Genetic regulation of Kv4.2 expression level alters dendritic Ca2+ influx during the back-propagation of APs, suggesting that dynamic regulation of Kv4.2 channel expression may define physiologically relevant microdomains in dendrites (Chen et al., 2006; Kim et al., 2005). For instance, the application of 4-AP increases the amplitude of back propagating APs and the occurrence of Ca2+ spikes in dendrites, particularly in oblique apical dendrites (Frick et al 2003). These Ca2+ signals are compartmentalized at distal dendrites by branch points, which are important for establishing dendritic microdomains (Cai et al, 2004). The physiological relevance of activity-dependent Kv4.2 channel down-regulation, combined with observations that Kv4.2-dependent A-type K+ current amplitude increases in a distant-dependent manner lead us to hypothesize that Kv4.2 channels are differentially cycled at different regions along the apical dendrite in CA1 pyramidal neurons.
In order to test this hypothesis we used the FRAP technique to bleach primary apical and primary oblique dendrites at various regions in neurons expressing Kv4.2g construct in organotypic hippocampal slice cultures. Slice cultures were used because they offer excellent optical properties, ease of infection, and good control of the experimental conditions while preserving much of the cytoarchitecture of intact tissue (Gähwiler, 1984; Gähwiler et al., 1997). The FRAP technique is an established optical method for quantifying the two dimensional lateral diffusion of lipid bound proteins is mammalian cells (Axelrod et al., 1976
). Our measurements of FRAP recovery curves in dendrites were divided into a mobile fraction and an immobile fraction (Star et al., 2002
; Sprague and McNally, 2005
It has been shown that Kv channels have a stable immobile fraction, and a highly mobile fraction (O'Connell et al., 2006
). Because we have observed that Kv4.2 trafficking is coincident with GluR1 receptor trafficking (Kim et al., 2007
), which has a recovery time constant on the order of ~ 50 seconds in dendrites (Sharma et al., 2006
), we decided to measure the mobile component of the FRAP recovery curve for Kv4.2 channels over a 1 minute period. Although it is possible that further, slower fluorescence recovery continues after this time-point, we wanted to measure Kv4.2 cycling during the time at which GluR1 receptors cycle in dendrites. Furthermore, in our system, using our scan and bleach parameters, we observed substantial photobleaching of the fluorescence recovery after 1 minute of imaging. We were able to observe the maximal amount of fluorescence recovery before photobleaching in this interval.
Our recovery curves consisted of an initial fast decay followed by slow exponential decay. Based on modeling studies fitting the FRAP recovery curve with a two-phase decay exponential function (Carrero et al., 2003
, Matsumoto et al., 2005
), we suggest that Kv4.2g channels transiently interact with immobile cellular structures (Phair & Misteli, 2001
). This is consistent with data showing that Kv4.2 interacts with the cytoskeletal protein filamin and large adaptor proteins like AKAPs and PSD-95 (Lin et al., 2010
; Petrecca et al., 2000
; Wong and Schlichter, 2004
). Thus, in our experiments, the total mobile fraction is comprised of both fast and slow mobile components of the recovery curve and is the total normalized fluorescence recovery recorded in 1 minute, fit with a two-phase decay exponential function (Zheng et al., 2010)
Kv4.2g constitutive cycling rate is faster at distal dendrites
CA1 pyramidal neurons were bleached at 50 µm increments along primary apical and primary oblique dendrites. After averaging, we found that, in our system, we could divide the main apical dendrite into a proximal (< 150 µm) and a distal (>200 µm) region (.) Within these general categories, we performed FRAP on both apical and oblique dendrites. In both groupings the total mobile fraction, measured as the plateau of the recovery curve, was not significantly different: 76.2% ± 1.6 for distal dendrites and 77.2% ± 1.7 for proximal dendrites (N = 20 dendrites/5 neurons/group; p >0.05, unpaired t-test). We also did not observe a significant difference in the recovery rate of Kv4.2g channels within these groups (N = 20 dendrites/5 neurons/group; τ½ = 21.6 s ± 0.8 for distal main apical vs. 28.5 s ± 0.7 for distal obliques; p >0.05, unpaired t-test, and τ½ = 43.2 s ± 0.4 for proximal main apical vs. 44.4 s ± 0.4 for proximal obliques; p >0.05, unpaired t-test)().
Constitutive cycling of Kv4.2g is slower in proximal dendrites as compared to distal dendrites
However, when the average normalized FRAP recovery curves were compared between groups, we observed a significant increase in the recovery rate at distal primary and oblique dendrites as compared to proximal primary and oblique dendrites (N = 20 dendrites/5 neurons/group; τ½ = 24.5 s ± 0.2 for distal vs. 51.4 s ± 0.3 for proximal; p < 0.05, unpaired t-test) ().
