can be identified by a histochemical stain based on kainate-stimulated uptake of Co²⁺ ions. Cobalt-labelled (top) and unlabelled (bottom) neurones are shown along with I-V curves generated from kainate exposures to outside-out patches pulled from each neurone (insets). B, Ca-A/K(+) neurones can be identified using Ca²⁺ imaging techniques. A hippocampal culture was loaded with the fluorescent Ca²⁺ probe, Fura-2; pseudocolour images convey relative intracellular Ca²⁺ concentrations ([Ca²⁺]_i) before (i) and after (ii) brief exposure to kainate in sodium-free, high Ca²⁺ buffer (10Ca-EB). Traces (iii) depict the time course of fluorescence ratio changes in individual neurones; coloured lines correspond to specific neurones indicated by coloured dots in (ii). I-V curves (iv) are from outside-out patches pulled from the same two neurones. Note that the neurone with a rapid and high [Ca²⁺]_i rise (red) displays strong inward rectification (RI = 0.13), whereas the one with little [Ca²⁺]_i rise (blue) does not (RI = 1.65).

Figure 7. The presence of extracellular Ca²⁺ has little apparent effect on Zn²⁺ entry through Ca-A/K channels

A, kainate-triggered [Zn²⁺]_i rises in individual neurones. Cultures were loaded with the zinc-sensitive probe Newport Green, and imaged before, during and after a 5 min exposure to kainate (100 μM) with Zn²⁺ (0.3 mm); the extracellular medium was either calcium-free (i, iii) or contained 1.8 mm Ca²⁺ (ii, iv). Pseudocolour fluorescence images (i, ii) depict the Newport Green fluorescence increases (ΔF) at the end of the 5 min exposure. Zinc-dependent signal increases are shown as pseudocolour ratios of peak to basal fluorescence. Traces (iii, iv) show the time course of ΔF in individual neurones, expressed as the ratio of fluorescence at each time point (F_x) to its own baseline fluorescence (F₀). Note that the marked segregation in [Zn²⁺]_i rises among neurones; those with abrupt ΔF (continuous traces) are considered to be Ca-A/K(+) (see Methods). B, summary data. Bars depict mean increase in Newport Green fluorescence (F_x/F₀) at the termination of 5 min kainate exposures, in the additional presence of the indicated cations. In each experiment, fluorescence changes were averaged within each culture (4-13 Ca-A/K(+), 6–24 Ca-A/K(-) neurones per dish) before compiling results across experiments (n = 4 matched sets of cultures). *ΔF different from the same exposure to Ca-A/K(+) neurones (P < 0.001); # ΔF not different from Ca-A/K(+) neurones upon exposure in Zn²⁺ alone (P > 0.05); P values determined by one-way ANOVA with Tukey's multiple comparison test.

Figure 1. Some hippocampal neurones display inwardly rectifying kainate-activated currents

A, recording protocol. Current-voltage (I-V) relationships were elicited in both whole-cell and outside-out patch configurations, from a holding potential of −70 mV, by applying ascending and descending voltage ramps (top). Applying this protocol to an outside-out patch in normal extracellular buffer (Na-EB), in the presence and absence of kainate (100 μM), yielded the currents shown (middle). The I-V curve was derived by subtracting the current in the absence of kainate from that in its presence (bottom); the descending limb of the curve was used for analyses. B, kainate elicits inwardly rectifying currents in some neurones. Outside-out patches were pulled and subjected to the recording protocol illustrated in A, and rectification quantified by calculating a rectification index (RI; see Methods). While some patches yielded strongly inwardly rectifying I-V curves (left; RI = 0.22), others showed near-linear or outwardly rectifying responses (right; RI = 1.49). C, evidence for synaptic calcium-permeable AMPA/kainate receptor-gated (Ca-A/K) channels on some neurones. Rectification of synaptic AMPA/kainate channels was assessed by comparing the mean amplitude of mEPSCs at +40 and −60 mV, recorded in the presence of NMDA antagonists. Neurones with low whole-cell RI values, indicative of the presence of somatic Ca-A/K channels, generally also showed inward rectification of miniature EPSCs (mEPSCs; left), whereas other neurones did not (right). Insets depict the corresponding averaged mEPSC traces at +40 and −60 mV (scale bar = 5 pA, 5 ms).

