cleaves and activates the TRPC5 channel to participate in semaphorin 3A-induced neuronal growth cone collapse

J. Stefan Kaczmareka,1, Antonio Ricciob,c,1, and David E. Claphamb,c,2

aProgram in Neuroscience, Division of Medical Sciences, Harvard University, Boston, MA 02115; bHoward Hughes Medical Institute, Department of Cardiology, Manton Center for Orphan Disease, Children’s Hospital Boston, MA 02115; and cDepartment of Neurobiology, Harvard Medical School, Boston, MA 02115

Contributed by David E. Clapham, April 6, 2012 (sent for review March 17, 2012) The nonselective cation channel transient potential can- on growth cones at the tip of neurites are thought to sense cues onical (TRPC)5 is found predominantly in the brain and has been that guide the extension of axons and dendrites (23). One current proposed to regulate neuronal processes and growth cones. Here, model proposes that sema3A signaling activates the MAPK family we demonstrate that semaphorin 3A-mediated growth cone col- of ; these kinases directly phosphorylate and activate cal- lapse is reduced in hippocampal from TRPC5 null mice. pain-2 (24). have numerous proteolytic targets in the This reduction is reproduced by inhibition of the calcium-sensitive synapse, processes, and soma of neurons, including ion channels − − calpain in wild-type neurons but not in TRPC5 / neu- and receptors (25). We investigated molecular mechanisms that rons. We show that calpain-1 and calpain-2 cleave and functionally might link neuropilin–plexin receptor activation to TRPC5 gating. activate TRPC5. Mutation of a critical threonine at position 857 We demonstrate that TRPC5 and calpain influence sema3A sig- inhibits calpain-2 cleavage of the channel. Finally, we show that naling in cultured hippocampal neuronal growth cones. We also the truncated TRPC5 predicted to result from calpain cleavage is demonstrate that calpains cleave and activate TRPC5. We propose functionally active. These results indicate that semaphorin 3A that calpains activate TRPC5 downstream of sema3A signaling to initiates growth cone collapse via activation of calpain that in contribute to growth cone collapse and neuronal development. turn potentiates TRPC5 activity. Thus, TRPC5 acts downstream of semaphorin signaling to cause changes in neuronal growth cone Results morphology and nervous system development. TRPC5 and Calpain Are Critical to Sema3A-Induced Growth Cone Collapse. We first compared representative growth cone collapse hippocampus | TRP channel | calcium signaling | neuronal development frequency at the neurite tips of cultured hippocampal neurons −/− from WT and TRPC5 null (Trpc5 ) mice (Fig. 1 A and D). WT −/− multitude of signals regulate development of the nascent and Trpc5 neurons had comparable levels of collapse (21 ± 3% Amammalian nervous system. Secreted and contact-mediated vs. 22 ± 4%). Sema3A treatment (1 nM for 5 min) significantly extracellular cues may be transduced to the interior of the cell by increased the proportion of collapsed growth cones to 61 ± 5% in − − surface receptors, initiating both short and long-term changes in WT neurons, but to only 38 ± 2% in Trpc5 / neurons (Fig. 1 B cellular physiology. Proper formation of the nervous system and D). This represents a significant (P = 0.007) reduction in the −/− requires axons, dendrites, and even the soma of neurons to ac- ability of sema3A to collapse growth cones in Trpc5 neurons. curately traverse great distances and connect with specific tar- The downstream targets of the sema3A receptor complex of gets. Such movements require spatially and temporally regulated neuropilin-1/plexin A1 have not been fully elucidated. Recent expression of extracellular guidance cue molecules by the sur- reports suggest that sema3A can activate the calcium-sensitive rounding tissues or distant targets that bind to surface receptors cysteine protease, calpain, via direct phosphorylation by MAPK- and attract or repel the developing (1, 2). family kinases (24). To determine whether the residual sema3A −/− Several members of the semaphorin class of secreted guidance response we observed in Trpc5 neurons was attributable to cal- −/− peptides cause intracellular calcium elevations upon binding to pain, we also preincubated WT and Trpc5 neurons with the cell- their cognate receptors (3, 4), but how they effect this change is permeant calpain inhibitors calpeptin (10 μM) and calpain inhibitor unclear. Transient receptor potential (TRP) , a family of III (5 μM) for 30 min and then treated those neurons with sema3A calcium-permeable nonselective cation channels, are widespread (Fig. 1 C and D). Preincubation with calpain inhibitors diminished in the nervous system and affect neuronal development and ner- the effect of sema3A on WT neurons, reducing the collapsed vous system function (5). In particular, studies of TRP canonical fraction from 61% to 38 ± 4% (P = 0.012 compared with sema3A fl (TRPC)5 suggest that it in uences the growth of neuronal pro- alone). However, calpain inhibition did not further reduce the ef- −/− cesses (axons or dendrites). TRPC5 mRNA is found at high levels fect of sema3A on Trpc5 neuronal growth cones; the collapsed in several brain regions, including the hippocampus, olfactory fraction remained nearly constant at 36 ± 1% (P =0.98compared bulb, amygdala, and cerebellum, and may also be detected dif- with sema3A alone). These results suggest that calpain and TRPC5 fusely throughout the cortex (6, 7). siRNA-mediated knockdown function in the same pathway downstream of sema3A signaling. or pharmacologic inhibition of this channel has been reported to both stimulate and inhibit neurite sprouting and extension, Calpains Activate TRPC5 Channels. Calpains cleave and alter the – depending upon the cell type and assays used (8 10). In addition, activity of several ion channels, receptors, and synaptic proteins the TRPC5 null mouse displays deficits in dendritic development in the cerebellum (11) and abnormal amygdalar function (12).