When we analyzed the recovery curves for fast mobile fractions (first exponential of the double exponential fit) of Kv4.2g at distal and proximal dendrites we observed no significant difference (τ½
= 1.1s ± 0.02 for distal vs. 1.6 s ± 0.01 for proximal; p > 0.05, unpaired t-test). This likely represents freely diffusible Kv4.2g since this recovery time constant is similar to that for neurons that express GFP alone (τ½
= 0.8 s) (Star et al., 2002
) and is a small fraction of the total mobile Kv4.2g we observed in dendrites (35% in proximal dendrites and 37 % in distal dendrites). Thus, Kv4.2g channels cycle at a slower rate in proximal dendrites, primarily due to a differences in a slower mobile fraction which may represent channels transiently interacting with immobile structures.
Activity-dependent Kv4.2g cycling rate is faster at distal dendrites
In addition to basal Kv4.2 turnover rates, Kv4.2 channels can mobilize in response to neuronal activity. Recently, it has been shown that stimulation of AMPA receptors in primary neuronal cultures expressing Kv4.2 can induce internalization of the channel in an NMDAR-dependent manner (Kim et al., 2007
). The application of AMPA increases network activity activating NMDA receptors which results in a Ca2+
-dependent internalization of Kv4.2 channels out of spines, thus presumably increasing Kv4.2 concentration in dendritic compartments. We therefore asked whether AMPA application to neurons expressing Kv4.2g in slice cultures would affect activity-dependent cycling rates. Application of 100 µM AMPA for 5 minutes induced a significant increase in the recovery rate of Kv4.2g channels in distal dendrites as compared to proximal dendrites, where the application of AMPA had no significant effect (N = 15 dendrites/5 neurons/group; τ½
= 24.5 s ± 0.2 for distal vs. 17.9 s ± 0.5 for distal + AMPA; p < 0.05, unpaired t-test) (). This suggests that activity-dependent Kv4.2g cycling preferentially occurs at distal apical dendrites and is partially dependent on NMDAR activity. Kv4.2 channels are constituents of a synaptic complex that regulates Ca2+
influx through NMDARs (Jung et al. 2008
; Kim et al., 2007
), suggesting that activity-dependent regulation of Kv4.2 expression in distal dendrites contributes to dendritic excitability and synaptic plasticity (Shah et al, 2010).
Kv4.2g cycling rate increases at distal dendrites after AMPA stimulation
Kv4.2g cycling at distal dendrites is dependent on PKA site Ser552
Hammond et al., (2008)
found that AMPA application in primary neuronal cultures induced Kv4.2 internalization, which was blocked with a Kv4.2-PKA mutant lacking a phosphorylation site at Ser552 (Kv4.2gS552A). It is possible that neuronal stimulation and subsequent NMDA receptor activation leads to increased PKA levels (Roberson and Sweatt, 1996
) and results in membrane-bound Kv4.2 channels being internalized to enhance the excitability of dendrites. Therefore, we asked whether the dynamic cycling of Kv4.2 channels at distal dendrites was dependent on PKA phosphorylation. In neurons expressing the PKA mutant Kv4.2gS552A, the rapid constitutive cycling of Kv4.2g channels at distal dendrites was diminished, and the recovery rate after FRAP was not significantly different than what was observed at proximal dendrites expressing Kv4.2gS552A (N = 10 dendrites/5 neurons/group; τ½
= 48.1 s ± 0.4 for proximal + Kv4.2gS552A vs. 49.8 s ± 0.5 for distal + Kv4.2gS552A; p > 0.05, unpaired t-test) (). Thus, the bifurcation of dynamic Kv4.2g cycling we observed above was abolished by the expression of channels that lack the PKA phosphorylation site at Ser552. In addition, Kv4.2gS552A expression had no effect on the cycling rate of channels at proximal dendrites compared with Kv4.2g, suggesting that PKA activity at distal dendrites is primarily responsible for enhanced constitutive cycling rates of Kv4.2g channels at these sites (). In fact, expression of Kv4.2gS552A resulted in cycling rates in distal dendritic regions equivalent to those found for Kv4.2g in proximal regions (N = 10 dendrites/5 neurons/group; τ½
= 51.4 s ± 0.3 for proximal vs. 49.8 s ± 0.5 for distal + Kv4.2gS552A; p > 0.05, unpaired t-test) ().
Distance-dependent Kv4.2 cycling requires Kv4.2 PKA phosphorylation site Ser552
PKA modulates hippocampal synaptic plasticity (Lin et al., 2008
,Otmakhov et al., 2004). It has been shown that PKA activation induces a downregulation of Kv4.2 currents in Xenopus oocytes (Schrader et al., 2002
). In addition, activation of PKA increases the amplitude of back propagating action potentials in hippocampal dendrites (Hoffman and Johnston, 1998
). Therefore, we hypothesized that activity-dependent changes in Kv4.2g channels were dependent on PKA phosphorylation of the channel. To test this, we expressed Kv4.2gS552A in CA1 pyramidal neurons and then applied 100 µM AMPA for 5 minutes. The application of AMPA had no significant affect on the cycling rate of Kv4.2gS552A at distal dendrites. This suggests that the distance-dependent cycling of Kv4.2g that we have observed is regulated by activity-dependent phosphorylation of Kv4.2 channels by PKA (N = 10 dendrites/5 neurons/group; τ½
= 49.8 s ± 0.5 for distal + Kv4.2S552A vs. 50.6 s ± 0.5 for distal + Kv4.2gS552A + AMPA; p > 0.05, unpaired t-test) ().
