A non-conducting role of the Ca_v1.4 Ca²⁺ channel drives homeostatic plasticity at the cone photoreceptor synapse

Joint Public Review

Cav1.4 calcium channels control voltage-dependent calcium influx at photoreceptor synapses, and congenital loss of Cav1.4 function causes stationary night blindness CSNB2. Based on a broad portfolio of methodological approaches - genetic mouse models, immunolabeling and microscopic imaging, serial block-face-SEM, ERGs, and electrophysiology - the authors show that cone photoreceptor synapse development is strongly perturbed in the absence of Cav1.4 protein, and that expression of a nonconducting Cav1.4 channel mitigates these perturbations. Further data indicate that Cav3 channels are present, which, according to the authors, may compensate for the loss of Cav1.4 calcium currents and thus maintain cone synaptic transmission. These data, which are in agreement with a similar study by the same authors on rod photoreceptor synapses, help to explain what functional defects exactly cause CSNB2 and why it is accompanied by only mild visual impairment.

The strengths of the present study are its conceptual and experimental soundness, the broad spectrum of cutting-edge methodological approaches pursued, and the convincing differential analysis of mutant phenotypes. Weaknesses mainly concern the mechanism by which Cav3 channels might partially compensate for the loss of Cav1.4 calcium currents.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

The authors should perform experiments to answer this question: does Cav3 transcription increase in the G369i-KI, or is there instead some post-transcriptional modulation that permits surface expression of functional Cav3-containing channels in the absence of typical HVA Ca conductances? Also, the authors should determine whether G369i-KI can mediate Ca2+ release from intracellular stores and whether release from stores is upregulated as Cav3-containing channel expression (or function) is increased.

We performed transcriptomic (drop-seq) analysis to test whether a Cav3 subtype is upregulated in cones of G369i KI mice. These experiments show that, consistent with previous studies (PMID 35803735, 26000488), Cacna1h appears to be the primary Cav3 subtype expressed mouse cones. However, as shown in new Supp.Fig.S3, there was no significant difference in the levels of Cacna1h transcripts in WT and G369i KI cones. Therefore, we propose that there may be some post-transcriptional modification, or alteration in a pathway that regulates channel availability, that enables the contribution Cav3 channels to the whole-cell Ca2+ current in the absence of functional Cav1.4 channels cones.

We also performed Ca2+ imaging experiments in WT vs G369i KI cone terminals to assess whether the diminutive Cav3 current in G369i KI cone terminals may be compensated by upregulation of a Ca2+ signal such as from intracellular stores. Arguing against this possibility, depolarization-evoked Ca2+ signals in G369i KI cones were dramatically reduced compared to WT cones (new Fig.9).

Reviewer #2 (Recommendations For The Authors):

Major points-

(1) It is stated in too many places that cone features in the Cav1.4 knock-in are "intact", preserved, or spared, but this representation is not accurate. There are two instances in this study that qualify as intact when comparing KI to WT: 1) the photopic a-waves in the Cav1.4 knock-in (also demonstrated in Maddox et al 2020) and 2) latency to the platform (current MS, Figure 7f). However, in the numerous instances listed below, the authors compared the Cav1.4 knock-in to the Cav1.4 knock-out, and then referred to the KI as exhibiting intact responses. The reference point for intactness needs to be wildtype, as appropriately done for Figures 2 and 3, and when comparing the KI to the KO the phrasing should be altered; for example: "the KI was spared from the extensive degeneration witnessed in the KO....".

In most cases, we clearly note that there are key differences in the WT and the G369i KI cone synapses, which highlight the importance of Cav1.4-specific Ca2+ signals for certain aspects of the cone synapse. We disagree with the reviewer on the point that we did not often use the WT as a reference since most of our experiments involved comparisons of only WT and G369i KI (Figs. 3-6) or WT, G369i KI, and Cav1.4 KO (Figs.1,7—and in these cases comparisons specifically between WT and G369i KI mice were included). We used “intact” as a descriptor for G369i KI cone synapses since these are actually present, albeit abnormal in the G369i KI retina, whereas cone synapses are completely absent in the Cav1.4 KO retina. To avoid confusion, we modified our use of “intact” and “preserved” where appropriate.