Little is known about normal modes of TRPC5 activation, al- Author contributions: J.S.K. and D.E.C. designed research; J.S.K. and A.R. performed re- though G -coupled receptor (GPCR-Gαq) signaling (13) search; J.S.K. and A.R. contributed new reagents/analytic tools; J.S.K., A.R., and D.E.C. and other molecules (14–19) strongly potentiate TRPC5 current. analyzed data; and J.S.K. and D.E.C. wrote the paper. Semaphorin (sema)3A is a secreted guidance peptide that binds The authors declare no conflict of interest. to the neuropilin-1/plexin A1 receptor complex and may cause ei- 1J.S.K. and A.R. contributed equally to this work. ther attraction, repulsion, or collapse of neuronal growth cones 2To whom correspondence should be addressed. E-mail: [email protected]. depending upon the intracellular milieu (20–22). Multiple filopodia edu.

7888–7892 | PNAS | May 15, 2012 | vol. 109 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1205869109 Downloaded by guest on October 1, 2021 uninduced, GFP-positive cells (P = 0.004) and −7 ± 1 pA/pF from induced, GFP-negative cells (P = 0.003; Fig. 2C). Next, we determined whether purified calpain-1 and calpain-2 could activate heterologously expressed TRPC5 channels. Cal- pain-1 requires micromolar Ca2+ concentrations for activation in vitro (1–20 μM), whereas calpain-2 requires near millimolar concentrations (0.3–0.8 mM) (25). Thus, we first used pipette solutions with free Ca2+ within or above these ranges to activate purified calpains. Because calpains are large proteins (∼110 kDa) and do not readily diffuse from the pipette into the cell during whole-cell patch clamp, we applied these purified calpains to the intracellular surface of excised inside-out patches from HEK cells stably expressing mTRPC5. Because TRPC5 is sen- sitive to intracellular calcium (18), we used high calcium sol- utions (5 μM free Ca2+ buffered with 5 mM (2-Hydroxyethyl) ethylenediaminetriacetic acid (HEDTA) for calpain-1; 2 mM unbuffered Ca2+ for calpain-2) throughout the experiment to first establish a baseline level of channel activity. Using a fast perfusion system, we then rapidly transitioned the patches into a solution stream containing purified calpain-1 or calpain-2. Fig. 1. TRPC5 knockout and calpain inhibition reduce sema3A-induced Both calpain-1 and calpain-2 induced a significant increase in hippocampal growth cone collapse. (A) Untreated, cultured hippocampal TRPC5 single-channel activity, with activity increasing to a peak −/− fi neuronal growth cones from WT and TRPC5 null (Trpc5 ) mice, xed and over several minutes (Fig. 3 A–D). Channel activity, as repre- stained with the -binding peptide phalloidin conjugated to Alexa Fluor 568. (B) Growth cones from neurons treated for 5 min with 1 nM sema3A sented by NPO (number of channels times the open probability before fixation. (C) Growth cones from neurons preincubated with the cal- of a channel) was collected in 30-s bins for each patch. Calpain-1 pain inhibitors calpain inhibitor III (5 μM) and calpeptin (10 μM) for 30 min increased average peak NPO by 7 ± 1-fold; calpain-2 increased and then treated with sema3A. (D) Growth cones from the indicated gen- channel activity 33 ± 10-fold (Fig. 3 A and C). Perfusion of a otypes and treatments were counted and the collapsed fraction quantified solution containing calpain-1 and a protease inhibitor mixture (three coverslips from two separate experiments; n =98–100 cones per including the cysteine protease inhibitor E-64 prevented activa- condition). Ambiguous growth cones not closely resembling the examples tion (1.5 ± 0.2-fold peak increase; P = 0.0003 compared with presented were excluded from analysis. P values in text are calculated using P B F Student t test. calpain-1 or = 0.009 to calpain-2 alone; Fig. 3 and ). Av- erage single-channel conductance observed for TRPC5 was 39.5 ± 0.5 pS. Importantly, we observed no activity of channels (26). The ubiquitous calpain-1 (μ-calpain) and calpain-2 with a conductance similar to that of TRPC5 in response to

