4.1 CalnI inhibits Ca2+ responses and neurotransmission
To characterize the functions of CalnI in modulating synaptic transmission in neuronal system, the Ca2+ imaging technique was applied. The primary cortical neurons were transfected with CalnI, CalnIΔHT or CalnIDE2AQ respectively (all cotransfected with mCherry), and loaded with Fluo-2 MA to detect the Ca2+ fluorescence intensity change. The cells overexpressing mCherry were selected as target cells, and a 405 nm laser spot was specifically illuminated on the target cell for 500 msec to locally uncage the MNI-caged glutamate in the bath to stimulate the target cell. The Fluo-2 fluorescence changes were recorded to reflect the Ca2+ responses in the stimulated neurons and neighboring neurons. This uncaging stimulation was repeated three times and the neighboring cells were selected randomly. As shown in Figure 1A, the region of interest (ROI) 1 is the selected target cell and ROI 2-9 are the neighboring cells. As laser illuminated on the target cell, Fluo-2 fluorescence intensity was recorded (Fig. 1B).
The laser illumination would cause an artificial peak in target cell recording which followed by a slow decline showing the real fluorescence intensity change (Fig. 1C). On the other hand, the fluorescence intensity recording of neighboring cells would be single peaks without the artifacts (Fig. 1D).
To compare the Ca2+ responses among different cells, fluorescence intensity
changes (ΔF) were normalized to the baseline fluorescence intensity before stimulation (Fbaseline). In CalnI-expressing neurons, the Ca2+ responses were significantly inhibited (Fig. 2A, red) and the neurotransmission to neighboring cells was also largely decreased (Fig. 2B, red). CalnIDE2AQ-expressing cells showed results similar to wild-type group (Fig. 2A and B, green). Target cells expressing CalnIΔHT had a certain level of decrease in Ca2+ response (Fig. 2A, blue) but showed normal neurotransmission to neighboring cells (Fig. 2B, blue).
To examine the effects of CalnI when the protein was expressed in post-synaptic neurons, CalnI-expressing cells were taken as neighboring cells to conduct the same glutamate stimulation experiments as above. Although receiving normal level of synaptic signals, CalnI-expressing neighboring cells showed declined Ca2+ responses (Fig. 2C and D, red) while CalnIDE2AQ-expressing neighboring cells were just partially inhibited (Fig. 2D, green) and CalnIΔHT-expressing neighboring cells had no significant decrease compared to control group (Fig. 2D, blue). Taken together, these results suggested that overexpressing of CalnI would impair the neurotransmission.
4.2 CalnI reduces CICR
To further confirm the effect of CalnI on the inhibition of neurotransmission, uncaging Ca2+ experiment was applied to directly evoke the intracellular Ca2+ signal.
The bath of neurons contained NP-EGTA, a cell-permeant photolabile chelator that exhibits a high selectivity for Ca2+ and releases Ca2+ upon UV illumination. When the Ca2+ concentration was forced to rise in the target cells expressing CalnI by laser illumination (Fig. 3A, red), the signals transmitted to their neighboring cells were significantly reduced (Fig. 3B, red). Compared to control group, CalnIDE2AQ-expressing neurons also had decreased Ca2+ responses in their neighboring cells (Fig. 3B, green) while CalnIΔHT-expressing target cells showed normal neurotransmission as control group (Fig. 3B, blue). Furthermore, the Ca2+
responses in the neurites of CalnI and CalnIDE2AQ-expressing neurons were also largely decreased as in the neighboring cells (Fig. 3C, red and green).
To verify if CalnI affects the calcium-induced calcium release, the neurons were then stimulated by caffeine (40 mM) and recorded the Ca2+ responses. In CalnI-expressing neurons, the Ca2+ responses induced by caffeine were significantly suppressed (Fig. 3D, red). Likewise, the Ca2+ responses in CalnIDE2AQ-expressing neurons were reduced during caffeine stimulations (Fig. 3D, green). There was also a slightly decrease in the Ca2+ responses of CalnIΔHT-expressing neurons compared to control group (Fig. 3D, blue) but was not as obvious as in CalnI and CalnIDE2AQ groups. These results indicated that CalnI may interfere with CICR pathway.