Figure 3. Correlation between two indices of the contribution of Ca-A/K channels to the total kainate-activated current

A, outside-out patch recording of kainate-activated currents from a Ca-A/K(-) neurone. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited first in Na-EB and then in 10Ca-EB. Note the near-linear I-V curve in Na-EB, and the lack of inward current in 10Ca-EB with very negative reversal potential (V_rev, arrow). B, outside-out patch recording of kainate-activated currents from a Ca-A/K(+) neurone. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited in Na-EB and then in 10Ca-EB. Note the strong inward rectification in Na-EB, and the inward current at −70 mV in 10Ca-EB with positively shifted V_rev (arrow). C, scatter plot of V_rev against RI from 20 outside-out patches. Note the strong correlation between a positive shift in V_rev and decreasing RI, both indicative of an increasing fraction of current through Ca-A/K channels (r = −0.84; P < 0.0001). Arrows indicate individual neurones illustrated in A and B.

Figure 4. Effects of the voltage-dependent Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) on kainate-induced currents

A, NAS blocks kainate-induced currents in Ca-A/K(+) neurones. After pulling an outside-out patch, the I-V relationship of kainate (KA)-activated currents was elicited in Na-EB alone, and in the presence of 300 μM NAS. Note that this strongly inwardly rectifying current (RI = 0.16) was blocked by NAS in a voltage-dependent manner, with greater block at negative potentials. Inset, effect of 300 μM NAS on the kainate response of a Ca-A/K(+) neurone at −70 mV. B, NAS has no effect on kainate-induced currents in Ca-A/K(-) neurones. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited without and with 300 μM NAS in Na-EB, as described earlier. Note the lack of effect of NAS on this outwardly rectifying current (RI = 1.57).

Figure 5. Kainate induces whole-cell Zn²⁺ currents in Ca-A/K(+) neurones

A, continuous current recordings in Ca-A/K(+) and Ca-A/K(-) neurones. After establishing a stable whole-cell voltage clamp (-70 mV) in Na-EB, neurones were microperfused with 3Zn-EB (containing 3 mm Zn²⁺ as the only permeant ion), before application of kainate (also in 3Zn-EB). Note that while the 3Zn-EB alone resulted in an increased outward current in both neurones, subsequent application of kainate (KA) induced an inward current in a Ca-A/K(+) neurone (top trace), while increasing the outward current in a Ca-A/K(-) neurone (bottom trace). B, NAS blocks inward Zn²⁺ currents in Ca-A/K(+) neurones. A Ca-A/K(+) neurone was patched in whole-cell mode and a kainate-induced I-V relationship elicited in the presence of 3Zn-EB alone, or in the additional presence of NAS (300 μM). Note that NAS caused a marked negative shift in V_rev, eliminating the inward Zn²⁺ current at −70 mV. The inset shows the effect of the NAS on a kainate-induced Zn²⁺ current at −90 mV. C, increasing extracellular Zn²⁺ causes a positive shift in V_rev in Ca-A/K(+) neurones: a Ca-A/K(+) neurone was patched in whole-cell mode and kainate I-V response elicited in the presence of Zn²⁺ (0.3 or 3 mm as indicated) as the only permeant cation. The inset shows a shift in V_rev of KA currents in six Ca-A/K(+) neurones upon switching from 0.3Zn-EB to 3Zn-EB.

Figure 6. Zn²⁺ does not block Ca²⁺ entry through Ca-A/K channels

A, comparative amplitude of currents carried by Zn²⁺ and Ca²⁺ through Ca-A/K channels. A Ca-A/K(+) neurone was patched in whole-cell mode and kainate response at −70 mV recorded sequentially in 3Zn-EB and 3Ca-EB. B, lack of effect of Zn²⁺ on Ca²⁺ currents. Whole-cell KA responses were elicited in Ca-A/K(+) neurones at −70 mV, in 3Ca-EB (left) or in 3Zn-EB (right) first alone, and then in the additional presence of the other cation, as indicated. Bars depict the mean amplitude of the resultant currents, normalized to those obtained in 3Ca-EB or 3Zn-EB alone (n = 7 and 8 neurones, respectively). Note the paucity of change in the Ca²⁺ current upon addition of 0.3 mm Zn²⁺, and the increase in the Zn²⁺ current upon addition of Ca²⁺ (*indicates difference from control condition, P < 0.01 by Student's paired t test).