(m-calpain) have the highest expression levels in brain. Calpain-2 calpain-1 or calpain-2 perfusion onto patches from naive HEK NEUROSCIENCE A Upper may be activated by phosphorylation at serine 50; mutation of cells. The peaks and troughs in NPO (e.g., Fig. 3 , ) rep- this serine to glutamic acid creates a constitutively active pro- resent burst-like channel activity, as the channel enters a long tease (27). We tested whether coexpression of constitutively series of open or closed states. Because there is significant active calpain-2 (S50E) could alter basal TRPC5 channel activity overlap between the substrates of calpain-1 and calpain-2 (26), it in a heterologous expression system. Calpain-2 S50E (large is not surprising that both proteases activate TRPC5. subunit; under the control of a dexamethasone-inducible pro- MAPK1 has been reported to directly phosphorylate and ac- moter), the calpain small subunit (CAPNS1), and eGFP were tivate calpain-2 (27). Therefore, we incubated purified, consti- cotransfected into HEK cells stably expressing mouse TRPC5 tutively active MAPK1 in the presence of ATP and Mg2+ with (mTrpc5). Dexamethasone-treated GFP-positive cells expressing calpain-2 in vitro. Perfusion of MAPK1-phosphorylated calpain- calpain-2 S50E exhibited a 2.5-fold increase in basal current 2 in 100 nM free Ca2+ [1,2-bis(o-aminophenoxy)ethane-N,N,N′, compared with uninduced, GFP-positive or induced, GFP-neg- N′-tetraacetic acid (BAPTA)] onto the intracellular surface of ative cells (Fig. 2 A and B); the whole-cell current density at TRPC5 channel-containing inside-out excised patches increased −100 mV shortly after break-in was −17 ± 3 pA/pF from in- channel activity by 24 ± 5-fold (Fig. 3 D and F). Boiling MAPK1 duced, GFP-positive cells, but only −7 ± 2 pA/pF from before incubation with calpain-2 increased channel activity by

Fig. 2. Coexpression of constitutively active calpain-2 S50E with TRPC5 increases basal channel currents. (A and B) Representative whole-cell current–voltage (I-V) relationships from HEK cells stably expressing TRPC5 cotransfected with calpain-2 S50E under the control of a dexamethasone-inducible promoter, CAPNS1, and eGFP as a marker. −Dex, uninduced; +Dex, dexamethasone-treated cells; GFP−, GFP-negative cells; GFP+, GFP-positive cells. The pipette con- tained 5 mM HEDTA and 5 μM free Ca2+ to accentuate basal TRPC5 currents. The bath contained the standard 2 mM Ca2+ extracellular solution. I-V rela- tionships were obtained shortly after break-in (<1 min). Both traces in B are from dexamethasone-treated cells. (C) Quantification of whole-cell current density at −100 mV from uninduced, GFP-positive (light gray column; n = 10), induced but GFP-negative (dark gray column; n = 10), and induced, GFP-positive (black column, n = 10) cells. **P < 0.01 (Student t test).

