Close appositions between the endoplasmic reticulum and plasma membrane (‘ER-PM junctions’) play important roles in membrane trafficking and cellular signaling within diverse tissues (Porter and Palade, 1957; Rosenbluth, 1962; Gardiner and Grey, 1983; Poburko et al., 2004; Wu et al., 2006), including neurons where there are large numbers of such junctions (Wu et al., 2017). Among the proteins that can induce the formation of ER-PM junctions are the junctophilins (‘JPH’), which have a single, short C-terminal segment that traverses the ER membrane and which bind to the inner surface of the plasma membrane by virtue of repeated ‘MORN motifs that are located toward the N-terminus (Takeshima et al., 2000).

Arguably, the most extensively characterized ER-PM junctions are those in skeletal and cardiac muscle. In both cell types, these junctions (triads and dyads) are the site at which functional coupling occurs between high-voltage-activated calcium channels in the plasma membrane (skeletal: Ca_V1.1; cardiac: Ca_V1.2) and calcium release channels in the ER (skeletal: RyR1; cardiac: RyR2). The formation of these junctions depends upon JPH2 in cardiac muscle and on both JPH2 and JPH1 in skeletal muscle (Takeshima et al., 2000; Ito et al., 2001). The closely related proteins, JPH3 and JPH4, are expressed in the nervous system (Nishi et al., 2003). Knockout of JPH4 does not produce an overt behavioral phenotype (Moriguchi et al., 2006), whereas knockout of only JPH3 causes motor discoordination (Nishi et al., 2002) that seemed to progress with aging (Seixas et al., 2012). However, a broad spectrum of changes occurs when both JPH3 and JPH4 are knocked out, including an aberrant hindlimb reflex, altered salivary secretion, and impaired memory (Moriguchi et al., 2006). Moreover, a triplet repeat expansion in exon 2A of human JPH3 causes reduced expression of JPH3 and results in Huntington disease-like 2 (Seixas et al., 2012). Thus, junctophilin-containing ER-PM junctions appear to be important for multiple neuronal functions.

Transcripts of JPH3 and JPH4 are present in diverse brain regions, with expression being highest in the hippocampal formation, isocortex, cortical subplate, striatum, and olfactory systems (Nishi et al., 2003; Lein et al., 2007). Groups of cells in other regions (e.g., cerebellar granule cells) also show high expression (Nishi et al., 2003; Lein et al., 2007). However, the electrophysiological consequences of the double knockout of JPH3 and JPH4 have only been investigated in slice recordings of hippocampal CA1 neurons and cerebellar Purkinje cells. In CA1 pyramidal neurons, a single action potential is followed by an afterhyperpolarization (AHP), which was found to be greatly reduced by apamin, SERCA inhibition, and ryanodine, and to be absent in neurons from JPH3/JPH4 double KO mice (Moriguchi et al., 2006). A similar pharmacological profile and absence in double KO cells was found for the slow AHP following complex spikes in Purkinje neurons (Kakizawa et al., 2007), which is perhaps surprising given that the expression of JPH3 and JPH4 is relatively low in Purkinje cells (Nishi et al., 2003). Based on their observations, Moriguchi et al., 2006 and Kakizawa et al., 2007 proposed that the AHP in CA1 neurons, and slow AHP in Purkinje cells, were generated at ER-PM junctions, which contained the neuronal junctophilins together with voltage- or ligand-activated Ca²⁺ channels and SK Ca²⁺-activated potassium channels in the plasma membrane and ryanodine receptors in the ER.

The molecular components of ER-PM junctions in CA1 neurons have also been probed with biochemical and immunolabeling approaches (Kim et al., 2007; Sahu et al., 2019). In particular, Kim et al., 2007 found that Ca_V1.3 and RyR2 interacted with one another and colocalized in CA1 neurons. Subsequently, Sahu et al., 2019 found with ultra-resolution microscopy that these neurons contained clusters of ryanodine receptors (specifically RyR2), SK channels (specifically KCa3.1), and L-type Ca²⁺ channels (Ca_V1.2 and Ca_V1.3), and found that this channel complex clusters with JPH3. Thus, the case can be made that the AHP in CA1 neurons can be generated at JPH3-containing ER-PM junctions by (1) Ca²⁺ entry across the plasma membrane via L-type channels, (2) Ca²⁺ release via RyR2 in the ER, and (3) activation of SK channels in the plasma membrane (Sahu et al., 2019), with entry of Ca²⁺ via NMDA receptors also being of importance (Moriguchi et al., 2006).