4.3 CalnI affects mGluR but not AMPA receptor
During glutamate stimulation, both metabotropic and ionotropic glutamate receptor would be activated at once. According to the results of glutamate stimulation experiment above, CalnI may affect not only the voltage-gated Ca2+ channels but also glutamate receptors. To distinguish the effect of CalnI on these two different types of glutamate receptors, group I mGluR agonist DHPG and AMPA were used individually to stimulate the target neurons expressing CalnI. When stimulated by DHPG (20 μM), the Ca2+ responses of CalnI-expressing target cells were significantly declined compared to control group (Fig. 4A, red), and also weaken the Ca2+ responses of downstream neighboring neurons (Fig. 4B, red). On the other hand, the Ca2+ responses of CalnIΔHT and CalnIDE2AQ-expressing target neurons upon DHPG stimulation were similar to control group, but the Ca2+ responses in their neighboring cells were increased (Fig. 4B, blue and green).
On the contrary, when stimulated by AMPA (10 μM), the Ca2+ responses of neurons showed no significant difference among all the groups (Fig. 4C and D). these results suggested that CalnI may inhibit mGluR signaling pathway but have no obvious effect on AMPA receptor.
4.4 CalnI reduces the distal neurite number of primary cortical neurons
Since CalnI would affect the synaptic transmission, it may also influence the neuronal morphology. To understand the effects of CalnI on neuronal morphology, the images of mCherry-expressing neurons were analyzed with Sholl analysis to examine the morphological differences between CalnI-expressing and non-expressing neurons.
In CalnI, CalnIΔHT and CalnIDE2AQ-expressing neurons, the neurites at around 35-75 μm from the center of soma was significantly reduced compared to control group (Fig.
6A, B and C). but there was no significant difference among the three groups of CalnI wild type and mutants at any distance (Fig. 6D).
To confirm that the decrease of distal neurites was not a general result caused by overexpressing and was actually caused by CalnI, calmodulin and CaM1234 were also coexpressed with mCherry in neurons, respectively (Fig. 5B). CaM-expressing neurons showed a similar reduction of distal neurites as CalnI and its mutants at 55-75μm from the center of soma (Fig. 7A), but CaM1234 group had almost no significant difference compared to control group (Fig. 7B).
Although CalnI and CaM-expressing neurons showed similar morphological changes compared to control group, there was significant difference between them on the neurite number around 20-40 μm from the center of soma (Fig. 8, left panels).
CalnI-expressing neurons and CaM1234-expressing neurons had significant differences in neurite number at 15-75 μm from the center of soma (Fig. 8, right panels). These results
indicated that overexpression of CalnI and its mutants would affect the neuronal morphology and reduce the distal neurite number.
4.5 CalnI affects the localization of CaMKIIβ in HEK293T cells
Previous studies in our lab showed a possible interaction between CalnI and CaMKIIβ by Yeast Two-Hybrid screening. To further confirm the interaction between these two proteins, three fusion proteins CFP-CalnI, YFP-CaMKIIα and YFP-CaMKIIβ were expressed in HEK293T cells to examine the localization of them. CaMKIIα was considered as a comparative since its structure is similar to CaMKIIβ. When expressed seperately, CFP-CalnI distrbuted in the cytosol and plasma membrane, while YFP-CaMKIIα evenly distributed in the whole cell and YFP-CaMKIIβ localized in several aggregation spots (Fig. 9A).
When CFP-CalnI and YFP-CaMKIIα coexpressed in HEK cells, their distribution patterns were very different and had no colocalization (Fig. 9B). On the other hand, CFP-CalnI changed the distribution of YFP-CaMKIIβ in HEK cells from aggregation spots into a more disperse distrbution pattern when they were coexpressed, and they also obviously colocalized (Fig. 9C). These results suggested that CalnI and CaMKIIβ interact with each other and affect both their localization in cells.