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. 2007 Feb 15;579(Pt 1):85-99.
doi: 10.1113/jphysiol.2006.123901. Epub 2006 Dec 14.

Presynaptic Plasma Membrane Ca2+ ATPase Isoform 2a Regulates Excitatory Synaptic Transmission in Rat Hippocampal CA3

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Free PMC article

Presynaptic Plasma Membrane Ca2+ ATPase Isoform 2a Regulates Excitatory Synaptic Transmission in Rat Hippocampal CA3

Thomas P Jensen et al. J Physiol. .
Free PMC article

Abstract

Plasma membrane calcium ATPase isoforms (PMCAs) are expressed in a wide variety of tissues where cell-specific expression provides ample opportunity for functional diversity amongst these transporters. The PMCAs use energy derived from ATP to extrude submicromolar concentrations of intracellular Ca2+ ([Ca2+]i) out of the cell. Their high affinity for Ca2+ and the speed with which they remove [Ca2+]i depends upon splicing at their carboxy (C)-terminal site. Here we provide biochemical and functional evidence that a brain-specific, C-terminal truncated and therefore fast variant of PMCA2, PMCA2a, has a role at hippocampal CA3 synapses. PMCA2a was enriched in forebrain synaptosomes, and in hippocampal CA3 it colocalized with the presynaptic marker proteins synaptophysin and the vesicular glutamate transporter 1, but not with the postsynaptic density protein PSD-95. PMCA2a also did not colocalize with glutamic acid decarboxylase-65, a marker of GABA-ergic terminals, although it did localize to a small extent with parvalbumin-positive presumed inhibitory terminals. Pharmacological inhibition of PMCA increased the frequency but not the amplitude of mEPSCs with little effect on mIPSCs or paired-pulse depression of evoked IPSCs. However, inhibition of PMCA activity did enhance the amplitude and slowed the recovery of paired-pulse facilitation (PPF) of evoked EPSCs. These results indicated that fast PMCA2a-mediated clearance of [Ca2+]i from presynaptic excitatory terminals regulated excitatory synaptic transmission within hippocampal CA3.