Kaczmarek et al. PNAS | May 15, 2012 | vol. 109 | no. 20 | 7889 Downloaded by guest on October 1, 2021 Fig. 4. Calpain-1 and calpain-2 cleave TRPC5 at its C terminus, generating a fragment likely to retain function, and this cleavage is sig- nificantly reduced by mutation of threonine 857 to alanine. (A and B)We briefly sonicated HEK cells stably expressing an N-terminally HA-tagged mTRPC5 protein and treated the resulting homogenates with 5 μg/mL pu- rified calpain-1 (lane 2), calpain-1 plus 10 μM calpeptin and 5 μM calpain Fig. 3. Purified calpains activate TRPC5 channels in excised patches. Aver- inhibitor III (lane 3), 5 μg/mL purified calpain-2 (lane 4), or calpain-2 plus the age single channel conductance observed for TRPC5 was 39.5 ± 0.5 pS (n = aforementioned inhibitors (lane 5) in a 2 mM Ca2+ buffer (control untreated; 20). (A, Upper) Activity (NPO) of TRPC5 channels in a representative inside- lane 1). (A, Upper) Western blot of calpain-treated HA-TRPC5 probed with out excised patch from a HEK cell stably expressing mTRPC5, activated by 15 a TRPC5 C-terminal monoclonal antibody. The large arrow below the 118- μg/mL purified calpain-1 (indicated by dashed line and open bar), collected kDa marker indicates full-length HA-TRPC5 protein. (B, Upper) Western blot in 30-s bins. (A, Lower) Currents at the time points indicated by the small of calpain-treated HA-TRPC5 probed with an HA monoclonal antibody. (A, letters above showing individual TRPC5 channel openings at the indicated Lower; and B, Lower) A monoclonal antibody to β-actin shows that equal voltage. C, closed (no open channels); O or 1, one open channel; 2, two open protein was loaded. (C) Western blot of calpain-2 treated homogenates channels. We used an intracellular solution with 5 mM HEDTA and 5 μM free from HEK cells transfected with an HA-TRPC5 wild-type construct or con- 2+ Ca throughout the experiment. (B) Same as A, except that we perfused structs containing the point mutations F843A, G847A, and T857A probed purified calpain-1 plus a protease inhibitor mixture including the cysteine with anti-HA. The large arrow below the 118-kDa marker indicates full- protease inhibitor E-64 (14 μM). (C) Same as A, except that we perfused length HA-TRPC5 protein. (D) Quantification of TRPC5 present in the cleaved purified calpain-2 onto the patch and used an intracellular solution con- form from the blot in C.*P < 0.05 (Student t test; n = 2 blots from in- 2+ taining 2 mM Ca .(D) Same as A, except that we perfused calpain-2, which dependent experiments). (E and F) A modified plasmid containing mTRPC5 had been phosphorylated in vitro with constitutively active MAPK1, onto the truncated at N854 was coexpressed with the muscarinic-type 1 receptor 2+ patch in an intracellular solution containing 100 nM free Ca .(E) Same as D, (M1R) and eGFP in HEK cells. (E) Representative whole-cell currents recorded except that MAPK1 was boiled before incubation with calpain-2 before the at −100 mV; application of the M1R agonist carbachol at 100 μM (vertical experiment. (F) Quantification of the effects of purified calpains on TRPC5. dashed line and open bar) significantly increased current density in five GFP- Fold increase is the ratio between the NPO of the peak bin following positive cells. The pipette contained a solution with 5 mM HEDTA and 5 μM 2+ treatment, divided by the average NPO preceding treatment. Each patch free Ca to potentiate any TRPC5-like currents. (F) Current–voltage re- is indicated by its own marker; the solid bar is the mean. *P < 0.05; **P < lationship (I-V) taken at the point indicated by a in E. 0.01 ( Student t test; n =6–8 for each experiment). Burst activity shown as