Although a picture is emerging about junctophilins in CA1 neurons, many questions remain unanswered about the neuronal junctophilins in other neuronal populations. For example, the presence of RyR2 in ER-PM junctions of hippocampal CA1 neurons is consistent with its being the most prominent RyR isoform in those cells, based on in situ hybridization (Mori et al., 2000). However, the RyR1 signal is comparable to, or exceeds that of, RyR2 in the hippocampal dentate gyrus, mitral cells of the olfactory bulb, olfactory tubercle, and cerebellar Purkinje cells (Mori et al., 2000). Moreover, RyR3 is a significant fraction of, or exceeds, RyR2 in hippocampal CA1 neurons and olfactory tubercle (Mori et al., 2000). Thus, the question arises: is relative expression the only factor influencing the junctional recruitment of RyR isoforms by the neuronal junctophilins or is there preferential recruitment of one or more isoforms?

There are similar questions about which voltage-gated calcium channels are recruited by the neuronal junctophilins. For example, both P/Q (Ca_V2.1) and T-type (Ca_V3.1) channels are highly expressed in cerebellar Purkinje cells (Lein et al., 2007). However, only Ca²⁺ influx through the P/Q type causes activation of the SK channels that produces the AHP following simple spikes (Womack et al., 2004). An obvious question based on these results, and those of Kakizawa et al., 2007, is whether the neuronal junctophilins cause junctional recruitment of Ca_V2.1 and not of Ca_V3.1. A related question is whether Ca_V2.2 is recruited by JPH3 and/or JPH4, a possibility that has not been suggested by previous studies of the junctophilins. Nonetheless, the recruitment of Ca_V2.2 seems plausible because Ca_V2.2 (Lein et al., 2007), JPH3, and JPH4 (Nishi et al., 2003) are all expressed at moderate levels in the paraventricular nucleus of the thalamus.

The goal of the work described here was to address the ability of the neuronal junctophilins to cause the junctional accumulation of specific Ca_V and RyR isoforms. An additional goal was to determine whether such junctional accumulation differs between JPH3 and JPH4 since such a difference could help explain why single knockout of JPH3, but not JPH4, results in a neurological phenotype (Moriguchi et al., 2006). For this work, we chose to use heterologous expression in tsA201 cells to provide a uniform basis for comparing the Ca_V, RyR, and JPH isoforms. Our work demonstrates that JPH3 and JPH4 both fail to cause Ca_V3.1 to accumulate at junctions, but that both cause the accumulation of P/Q (Ca_V2.1) and N (Ca_V2.2) channels. In addition, both junctophilins cause a slowing of the inactivation of Ca_V2.1 and Ca_V2.2. JPH3 and JPH4 differ substantially in their ability to recruit RyRs. Whereas JPH4 causes some accumulation of RyR3, JPH3 causes accumulation of all three RyR isoforms, especially RyR1. An important contributor to the accumulation of RyR1 arises from a binding interaction between its cytoplasmic domain and a region of JPH3 that is highly divergent from the corresponding region of JPH4.

Here, we used colocalization of fluorescently tagged proteins expressed in tsA201 cells, together with electrophysiological measurements, to obtain insight on likely constituents of ER-PM junctions induced by the neuronal junctophilins JPH3 and JPH4. After verifying that these two proteins retained their ability to form ER-PM junctions when N-terminally tagged with fluorescent proteins (Figure 1), we tested three HVA Ca²⁺ channels (Ca_V1.2, Ca_V2.1, and Ca_V2.2), and one LVA channel (Ca_V3.1) as potential constituents of the PM side of these junctions. The three HVA channels colocalized with both JPH3 and JPH4 (Figure 2), with mean Pearson’s coefficients ranging from ~ 0.5 (Ca_V2.1/JPH3) to ~ 0.8 (Ca_V2.2/JPH4). By contrast, significantly less colocalization occurred between the LVA channel and either JPH3 or JPH4 (Figure 2, Pearson’s coefficients < 0.4).

