A, Western blot with CaMKII antibodies showing CaMKII expression in HEK293T cells transfected with CaMKIIα alone or +Ca_v1.3 (α₁1.3, β_1b, α₂δ) but not untransfected cells (U). B, Ba²⁺ currents (I_Ba) were evoked by 50-ms test pulses from −90 to various voltages in HEK293T cells transfected with Ca_v1.3 alone (n=18, open circles) or cotransfected with CaMKII (n=16, closed circles). Representative current traces (left) and normalized I-V relationship (right) are shown. For Ca_v1.3 alone, V_1/2=−26.3±2.4 mV, k=−6.7±0.5. For Ca_v1.3+CaMKII, V_1/2=−26.6±1.5 mV, k=−7.2±0.3. C, I_Ba was evoked by 10-ms test pulse from −90 to −20 mV before (P1) and after (P2) a 20-ms prepulse (Pre) to various voltages. Left, Voltage protocol and representative test currents using +60-mV prepulse. Dashed line indicates initial P1 current amplitude. Right, Facilitation was measured as the ratio of the P2 and P1 test currents and plotted against prepulse voltage for cells transfected with Ca_v1.3 alone (n=10) or cotransfected with CaMKII (n=12). By two-way ANOVA, there was no difference in facilitation between groups.

A, Top, rat α₁1.3 with C-terminal type I PDZ-domain indicated with italics. Bottom, rat densin with leucine rich region (LRR), CaMKII-binding domain (CBD) and PDZ domain (not drawn to scale). Numbers indicate amino acid boundaries. B, Coimmunoprecipitation of GFP-densin with FLAG-tagged α₁1.3. Lysates from HEK293T cells cotransfected with Ca_v1.3 (FLAG-α₁1.3, β_1b, α₂δ) and GFP-densin (lane 1) or transfected with GFP-densin alone (lane 2) were incubated with goat α₁1.3 antibodies. Immunoprecipitated proteins were detected by western blotting with FLAG- (top) or GFP- (bottom) antibodies. Asterisk indicates nonspecific band detected by GFP antibody. C, Pull-down of GFP-tagged densin by GST-tagged α₁1.3 CT (GST-CT). GST (lane 2) or GST-CT (lane 3) was immobilized on glutathione-agarose beads and incubated with lysates from HEK293T cells transfected with GFP-densin. Top, western blot with GFP antibody. Bottom, GST-CT and GST protein used for pull-down are indicated in the ponceau-stained blot. Lane 1 (In) shows ~5% of the GFP-transfected cell lysate used in the assay. D, Binding of His-tagged densin PDZ-domain to GST-CT, but not GST or CT with mutation in PDZ-binding sequence (GST-CT_L-A) in protein overlay assay. GST proteins were separated by SDS-PAGE and transferred to nitrocellulose, which was incubated with His-Densin. Left panel, Ponceau staining shows levels of GST-proteins used in assay. Right panel, western blot with anti-His antibodies shows binding of His-densin PDZ only to GST-CT.

A,B, Epifluorescence images of immunofluorescently labeled transfected mouse hippocampal neurons in low-density culture. Greyscale images show fluorescence due to individual fluorophores; merged images are shown in color with regions of colocalization appearing yellow. A, Dendritic tree of neuron (17 DIV) co-transfected with HA-α₁1.3 and eGFP after live-cell staining with HA antibody (anti HA). HA-α₁1.3-positive puncta (left, red) are present on the neuronal surface throughout the dendrites. A dendrite segment shown at higher magnification (right) reveals HA-α₁1.3 puncta on the plasma membrane of dendritic spines (arrowheads). B, Neuron (20 DIV) transfected with GFP-densin, double-labeled with antibodies against GFP (anti GFP, green) and the postsynaptic density protein 95 (anti PSD95, red). Immunofluorescence for GFP was required due to the weak intrinsic fluorescence of GFP-densin. GFP-densin and PSD95 display a similar clustered distribution throughout the dendrites. At higher magnification (right), there is strong colocalization of GFP-densin (green) with PSD95 (red; arrowheads). Scale bars, 20 μm (left), 5 μm (right). C-E, Coimmunoprecipitation of densin, CaMKII and α₁1.3. Mouse hippocampal lysates were incubated with control goat IgG or affinity purified goat antibodies against α₁1.3 (C), densin (D) or CaMKII (E). Coimmunoprecipitated proteins are indicated by arrows in western blots for α₁1.3 (top), densin (middle), or CaMKII (bottom). Input lanes (In) represent 5% of lysates used in assay.