rising and falling NPO is characteristic of TRPC5. TRPC5 to an in vitro calpain cleavage assay, using a HEK cell only 1.3 ± 0.4-fold (Fig. 3 E and F; P = 0.012 compared with line stably expressing mouse (m)TRPC5 fused to an N-terminal nonboiled MAPK1-treated calpain-2). Finally, we recorded sema- HA tag. We homogenized cells with a brief sonication and then phorin responses from cultured hippocampal neurons using whole- subjected these homogenates to digestion by calpain-1 or cal- cell patch clamp at the soma but observed no large immediate pain-2. We performed Western blot analysis on the homogenates A current changes in response to 1 nM semaphorin application; and probed the blots with an anti-TRPC5 antibody (Fig. 4 ; this may be attributable to space clamp limitations in these cells directed against the extreme C terminus) or an anti-HA antibody B and the localization of TRPC5 to distal processes. (Fig. 4 ). The TRPC5-directed antibody revealed a substantial reduction in full-length TRPC5 protein in those homogenates Calpain-1 and Calpain-2 Cleave TRPC5 in Vitro. Thus far, the ex- digested with calpain-1 and calpain-2; this reduction was pre- periments have shown that calpain strongly potentiates TRPC5 vented by the inclusion of calpain inhibitors. The HA-directed current but do not identify the substrate. Next, we subjected antibody produced the same result but also revealed a cleavage

7890 | www.pnas.org/cgi/doi/10.1073/pnas.1205869109 Kaczmarek et al. Downloaded by guest on October 1, 2021 product ∼15 kDa smaller than full-length TRPC5 in those homog- sema3A, calpain, calcium, and TRP channel function to define enates digested with calpain-1 and calpain-2. We also observed a mechanism for the regulation of growth cones. this product, although to a lesser extent, in untreated homoge- Site-directed mutagenesis indicates that threonine 857 of nates, likely reflecting endogenous calpain digestion activated by TRPC5 is likely to represent one, but not necessarily the only, the high-calcium digestion buffer. These results indicate that cleavage site of the channel. Alternatively, mutagenesis at this calpain cleaves an ∼15-kDa segment of the C terminus of position may not fully disrupt the tertiary structure recognized by TRPC5, leaving an ∼100-kDa N-terminal fragment that includes the protease; such observations are not uncommon (28). There is the HA tag and the transmembrane regions responsible for ion significant overlap in calpain substrates between family mem- conduction. We attempted to perform the same analysis on bers, so it is possible that other calpains may activate TRPC5 homogenates from cultured hippocampal neurons, but the ex- as well (26). The data in Fig. 3 suggest that calpain-2 induces pression level of TRPC5 proved too low for accurate detection in a larger increase in channel activity (NPO) than calpain-1, but Western blots. this may result from changes in basal channel activity attributable A recent study proposed an in silico method for predicting to the differing calcium concentrations used. The data also in- the location of calpain digestion sites within putative substrates dicate a much larger relative increase in TRPC5 current fol- (28). Using the full-length mTRPC5 amino acid sequence, this lowing calpain cleavage in excised patches than that observed model predicted calpain cleavage of the channel in the C ter- from cotransfection of calpain-2 S50E with TRPC5. However, minus at threonine 857 (T857). Because our results from Fig. 4 A this discrepancy is likely attributable to either desensitization C D and B indicated that cleavage occurred close to the C terminus, (suggested by the data in Fig. 3 and ) or down-regulation of a cleavage site at T857 seemed plausible. Additionally, a molec- calpain-2-cleaved TRPC5 channels. Genetic titration of calpain ular mass prediction model (expasy.org/tools/pi_tool.html) sug- in transgenic mice might shed more light on some of these issues gested that the large fragment remaining after calpain digestion and on the links between semaphorin signaling, calpain, TRPC5, would be ∼13 kDa smaller than full-length, which agrees with and growth cone morphology. Unfortunately, deletion of the our results from Fig. 4 A and B. We tested this prediction by calpain-2 gene from mice is embryonically lethal (25). Intriguingly, mutating T857 to alanine and quantified the ratio of cleaved to genetic deletion of TRPC5 leads to irregularities in dendrite uncleaved HA-TRPC5. After treatment of homogenates with formation and brain function, supporting a role for the channel calpain-2 (Fig. 4C), 88% of wild-type TRPC5 was detected in the in neuronal development (12). cleaved state, but only 58% of TRPC5 T857A was cleaved (Fig. A number of groups have published seemingly incongruous 4D; P = 0.045), suggesting that this residue resides within the reports of TRPC5 exerting both positive and negative effects on calpain recognition site. TRPC5 channels with two unrelated neuritogenesis and development. Previous work from our labo- ratory indicated that TRPC1 was excluded from distal processes mutations (F843A and G847A) were cleaved ∼80% by calpain-2, but may form heteromeric channels with TRPC5 in and near the similar to WT. The amount of cleavage inhibited by mutation of soma (8). A recent study suggests that TRPC1 exerts a positive T857A (∼30%) suggests the possibility of either additional cal- effect, whereas TRPC5 negatively regulates neurites (35). There- pain cleavage sites or incomplete disruption of the local tertiary fore, we suggest that TRPC1/TRPC5 heteromeric channels, pri- structure recognized by the active site of the protease. To con- fi marily located near the soma and proximal processes, might

