CSPα co-expression alters BK channel whole cell current density in transiently transfected CAD cells.

<p>(A) Representative recordings of single CAD cells voltage clamped as depicted in the protocol using the whole-cell configuration of the patch clamp technique. The left-hand column displays macroscopic BK channel currents recorded from CAD cells transiently-transfected with BKα subunits alone. The middle and right-hand columns display BK currents recorded from cells co-transfected either a CSPα mutant (i.e. CSPα_HPD-AAA) or wild-type CSPα, respectively. Recordings were done 40–48 hours post-transfection. In all three conditions, whole cell currents were largely inhibited following addition of penitrem-A (100 nM). Penitrem-A sensitive currents, obtained by digital subtraction, are displayed in the bottom row of traces. The vertical and horizontal scale bars shown apply to all current tracings. The current-voltage plot shown in panel B quantifies the average penitrem A-sensitive peak currents observed under each transfection condition and highlights the significant differences in current density magnitude found between CAD cells transiently-transfected with BKα subunits alone, and those co-transfected with either WT or a mutant form of CSPα (Dunnett post-hoc ANOVA, one-way Vs BK channel alone; *p<0.05, **p<0.01). Inset shows BK channel expression in CAD cells transiently co-transfected with 0.75 µg cDNA encoding either CSPα or CSPα_HPD-AAA. For detection of surface expressed BK channel, transfected CAD cells were labeled for 30 min at 4°C with bath applied Biotin, as described in the methods. Following lysis, 1 mg of soluble protein lysate was subjected to overnight streptavidin pull-down at 4°C. Proteins isolated by streptavidin pull down (top panel) and 30 µg of soluble cellular lysate (bottom panel) were analyzed by Western Blot with an anti-BKα subunit antibody. Data is representative of 3 similar experiments.</p

Hsc70 does not reduce BK channel expression in the absence of CSPα.

<p><b>A.</b> CAD cells were transiently co-transfected with 1 µg cDNA encoding BKα subunit and either 1 µg HA-tagged, wild-type Hsc70 or the Hsc70 ATPase domain. Empty pCMV vector (1 µg) was co-transfected with 1 µg cDNA encoding BKα subunit as a transfection control. 24 h post-transfection, the cells were lysed and BK channel and β-actin expression was analyzed. <b>B</b>. The histogram quantifies the relative changes in cellular BK channel expression in the presence of WT Hsc70 and the Hsc70 ATPase domain, which were not statistically different than the control (i.e. pCMV vector alone).</p

CSPα alters BK channel expression.

<p><b>A.</b> Native CAD cells were transiently transfected with 1 µg cDNA encoding a neuronal BKα subunit, along with different amounts of myc-tagged CSPα (0.25 µg, 0.5 µg and 0.75 µg). Empty pCMV expression vector (0.75 µg) was co-transfected with 1 µg BKα subunit cDNA as a transfection control. 24 h post-transfection, the cells were lysed and the expression of BKα subunit and myc-tagged protein was analyzed by Western Blot. β-actin detection is shown to verify comparable sample loading. <b>B.</b> Histogram depicting quantification of BKα subunit levels in CAD cells co-transfected with increasing amounts of CSPα cDNA. Data are presented as mean ± SE of 5 similar experiments; *p<0.05 vs. pCMV vector control. <b>C.</b> Cells were transfected with 1 µg cDNA encoding BKα subunit along with either 0.25 µg or 0.75 µg of myc-tagged CSPα or 1 µg of pCMV. 24 h and 48 h post-transfection, BKα subunit expression was analyzed by Western Blot. <b>D.</b> Histograms depicting quantification of immunoreactive BKα subunit observed in the presence of co-transfected CSPα, as displayed in panel C. BKα subunit immunoreactivity detected at 48 h is expressed relative to the level of BKα subunit observed at 24 h; data are presented as mean ± SE of 4 similar experiments. Statistical significance was determined using one way ANOVA, *p<0.05; **p<0.01.</p

BK channelβ-subunits (BKβ4 and BKβ1) do not alter the CSPα-mediated reduction in BK channel expression.

<p>CAD cells were transiently transfected with 1 µg BKα subunit cDNA plus 1 µg BKβ4 subunit cDNA (<b>A</b>) or 1 µg BKβ1 subunit cDNA (<b>B</b>), along with either 0.75 µg CSPα or pCMV vector (control). 24 h post-transfection, protein expression was analyzed by Western Blot with primary antibodies recognizing BKα subunit, BKβ4 subunit, BKβ1 subunit, myc epitope tag, and β-actin. <b>C</b> and <b>D,</b> Quantification of BKα subunit expression in CAD cells in the absence or presence of co-transfected CSPα, along with the presence of either BKβ4 (panel C) or BKβ1 (panel D). <b>E</b> CAD cells were transfected with cDNAs encoding pCMV vector alone, BKα subunit or BKα subunit+CSPα and the indicated proteins were evaluated by Western blot analysis. <b>F</b> CAD cells were transfected with TRPC6 channel or syntaxin1A cDNA in either the absence of presence of CSPα cDNA. Detection of β-actin was utilized to verify equal protein sample loading. Data are presented as mean ± SE of 3 similar experiments; statistical significance was determined using one way ANOVA, *p<0.05; **p<0.01. Data shown in E and F are representive of 4 independent experiments.</p

CSPα chimeras with substituted J domains alter BK channel expression.