Ca²⁺ currents (I_Ca, A) and Ba²⁺ currents (I_Ba, B) were evoked by 5-ms pulses from −90 to −10 mV at 100 Hz in cells transfected with Ca_v1.3 alone (α₁1.3, β_1b, α₂δ; n=9 both for I_Ca and I_Ba) or cotransfected with densin+CaMKII (n=10 for I_Ca; n=12 for I_Ba). Test current amplitudes were normalized to the first in the train (Fractional current) and plotted against time (right). C, Quantitation of data in A and B. Fractional I_Ca and I_Ba for the last 11 pulses of the train were averaged for cells transfected with Ca_v1.3 alone, +densin+CaMKII, +densin, or +CaMKII. Parentheses indicate numbers of cells. *p<0.05 compared to each group, by one-way ANOVA and Bonferroni post-hoc test.

A,B, Fractional current for I_Ca was measured as in Fig.4 for: A, Ca_v1.3 (α₁1.3_L-A, β_1b, α₂δ) with mutated PDZ-binding site (Ca_v1.3_L-A, n=8) alone or cotransfected with densin+CaMKII (n=7); B, wild-type Ca_v1.3 alone (α₁1.3, β_1b, α₂δ; n=9) or cotransfected with densin+CaMKII (n=11), or with densin lacking PDZ domain (ΔPDZ) +CaMKII (n=8). C, Coimmunoprecipitation of CaMKII with densin but not densin with internal deletion (Δ483-1377). Transfected HEK293 cell lysates were incubated with control IgG (lane 3,4) or antibodies against densin (lane 5,6). Input lanes (1,2) represent ~5% of cell lysate used for coimmunoprecipitation. Western blotting was with antibodies against densin (upper panel) or CaMKII (bottom panel). D, Same as in A,B but for cells transfected with Ca_v1.3 alone (n=9) or cotransfected with densin+CaMKII (n=11), or +Δ483-1377+CaMKII (n=7). Statistical differences described in text were determined by t-test (A) or one-way ANOVA with post-hoc Bonferroni post-hoc test (B,D).

A-C, Left, Fractional I_Ca was measured as in Fig.4 for I_Ca for cells transfected with Ca_v1.3 alone (α₁1.3, β_1b, α₂δ) or cotransfected with densin and CaMKII. Right, Average fractional I_Ca for the last 11 pulses. A,B, KN-93 (10 μM) but not KN-92 (10 μM) in the intracellular recording solution inhibited CDF (A) as did CaM-KIINtide (B, 10 μM). For B, *, p<0.05 by Kruskal-Wallis ANOVA on ranks and Dunn’s post-hoc test. Results for +CaM-KIINtide were not significantly different from Ca_v1.3 alone. C, CaMKII autophosphorylation mutant (T286A) inhibited CDF. Parentheses indicate number of cells. For A and C *, p<0.01 by one-way ANOVA and Bonferroni post-hoc analysis.

Fractional I_Ca was measured as in Fig.4 for I_Ca for cells transfected with Ca_v1.3 alone (α₁1.3, β_1b, α₂δ) or cotransfected with densin and CaMKII for I_Ca with amplitude >250 pA (A) or <250 pA (B) or with EGTA or BAPTA in the intracellular solution (C). Average fractional I_Ca for last 11 pulses was compared by t-test (*, p<0.001; in B, p=0.14).

A, I_Ca was evoked by 300-msec step pulses from −90 mV to various voltages. Residual current amplitude normalized to peak current (I_res/I_peak) was plotted against test voltage (bottom) for Ca_v1.3 (α₁1.3, β_1b, α₂δ) alone (n=7) or Ca_v1.3+densin+CaMKII (n=13). Except for at +30 mV, there was no difference in I_res/I_peak for Ca_v1.3 alone or cotransfected with densin and CaMKII by two-way ANOVA and Bonferroni post-hoc test. B, Fractional I_Ca obtained from 50-Hz depolarizations (5-ms pulses from −90 to −10 mV) for Ca_v1.3 alone (n=14) or cotransfected with densin+CaMKII (n=18). By t-test, there was no difference in Fractional I_Ca for last 11 pulses for Ca_v1.3 alone (0.54±0.03) or +densin+CaMKII (0.55±0.02; p=0.62). C-D, Intracellular Ca²⁺ signals in HEK293T cells cotransfected with Ca_v1.3, densin, and CaMKII measured by Fura-2. Transfected cells were subject to simultaneous whole-cell patch clamp recording of I_Ca and imaging of Fura-2 fluorescence. C, Representative traces show Ca²⁺ signal measured as ratio of Fura-2 fluorescence from excitation at 340 and 380 nm following 50 or 100 Hz stimulation. D, The signal amplitude at 0.3 s (indicated by arrows in C) was normalized to whole-cell current amplitude to control for differences in current density between cells and compared by t-test (*, p=0.02 by t-test). The slope of the traces was compared by Mann Whitney rank sum test (*, p=0.007).