rm that a fragment of TRPC5 generated by cleavage near T857 NEUROSCIENCE fi promote neuritogenesis, whereas TRPC5 homomeric channels could function as an ion channel, we generated a modi ed present in distal processes respond to inhibitory signals, such as TRPC5 protein truncated at asparagine 854. Using whole-cell fi sema3A. Another possibility is that TRPC5 could serve as a patch clamp, we con rmed that this truncated channel was still common calcium influx pathway downstream of the neuropilin-1/ functional by coexpressing it with the muscarinic type-1 G pro- E F plexin A1 receptor complex and that other changes in the in- tein coupled receptor (M1R) in HEK cells (Fig. 4 and ). The tracellular milieu, such as cyclic nucleotide levels, determine the truncated channel produced constitutive TRPC5-like current sign (positive or negative) of sema3A and TRPC5 signaling. In- − ± − − − in all cells analyzed ( 10 1 pA/pF at 100 mV); carbachol triguingly, although inhibition of calpain in TRPC5 / neurons fi − ± signi cantly increased the average current density to 62 13 did not change the overall growth cone collapse rate, the ap- P pA/pF ( = 0.005). pearance of growth cones from this condition was more delicate, with less intense actin staining and many small filopodia (although Discussion not always arranged in the bulb-like structure shown in Fig. 1C). fi The primary nding of this work is that TRPC5, a calcium-per- This might suggest calpain-independent effects of TRPC5 on meant ion channel, and calpain, a calcium-activated protease, growth cones or compensation of calpain and TRPC5 for one contribute to sema3A-mediated growth cone collapse. Taking another in a common signaling pathway. − − into account what is known about semaphorin signaling, we pro- Sema3A / mice exhibit nerve defasciculation (36), abnormal pose a model in which sema3A binds to the neuropilin-1/plexin A1 patterning in the olfactory bulb (37), mistargeting within the receptor complex, activating MAPK that in turn phosphorylates hippocampus (38), and several other embryonic abnormalities that − − and activates calpain (29). Calcium flowing into the cell through resolve by birth (39). TRPC5 / mice also display abnormalities in − − the cleaved potentiated TRPC5 leads to growth cone collapse. nervous system development, but similarly to sema3A / mice, Calcium influx has been linked to changes in growth cone these effects are not lethal. It should be noted that TRPC5 is morphology, collapse, and neurite outgrowth in a number of expressed at the highest levels in hippocampus and olfactory bulb 2+ different pathways (30–32), such as those involving Ca /cal- (40, 41), two of the regions implicated in sema3A function. Direct modulin-dependent protein (CaMK)II (33), calcineurin, and specific block of these two molecules in postnatal mice could and Rho-Rho kinase (4). Semaphorin signaling has been shown to reveal more details about their functions. Unfortunately, although cause calcium influx in both a neuronal-derived cell line (3) and inhibitors of sema3A have been reported (42), no specific blocker native neuronal growth cones (4). Other TRP channels have also of TRPC5 is available. In addition its influence on brain develop- been implicated in growth cone collapse, such as TRP vanilloid ment, TRPC5 might also be a target for nerve regeneration thera- (TRPV)1 (34). Although the connection between in vitro growth pies; intriguingly, inhibition of sema3A improved regenerative cone collapse and in vivo axon path-finding is not entirely clear, responses and recovery in a model of spinal cord injury (43). the collapse assay nevertheless provides evidence that func- Regenerating neurons do establish new growth cones (44), and if tionally links semaphorin-dependent signals with calpain and TRPC5 is present as a negative regulator of their development, TRPC5. Our data agree with and extend similar studies on pharmacologic inhibition of TRPC5 may enhance CNS regeneration.