Based on its colocalization with JPH3 and JPH4 (Figure 2), Ca_V2.2 may be a hitherto unrecognized constituent of neuronal ER-PM junctions. Thus, it is important to take into account that Ca_V2.2, and also Ca_V2.1, may have been present in the plasma membrane at relatively low densities compared to those of Ca_V1.2. In particular, single-channel measurements have yielded maximum open probabilities of 0.6 for Ca_V2.1 (expressed in HEK293 cells; Luvisetto et al., 2004) and 0.5 for Ca_V2.2 (in frog sympathetic ganglion neurons; Delcour and Tsien, 1993; Lee and Elmslie, 1999) compared to 0.03 for Ca_V1.2 (in rabbit ventricular myocytes; Lew et al., 1991). Under the assumption that the same open probabilities are applicable to these channels co-expressed with JPH3 or JPH4, and ignoring small differences in unitary conductance, one would predict that the plasma membrane densities of Ca_V2.1 and Ca_V2.2 would be about 20- to 25- fold lower than those of Ca_V1.2 in order to produce the peak current densities illustrated in Figure 3. Thus, even though the analysis of colocalization was based on confocal scans near the surface, such scans would have included some contribution from channels that were near to, but not yet inserted into, the plasma membrane, and this could have been more significant for Ca_V2.1 and Ca_V2.2 than for Ca_V1.2. However, the slowing of inactivation (Figure 3B, C) provides evidence that JPH3 and JPH4 altered the functional environment of the majority of the Ca_V2.1 and Ca_V2.2 channels actually inserted into the plasma membrane, presumably because these channels were localized at the junctions induced by these neuronal junctophilins. Although we do not know the mechanism responsible for the slowing of inactivation, one possibility is that it involves an interaction between the junctophilins and Ca_V2 channels because the slowing of inactivation also occurred with truncated variants of JPH3 and JPH4, which do not induce ER-PM junctions (Figure 4). Whatever the exact mechanism may be, the slowing of inactivation may function to increase calcium entry via Ca_V2.1 and Ca_V2.2, and raises the possibility that JPH3 and JPH4 function not only to organize ER-PM junctions but also to modify the behavior of the signaling molecules present in those junctions.

The localization of Ca_V1.2 at junctions induced by JPH3 and JPH4 seems likely to depend on regions homologous to those that cause Ca_V1.1 to localize at triad junctions in skeletal muscle. In skeletal muscle, Ca_V1.1 co-IPs with both JPH1 and JPH2, an interaction for which residues 230 – 369 of JPH1 and 216 – 399 of JPH2 (human sequences) were found to be important (Golini et al., 2011). These sequences are reasonably well conserved for all the members of the junctophilin family, with a percentage of residue identity ranging from a minimum of 48 % (JPH1 versus JPH4) to a maximum of 66 % (JPH1 versus JPH3). In Ca_V1.1, a 15-residue segment within the C-terminus (amino acids 1595 – 1606) was found to bind to both JPH1 and JPH2 and to contain a motif IFFRxGGLFG that is also present in the Ca_V1.2 C-terminus (Nakada et al., 2018). A partially conserved sequence (five identical and three conserved residues out of nine) is also found in the corresponding region of Ca_V1.3 (Fujita et al., 1993). Thus, it may be that Ca_V1.2 and Ca_V1.3 participate in interactions with JPH3 and JPH4, which are similar to those occurring between the Ca_V1 channels and muscle junctophilins. The search for potential sites of interaction between Ca_V2.1 or Ca_V2.2 and the neuronal junctophilins will have to proceed de novo because neither of the Ca_V2 C-termini contain a junctophilin-interacting motif like that in the Ca_V1 C-termini. For the Ca_V2 channels, it will also be important to determine whether a single site of interaction accounts for both selective retention at junctions and the slowing of inactivation.