Kv4.2g cycling rate is affected by clathrin-mediated endocytosis
AMPA mediated endocytosis of Kv4.2 channels in primary hippocampal neurons is a clathrin-regulated process, under the control of dynamin (Kim et al., 2007
). In order to isolate Kv4.2 channels inserted in the plasma membrane, we incubated slice cultures in a membrane-permeable dynamin-derived peptide (Kv4.2g + myr-DYN) or a non-functional scrambled peptide (Kv4.2g + myr-scram-DYN) for 10 minutes before applying 100 µm AMPA and performing FRAP at distal dendrites. The myr-DYN peptide blocks the recruitment of dynamin to clathrin-coated pits and thus inhibits clathrin mediated endocytosis of Kv4.2 channels at the dendritic membrane (Jung et al., 2009
; Kim et al., 2007
; Nong et al., 2003,).
Incubation of slice cultures with myr-DYN resulted in a significant decrease in the recovery rate of Kv4.2g at both distal and proximal dendrites (N = 10 dendrites/5 neurons/group; τ½= 46.9 s ± .6 for Kv4.2g + myr-DYN + distal vs. 24.5 s ± 0.2 for distal; τ½= 61.6 s ± 0.5 for Kv4.2g + myr-DYN + proximal vs. 53.7 s ± 0.6 for proximal; p < 0.05, unpaired t-test)(). In addition, myr-DYN significantly decreased the distance-dependent differences in Kv4.2g cycling rate we observed in distal dendrites. Thus, cycling rates at distal dendrites were not significantly different than those at proximal dendrites (N = 10 dendrites/5 neurons/group; τ½= 46.9 s ± .6 for Kv4.2g + myr-DYN + distal vs. 61.6 s ± 0.5 for Kv4.2g + myr-DYN + proximal; p > 0.05, unpaired t-test) (). Taken together, these results suggest that constitutive distance-dependent Kv4.2g cycling in dendrites is mediated by both PKA phosphorylation of the Kv4.2 channel and subsequent clathrin-mediated endocytosis of the channel.
Clathrin-mediated endocytosis regulates distance-dependent Kv4.2 channel cycling rates
Interestingly, recovery rates at both distal and proximal dendrites treated with myr-DYN increased significantly after the application of AMPA when compared to those treated with myr-DYN alone (N = 10 dendrites/5 neurons/group; τ½ = 22.4 s ± 0.7 for Kv4.2g + myr-DYN + AMPA + distal vs. 46.9 s ± .6 for Kv4.2g + myr-DYN + distal; τ½ = 49.3 s ± 0.7 for Kv4.2g + myr-DYN + AMPA + proximal vs. 61.6 s ± 0.5 for Kv4.2g + myr-DYN + proximal ; p > 0.05, unpaired t-test) (). However, application of AMPA to proximal dendrites treated with myr-DYN resulted in Kv4.2g cycling rates that were not significantly different than dendrites expressing Kv4.2g without myr-DYN treatment (N = 10 dendrites/5 neurons/group; τ½= 49.3 s ± 0.7 for Kv4.2g + myr-DYN + proximal + AMPA vs. 53.7 s ± 0.6 for proximal; p > 0.05, unpaired t-test) ().
Activity-dependent changes in Kv4.2 cycling rate are modulated by clathrin-mediated endocytosis
Distal dendrites from neurons treated with myr-DYN and 100 µM AMPA showed a significant decrease in fluorescence recovery as compared to those treated with myr-scram-DYN (N = 10 dendrites/5 neurons/group; τ½ = 17.9 s ± 0.5 for distal + AMPA vs. 15.6 s ± 0.4 for Kv4.2g + myr-scram-DYN + AMPA + distal vs. 19.0 s ± 0.3 for Kv4.2g + myr-DYN + AMPA + distal; p < 0.05 ANOVA and Tukey's test) (). However, when distal dendrites expressing Kv4.2g were compared to dendrites treated with myr-DYN and AMPA, no significant difference was detected (N = 10 dendrites/5 neurons/group; τ½ = 19.3 s ± 0.5 for Kv4.2g + distal vs. 19.0 s ± 0.3 for Kv4.2g + myr-DYN + AMPA + distal; p > 0.05, ANOVA and Tukey's test) (). These results suggest that during synaptic activity, Kv4.2 is driven out of spines into the dendrite in a clathrin-mediated process. When clathrin-mediated endocytosis is blocked activity-mediated increases in Kv4.2 cycling rate at distal dendrites is inhibited.