Figures

Figure 1
Figure 1. PMCA2a is selectively enriched in forebrain synaptosomes and shows a punctate distribution within hippocampal CA3
A, representative Western blots from hippocampal homogenates probed with antibodies raised against total PMCA isoforms 1–4 (lanes 1–4, left to right) or PMCA1a–4a (lanes 5–8 left to right). B, Western blots from forebrain homogenates (H) and synaptosomes (Syn) probed for total PMCA1, PMCA1a, total PMCA2 and PMCA2a. For all blots, 15 μg of protein from homogenate or synaptosome protein samples were loaded into their respective lane, 205 and 116 bars represent the approximate positions of these standard molecular weight markers. C, representative montages of the hippocampal CA3 region (top) or zoomed images of the CA3 pyramidal cell layer (bottom) labelled with antibodies raised against total PMCA2 (left panel) or PMCA2a (right panel); scale bars indicate a distance of 100 μm or 20 μm (top and bottom, respectively), and arrowheads indicate the location of PMCA2a punctae. S/O, Pyr and SR indicate the stratum oriens, pyramidal cell layer and stratum radiatum, respectively.
Figure 2
Figure 2. PMCA2a colocalizes with the presynaptic marker synaptophysin but not the postsynaptic marker PSD-95
A and B, representative confocal micrographs from the CA3 pyramidal cell layer showing typical colocalization of PMCA2a labelling (centre, red) with the presynaptic marker synaptophysin (Syn) and the postsynaptic marker PSD-95 (B) (left, green); merged images (A and B, right third panel) show colocalized pixels as yellow. Scale bars represent 20 μm, arrowheads indicate colocalized punctae in the third panel of A and arrows in the third panel of B indicate apposed PMCA2a and PSD-95 expression. C, quantification of colocalization using Pearson's correlation; the left bar shows mean Pearson's correlation values for PMCA2a and synaptophysin (Syn) and the right bar shows mean correlation values for PMCA2a and PSD-95. Values are means ±s.e.m. for n = 9, images from a minimum of nine images from four slice cultures prepared from four animals; ***P < 0.0001.
Figure 3
Figure 3. PMCA2a is present within VGLUT1-containing excitatory presynaptic terminals and inhibition of PMCA activity increases the frequency but not the amplitude of mEPSCs
A and B, representative confocal micrographs from the CA3 pyramidal cell layer (Pyr, A) and the CA3 stratum radiatum (SR, B) showing typical colocalization of PMCA2a labelling (centre, red) with the vesicular glutamate transporter 1 (VGLUT1; left, green); merged images (A and B, third panel right) show colocalized pixels as yellow. Scale bars represent 20 μm, arrowheads indicate colocalized punctae. C, quantification of colocalization using Pearson's correlation; the left bar shows mean Pearson's correlation values for PMCA2a and VGLUT1 in the pyramidal cell layer (Pyr) and the right bar shows mean correlation values for PMCA2a and VGLUT1 in the stratum radiatum (SR), values are means ±s.e.m. from a minimum of nine images from four slice cultures prepared from four animals; *P < 0.0001. C, representative current traces recorded from a CA3 pyramidal cell held at −70 mV in the presence of 10 μm bicuculline and 1 μm TTX before (top, black line) and after 15 min of 10 μm CE treatment (bottom, red line); events accepted for analysis are highlighted by black vertical bars, events that were detected but did not fit criteria for analysis (see Methods) are indicated by a circle, scale bars represent 10 pA vertical and 1 s horizontal. D, a cumulative histogram of mEPSC inter-event intervals. The bar chart (inset) shows the mean median inter-event interval of mEPSCs in untreated cells (left, black) and after 10 μm CE treatment (right, red). E, cumulative histogram of mEPSC peak amplitudes (pA). The bar chart (inset) shows the mean median amplitude of mEPSCs in untreated cells (left, black) and after CE treatment (right, red). All error bars are ±s.e.m., n = 9 for control and CE-treated cells. *P < 0.01 tested with paired, two-tailed t test.
Figure 4
Figure 4. PMCA2a is present within a subset of PV-containing inhibitory presynaptic terminals but inhibition of PMCA activity does not significantly influence the frequency or amplitude of mIPSCs
A and B, representative confocal micrographs from the CA3 pyramidal cell layer of GAD-65 (A) and PV (B), green, upper panels, colocalized with PMCA2a (red); merged images (A and B, third panel) show colocalized pixels as yellow and some are identified with arrowheads, whereas arrows indicate PMCA2a punctae that are not colocalized. Scale bars represent 20 μm. Below the confocal micrographs the bar graph shows quantification of colocalization using Pearson's correlation; the left bar shows mean Pearson's correlation values for PMCA2a and GAD-65, and the right bar shows mean correlation values for PMCA2a and PV, values are means ±s.e.m. from a minimum of six slices. C), representative current traces of mIPSCs recorded from a CA3 pyramidal cell held at −70 mV in the presence of TTX before (top, black line) and after 15 min of 10 μm CE treatment (bottom, red line); events accepted for analysis are highlighted by black vertical bars (see Methods), scale bars represent 15 pA vertical and 1 s horizontal. D, cumulative histogram of mIPSC inter-event intervals. The bar chart (inset) shows the mean median inter-event interval of mIPSCs in untreated cells (left, black) and after 10 μm CE treatment (right, red). E, cumulative histogram of mIPSCs peak amplitudes (pA). The bar chart (inset) shows the mean median amplitude of mIPSCs in untreated cells (left, black) and after CE treatment (right, red). All error bars are ±s.e.m., n = 9 for control and CE-treated cells.