Although JPH3 and JPH4 have largely overlapping abilities with respect to the recruitment of voltage-gated calcium channels in the plasma membrane (Figures 2–4), they differ substantially with regard to ryanodine receptors in the ER (Figure 5). Specifically, all three RyR isoforms colocalized with JPH3, with mean Pearson’s coefficients of about 0.5 (RyR2), 0.7 (RyR3), and 0.8 (RyR1). Thus, in brain regions expressing all three RyR isoforms, RyR1 and RyR3 may be preferentially recruited to junctions containing JPH3. Examples of such regions include dentate gyrus, caudate putamen, olfactory bulb (mitral cell layer), and olfactory tubercle (Mori et al., 2000). By contrast with JPH3, JPH4 colocalized only with RyR3 with a mean Pearson’s coefficient slightly greater than 0.5, whereas the mean coefficients were only about 0.3 for RyR1 and 0.2 for RyR2. This differential recruitment of RyRs may at least partially account for why there was no apparent behavioral phenotype observed for knockout of JPH4 only (Moriguchi et al., 2006), a detectable phenotype (motor discoordination) for knockout of JPH3 only (Nishi et al., 2002), and a broad range of neurological deficits for knockout of both JPH3 and JPH4 (Moriguchi et al., 2006).

We tested whether the additional expression of Ca_V1.2 would increase the colocalization of RyR2 with JPH3, and that of RyR3 with JPH4. No increase of colocalization occurred (Figure 6), and Pearson’s coefficients (~ 0.5) remained lower than those of either RyR1/JPH3 or RyR3/JPH3. However, the lower colocalization cannot be taken as indicating a lesser functional importance in neuronal calcium signaling. The lower colocalization could be a consequence of the presence of RyRs in both junctional and non-junctional ER. Those at the junctions would be required for the initial calcium release triggered by calcium entering across the plasma membrane, whereas those in non-junctional ER could increase the spatial spread of the cytoplasmic calcium transient. Precedent for this idea is provided by contractile cells of the heart in which RyR2 is located not only in the junctional SR, where it is activated by calcium entry via Ca_V1.2, but also in non-junctional (‘corbular’) SR (Dolber and Sommer, 1984; Jewett et al., 1971).

We also characterized the ER-PM junctions in cells transfected with YFP-tagged Ca_V2.1 or GFP-tagged Ca_V2.2 together with CFP-RyR1 and either mCherry-JPH3 or mCherry-JPH4. In the presence of JPH3, but not of JPH4, both Ca_V2.1 and Ca_V2.2 colocalized with RyR1 (Figure 7A–D). Furthermore, it appeared that ER calcium release could occur in cells expressing JPH3, RyR1, and either Ca_V2.1 or Ca_V2.2 (Figure 7E–J). More specifically, of the cells stably transfected with RyR1, and transiently transfected with Ca_V2.1 or Ca_V2.2, R-CEPIA-er and CFP-JPH3, about a third that produced cytoplasmic calcium transients in response to KCl depolarization, also showed a concomitant release of calcium from the ER. One likely contributor to this variability was variable expression of RyR1 because in cells stably transfected only with RyR1 the amplitude of caffeine transients varied substantially from cell to cell, resulting in a large standard error of the mean (Figure 5—figure supplement 1). Second, the cells selected for the presence of CFP-JPH3 would be expected to have had variable colocalization between the voltage-gated calcium channels and RyR1 (Figure 7B, D). Third, at sites of junctional contact with the plasma membrane, the depth of the ER lumen (perpendicular to the cell surface) is on the order of 100 nm or less (Figure 1B, bottom panels), meaning that ROIs for measurement of fluorescence (e.g., Figure 7E, H) would have included both junctional and non-junctional ER. Thus, a detectable change of R-CEPIA-er fluorescence would have required calcium release from both compartments. Given these limitations, the strongest statement that can be made is that the data in Figure 7D–J are consistent with, but do not prove, that both Ca_V2.1 or Ca_V2.2 can trigger activation of RyR1 at junctions induced by JPH3.