Kaczmarek et al. PNAS | May 15, 2012 | vol. 109 | no. 20 | 7891 Downloaded by guest on October 1, 2021 Materials and Methods EDTA, we added an additional 0.8 mM CaCl2. The 2 mM CaCl2 solution 2+ Growth Cone Analysis. Primary hippocampal neurons from P1 TRPC5−/− mice contained no chelator. Free [Ca ] was calculated using WebMaxChelator and WT littermates were prepared as previously described (45) and cultured (http://maxchelator.stanford.edu), assuming an ionic strength of 0.16 M. The for 40 h. After treatment at 37 °C, neurons on 12-mm coverslips were fixed standard external solution contained (in mM): 150 NaCl, 4 KCl, 2 CaCl2,

with 4% (vol/vol) paraformaldehyde (PFA) for 10 min, permeabilized with 1 MgCl2, 10 Hepes, 10 D-glucose (pH 7.40) with NaOH. Electrophysiological 0.1% Triton X-100 for 5 min, and stained with phalloidin conjugated to experiments were performed at room temperature. Alexa Fluor 568 (Invitrogen) for 20 min. Coverslips were mounted onto slides – with VectaShield medium (Vector Laboratories) containing DAPI, and 15 20 In Vitro Cleavage. We collected HEK cells stably expressing mTRPC5 tagged fi random cell-containing elds were acquired per coverslip using an Olympus with an N-terminal HA epitope in 150 mM NaCl with 10 mM Hepes (pH 7.5). We FV-1000 confocal microscope with a 60× objective. Neurons were identified sonicated the cells and added CaCl to the homogenates for a final concen- based on standard morphological characteristics (8). 2 tration of 2 mM. Purified calpain-1 or calpain-2 (EMD) was added at 5 μg/mL; calpeptin and calpain inhibitor III (EMD/Calbiochem) were added at 10 μM Electrophysiology. We filtered whole-cell currents using an AxoPatch 200A at μ 5 kHz (four-pole low-pass Bessel), sampled at 20 kHz, and used voltage ramps and 5 M, respectively. After 30 min, a standard lysis buffer containing 1% × consisting of a 40 ms step to −100 mV, a 200-ms ramp to +100 mV, and a 40- Triton X-100 and 1 protease inhibitors (Sigma) was added. For blotting, the ms step at +100 mV applied at 0.5 Hz from a holding potential of −40 mV. primary antibody was either mouse monoclonal to TRPC5 (1:500; clone: N67/ Series resistance was compensated by 85%. Voltages are corrected for the 15; NeuroMab), mouse monoclonal to HA (1:1,000; ), or mouse −11-mV junction potential between internal and external solutions. We monoclonal to β-actin (1:2,000; Santa Cruz). The secondary antibody was goat filtered single-channel currents at 2 kHz and sampled at 50 kHz. In record- anti-mouse HRP-conjugated (1:5,000; Jackson ImmunoResearch). 2+ ings using calpain-2 (Fig. 3C), the high free [Ca ] activated two other All statistical comparisons were calculated with Student’s t test. channels (12 pS and 28 pS); we excluded such openings from analysis. The standard internal solution contained (in mM): 150 Cs-aspartate, 2 MgCl2,0.3 ACKNOWLEDGMENTS. We thank Svetlana Gapon for cell culture and CaCl2, 1 BAPTA, 4 MgATP, 0.3 NaGTP, 10 Hepes (pH 7.20) with CsOH (100 nM Nathaniel T. Blair for electrophysiology advice and reagents. We also thank free Ca2+). For the 5 μM free Ca2+ solution, we replaced BAPTA with 5 mM Dr. Alan Wells for the constitutively active calpain-2 S50E clone. This work was HEDTA and added 0.95 mM CaCl2; when using inhibitors containing 1 mM supported by National Institutes of Health Grant 1 R01 MH090293 (to D.E.C.).

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