Figure 5
Figure 5. Inhibition of PMCA activity does not alter the amplitude or the kinetics of recovery of paired-pulse depression of evoked IPSCs
A, the mean paired-pulse ratio (IPSC2/IPSC1) of stimulus-evoked IPSCs from CA3 pyramidal neurones plotted against the interstimulus interval (ISI, ms), filled black squares indicate mean paired-pulse ratios from control cells, while filled red squares indicate the mean paired-pulse ratios from cells treated with 10 μm CE for 15 min, error bars are ±s.e.m., n = 6 for control and 10 μm CE-treated cells at all time points. Lower panel, representative current traces recorded from the same CA3 pyramidal cell held at −70 mV before (black lines) and after 15 min of 10 μm CE treatment (red lines). IPSCs were evoked with interstimulus intervals of 100 ms (top) and 1000 ms (bottom) at the time points highlighted by filled dots. Note the lack of any effect of CE on PPD at both short and long ISI, but note also the small (approx 5–10 pA) increase in the amplitude of the first evoked IPSC that was seen in 3/6 cells. Scale bars represent 150 pA vertical and 200 ms horizontal. B, mean half-time for recovery of the paired-pulse depression of evoked IPSCs determined from single exponential fits of the recovery of paired-pulse depression in individual cells before (filled black bar) and after (filled red bar) 10 μm CE.
Figure 6
Figure 6. Inhibition of PMCA activity increases paired-pulse facilitation of evoked EPSCs, by enhancing the second EPSC, in a reversible and specific manner
A, application of 10 μm CE (represented by the horizontal black bar) increases the paired-pulse ratio recorded every 30 s, grey triangles for individual data points. Filled circles and error bars represent a running average ±s.e.m. over 2.5 min intervals. Below the graph are representative current traces recorded from the same CA3 pyramidal cell held at −70 mV, where small filled black circles represent the time of delivery of the stimulus, before application of CE (left) during CE application (middle) and 15 min after removal of CE (right), dashed black lines represent amplitudes of the first EPSC (black) and the control second EPSC following 10 μm CE treatment. In three neurones, 10 μm CE application increased the 50 ms paired-pulse ratio from 2.0 ± 0.1 to 3.1 ± 0.2 (n = 3; P < 0.05; paired t test) that was partially reversed 15 min after the removal of CE back to 2.4 ± 0.1 (n = 3; P= 0.07 paired t test). All scale bars represent 50 pA vertical and 100 ms horizontal. B, in the same cell shown in A the amplitude of the first EPSC (top) and cell input resistance (lower) recorded every 30 s during treatment with CE (shown by the horizontal black bar in A).
Figure 7
Figure 7. Inhibition of PMCA activity slows the recovery of paired-pulse facilitation of evoked EPSCs in CA3 pyramidal neurones
A, the mean paired-pulse ratio (EPSC2/EPSC1) of stimulus-evoked EPSCs from CA3 pyramidal neurones plotted against the interstimulus interval (ISI, ms), open circles indicate mean paired-pulse ratios from control cells, while filled red circles indicate the mean paired-pulse ratios from cells treated with 10 μm CE for 15 min, error bars are ±s.e.m., n > 5 for control and 10 μm CE-treated cells at all time points. Curves, black, control and red, CE, represent an unconstrained single exponential fit to this mean data. Lower panel, representative current traces recorded from the same CA3 pyramidal cell held at −70 mV before (black lines) and after 15 min of 10 μm CE treatment (red lines). EPSCs were evoked with ISIs of 50 ms (top) and 500 ms (bottom) at the time points highlighted by small filled black circles. Note the lack of any major influence of CE at the longer ISI. All scale bars represent 50 pA vertical and 100 ms horizontal. B, the normalized recovery of PPF in six cells before (open black circles) and after 10 μm CE (closed red circles), where the recovery of the PPF was fitted in each individual cell with a single exponential and then normalized using the extrapolated maximum paired-pulse ratio obtained from the A1 value as described in Methods. The inset bar graph shows the mean half-time for PPF decay for individual cells based upon individual single exponential fits before and after 10 μm CE, black and red bars, respectively. **P < 0.01 tested with paired, two-tailed t test.

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