Evidence of binding interactions that may be important for the localization of RyR1 and RyR3 at JPH3-induced junctions is provided by the behavior of YFP-RyR1_1:4300 and YFP-RyR3_1:4032, which lack the C-terminal, pore-forming regions that anchor the full-length proteins in the ER. In particular, the fluorescence associated with YFP-RyR1_1:4300 and YFP-RyR3_1:4032 accumulated at JPH3-induced junctions, which caused their fluorescence to be increased relative to regions lacking junctions (Figure 9). The accumulation of YFP-RyR1_1:4300 and YFP-RyR3_1:4032 at JPH3-induced junctions implies that these constructs bound to a component(s) present in these junctions, with JPH3 itself being an obvious candidate. If it is assumed that the binding of YFP-RyR1_1:4300 and YFP-RyR3_1:4032 indicates that they took on near-native conformations, it would imply that the cytoplasmic domains of the full-length RyR1 and RyR3 bind to JPH3 and thus help retain these RyRs at ER-PM junctions.

The failure of YFP-RyR2_1:4226 to accumulate at JPH3- or JPH4-induced junctions may have occurred because this construct failed to fold properly. For this reason, and because RyR2 has been reported to co-immunoprecipitate with JPH3 in pancreatic tissue (Li et al., 2016), we tested a second construct, YFP-RyR2_1:3991. This construct also failed to accumulate at junctions induced by JPH3 or JPH4 (data not shown). Possibly both YFP-RyR2_1:4226 and YFP-RyR2_1:3991 may not have folded correctly. Alternatively, it may be that the RyR2 cytoplasmic domain interacts weakly, or not at all, with JPH3 and that the co-immunoprecipitation of JPH3 and RyR2 in pancreatic tissue depends on the presence of C-terminal segments that are absent in RyR2_1:4226 and YFP-RyR2_1:3991.

Because colocalization of RyR1 with JPH3 appeared to depend on the JPH3 divergent domain (Figure 8), we tested for interactions between RyR1_1:4300 and constructs containing varying sized fragments of this divergent domain. We found that RyR1_1:4300 colocalized with the smallest fragment that we tested (Figure 10D), which consisted of JPH3 divergent domain residues 653 – 727 linked to the transmembrane domain. However, the observation that RyR1_1:4300 interacted with two different constructs containing JPH3 divergent domain residues 418 – 707 (Figure 10A, B) indicates that residues 708 – 727 are not required for this interaction. Additionally, full-length RyR1 colocalized with a JPH3 construct in which residues 681 – 725 had been deleted (Figure 10—figure supplement 1). Thus, we propose that JPH3 residues 653 – 680 contain the site that appears to be important for binding to the cytoplasmic domain of RyR1. This 28-residue segment is strongly conserved across a number of vertebrate species (Figure 12).

Figure 12

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Clearly, a major goal of future investigations will be to translate our work into new examinations of ER-PM junctions in neurons, for which the results from tsA201 cells provide useful conceptual and experimental tools. For example, based on our results, we think that it will be important to determine whether P/Q currents inactivate more rapidly in cerebellar Purkinje cells lacking both JPH3 and JPH4, and whether N-type Ca²⁺ channels are present in ER-PM junctions of the paraventricular nucleus of the thalamus where their transcripts are present together with those of JPH3 and JPH4. Similarly, we would like to determine whether specific functions can be assigned to RyR1 in neuronal ER-PM junctions. For this question, JPH3 constructs with altered sequence in the divergent domain may be a useful tool.

Raw data for peak current vs voltage, inactivation vs voltage, and Pearson's coefficients have been provided with the uploaded manuscript files.

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Author details

1. Stefano Perni

   Department of Physiology and Biophysics, Anschutz Medical Campus, University of Colorado, Aurora, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0591-4376

2. Kurt Beam

   Department of Physiology and Biophysics, Anschutz Medical Campus, University of Colorado, Aurora, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft

   For correspondence

   kurt.beam@cuanschutz.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-6902-085X

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