A. Abstract, line 34 to 35: ".......preserved in KI but not in KO.".

Abstract was rewritten and this line was removed.

B. Line 36: "....synaptogenesis remains intact". The MS documents many differences in the morphology of KI and WT cones (immunofluorescence and electron microscopy data), which is counter to an intact phenotype.

The sentence was: “In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the Ca2+-independent role of Cav1.4 in cone synaptogenesis remains intact.”

Here the meaning of “intact” refers to the Ca2+ -independent role of Cav1.4, not synapses. Thus, we have left the sentence unchanged.

C. This strikes the right balance, lines 67 to 68: "....although greatly impaired.....".

D. Line 149, "Cone signaling to a postsynaptic partner is intact in G369i KI mice". This description is inaccurate. Here there is only WT and KI, and the text reads as follows in line 162: "terminals (Figure 6b). The ON and OFF components of EPSCs in G369i KI HCs were measurable, although lower in amplitude than in WT (Figure 6a,b)." Neither "measurable" nor "lower in amplitude" meet the definition of "intact", and actual numerical values are lacking in the text.

We have added results showing that there are no light responses in the Cav1.4 KO horizontal cells and have modified the sentence to: “Cone synaptic responses are present in horizontal cells of G369i KI but not Cav1.4 KO mice”.

We have modified discussion of these results as (line 210-213): “Consistent with the lack of mature ribbons and abnormal cone pedicles (Fig.1), HC light responses were negligible in Cav1.4 KO mice (Fig.8a,b). In contrast, the ON and OFF responses were present in G369i KI HCs although significantly lower in amplitude than in WT HCs (Fig. 8a,b).”

E. Please add a legend to Figure 6a to indicate the intensities. The shape of the KI responses is different from the control which is worthy of discussion: i) there is no clear cessation of HC EPSCs in the KI during the light ON period (when release stops, Im fluctuations should be minimal), and ii) the "peaked" appearances of the initial 500ms of the On and Off periods are very similar in shape for the KI (hard to interpret in the same fashion as a control response). How were the On and Off amplitudes analyzed? Furthermore, the OFF current is not summarized in Figure 6D, but should not this be when Cav3 should be opening and triggering release: Off response-EPSC? Lastly, Figure 6b,d shows a ~70% reduction in On-current in the KI, and the KI example of 6b an 80% reduction in Off current compared to WT. Yet, the only place asterisks are used to indicate sig diff is the DNQX data within each genotype in Fig 6d. These data cannot be described as showing "intact" KI responses, and the absence of numerical and statistical values needs to be addressed.

New Fig.8a depicting the horizontal cell light responses has been modified to include the legend indicating light intensities. The ON and OFF amplitudes were analyzed as the peak current amplitudes. This information has been added to the legend.

The reviewer is correct in that the OFF response represents the EPSC whereas the ON response represents the decrease in the EPSC with light. To avoid confusion, we changed the y axis label for the averaged data to read ON or OFF “response” rather than “current” in new Fig.8b.

As the reviewer suggests, the more transient nature of the KI response during the light ON period could result from aberrant continuation of vesicular release during the light-induced hyperpolarization of cones in the KI mice, in contrast to the prolonged suppression of release by light which is evident in the WT responses. We speculated on this difference as follows (lines 237-241):

“In addition to its smaller amplitude, the transient nature of the ON response in G369i KI HCs suggested inadequate cessation of cone glutamate release by light (Fig.8b). Slow deactivation of Cav3 channels and/or their activation at negative voltages20 could give rise to Ca2+ signals that support release following light-induced hyperpolarization of G369i KI cones.”

We added astericks to new Fig.8b,d indicating statistical differences and description of the tests in the legend.

F. line 168 the section titled "Light responses of bipolar cells and visual behavior is spared in G369i KI but not Cav1.4 KO mice".

Changed to: “Light responses of bipolar cells and visual behavior are present in G369i KI but not Cav1.4 KO mice”

Last sentence of erg results, 189-190: "These results suggest that cone-to-CBC signaling is intact in G369i KI mice.". "Spared and intact" are not accurate descriptions. The ERG data presented here shows massive differences between WT and the KI, except in the instance of awaves.