<p><b>A.</b> Schematic of wild-type CSPα and CSPα chimeras in which the J domain of CSPα is replaced by the J domain of the related J proteins Hsp40, Rdj2 or Rme8. <b>B.</b> Western blot analysis of BK channel expression in CAD cells 24 h following transfection with cDNA encoding BKα subunit and myc-tagged CSPα or the indicated CSPα myc-tagged chimeric constructs. 0.75 µg of pCMV vector was co-transfected with 1 µg cDNA encoding BKα subunit as a transfection control. Expression of myc-tagged proteins is shown by western blot, along with β-actin detection to verify similar sample loading. <b>C.</b> Histogram showing quantification of BK channel expression in the presence of either wild-type or J-domain (JD) substituted CSPα chimeras relative to cells co-transfected with pCMV vector. Mean data were obtained from 3–4 independent experiments, and statistically significant differences were determined by one-way ANOVA; **p<0.001.</p

BK channel co-immunoprecipitates with CSPα but not CSPα_HPD-AAA.

<p><b>(A)</b> CAD cells were transiently transfected with 1.0 μg cDNA encoding either CSPα_L115R, CSPα_L116Δ or CSPα_HPD-AAA in the presence and absence of 0.75 μg cDNA encoding myc tagged wild type CSPα and lysed 48 hours post-transfection. Western analysis and quantification of BK channel is shown; *p<0.05. Detection of β-actin on the same blot was used to verify equal loading between the various lanes. <b>(B)</b> CAD cells were transiently co-transfected with 1 μg BK channel and 0.75 μg myc-tagged CSPα, 0.75 μg myc-tagged CSPα_HPD-AAA and 0.75 μg pCMV (negative control). 0.7 mg of soluble cell lysate was subjected to immunoprecipitations with anti-myc monoclonal followed by Western blot analysis with anti-BK channel polyclonal and anti-myc monoclonal. The 55 kDa and 26kDa represent the heavy and light chain of the monoclonal myc-tag antibody. The right panel shows total cellular protein (input). Data are representative of three experiments.</p

BKα channel expression is elevated in ANCL.

<p>Western analysis of BKα channel, CSPα, and Hsc70 detected in 20μg of crude synaptosome fraction prepared from <u>human cortex</u> Control (34 yrs) and ANCL (L 116 Deletion; 36 yrs) as indicated. Detection of β-actin on the same blot was used to verify equal loading between the various lanes.</p

CSPα expression is lower in ANCL and some AD patients.

<p><b>(A)</b>Western analysis of CSPα, Dynamin 1, SNAP25, Hsc70 and β-actin detected in synaptosome fractions prepared from <u>human cortex</u> as indicated. For the 10 human samples the CSPα values were 671000, 459000, 583000, 530000, 684000, 690000, 351000, 432000, 397000, 342000. SNAP25 values were 2540000, 1640000, 2880000, 3040000, 3210000, 2530000, 2460000, 2050000, 2430000, 2140000, dynamin values were 3290000, 2160000, 3060000, 2580000, 3410000, 3500000, 3010000, 3240000, 2530000, 2300000 (B) Immunoblot showing CSPα monomer expression (left panel) and longer exposure showing high molecular weight CSPα oligomers (right panel) (C) mRNA levels for the indicated proteins (fold change compared to control brain tissue).</p

Oligomerization of CSPα_L115R, CSPα_Δ116 mutants is concentration-dependent.

<p>CAD cells were transfected with 0.75μg of flag-tagged WT CSPα and 0.25, 0.5, 0.75, 1 μg of either CSPα_L115R, or CSPα_L116Δ as indicated and lysed 48 hours post transfection. Mutant CSPα was detected with anti-myc (upper panel) and WT CSPα was detected with anti-flag (lower panel). Detection of β-actin was used to verify equal loading amongst the various lanes. <b>(B)</b> Quantification of CSPα high molecular weight oligomers (starting from 70kDa) and CSPα monomers. Differences between CSPα_L115R and CSPα_L116Δ oligomers are not significant, *p < 0.02; **p < 0.001. Results are from 3 independent experiments.</p

Expression of DnaJA1, DnaJA2, DnaJA3, DnaJA4 and DnaJB1, in human cortex samples.

<p>Western analysis of the indicated proteins in synaptosome-enriched fractions prepared from <u>human cortex</u>. For the 10 human samples the DnaJA2 values were: 77500, 57900, 85900, 78500, 98100, 69700, 52500, 83800, 64300, 56400. The DnaJ3A values were: 298000, 224000, 356000, 416000, 554000, 509000, 375000, 342000, 234000, 181000.</p