This sentence was removed.

As for Figure 6, the results text related to Figure 7a-d does not present real numbers for ERG responses, and there is no indication of significant differences there or in the Figure panels. For instance, in Figure 7b, b-waves are KI are comparable to KO, except at the two highest-intensity flashes that show KI responses ~20% the amplitude of WT. Presentation of KI and KO data on a 6- to 10-fold expanded scale higher than WT can be misleading: a quick read of these Figure panels might make one incorrectly conclude that the KI is intact while the KO is impaired when compared to WT. The Methods section needs more details on the ERG analysis (e.g. any filtering out of oscillatory potentials when measuring b-wave, and what was the allowable range of time-to-peak for b-wave amplitude, etc..).

The vertical scaling of the ERG results in new Fig.10c,d has been changed so as to reflect clearly diminished responses of the KO and KI vs the WT. Further details regarding the ERG analysis was added to the Methods section.

G. Can you point to other studies that have used the "visible platform swim test" used in Figure 7e, f, and specify further how mice were dark/light adapted prior to the recordings?

As referenced in the Methods, original line 674, the methods we used for the swim test were described in our previous study (PMID 29875267). Other studies that have used this assay include PMIDs: 28262416, 26402607.

(2) The Maddox et al 2020 study does not safely address whether rods have a residual T-type Ca2+ current in the Cav 1.4 KO or KI. The study showed that membrane currents measured from rods in the KI and KO retina were distinct from WT, supporting their claim that L-type Ca2+ current is absent in the KI and KO. However, the recordings had shortcomings that challenge the analysis of Ca2+ currents: i) collected at room temp (22-24{degree sign}C), ii) at an unknown distance from the terminal (uncertain voltage clamp), iii) with a very slow voltage ramp rate that is not suitable for probing T-type currents (Figure 1d Maddox 2020, 140 mV over 1 sec: 7msec/1mV), and iv) at a signal-to-noise that does not allow to resolve a membrane current under 1 pA (avg wt rod Ca2+ current was -3.5 pA, and line noise ~1pA peak-to-peak in Maddox 2020). Suggestion: say T-type currents were not probed in Maddox et al 2020, but Davison et al 2022 did not find PCR signal for Cav3.2 in rods.

We disagree that recordings in the Maddox 2020 study were not sufficient to uncover a T-type current. The voltage ramps in that study were not much slower than that of the Davison et al. 2022 study (they used 0.19 mV/ms). Moreover, in new Supp. Fig.S1, we show that like the slower voltage ramp (0.15 mV/ms) used in the prior study of G369i KI rods, the voltage ramps we used in the present study (0.5 mV/ms), which clearly evoke currents with T-type properties in G369i KI cones (Fig.2a,b, Fig.3a,b) do not evoke currents in WT or G369i KI rods.

Minor comments.

(1) Suggestion: add an overview panel to Figure 1 that shows the rod terminals in the KI. The problem is that cropping out the ribbon and active zone signals from rods, to highlight cones, can give the impression that the cones are partially spared in the KI, and the rods are not spared at all. (yet you nicely clarify this in Figure 4 and in the legend and text, etc.).

We chose to modify the legend with this information as in Fig.4 rather than modify the figure.

(2) Mouse wt cone Ca2+ currents look like L-type currents, as do your monkey and squirrel cone recordings, and also much like those of mouse rods (see Figure S5, Hagiwara et al., 2018 or Grabner and Moser 2021). Your pharm data from mice and squirrels further supports your conclusion, and certainly took much effort. Davison et al 2022 J Neurosci showed PCR results that support their claim that a Cav3 current exists in wt cones. Questions: 1) have you tried PCR? 2) Can you offer more details on what Cav3 KO you tried and what antibodies failed to confirm the KO? As the authors know, one complication is that the deletion of one Cav can be compensated for by the expression of a new Cav. There are 3 types of Cav3s and removal of one type may be compensated for by another Cav3.

We have included drop-seq data (new Supp.Fig.S3) implicating Cav3.2 as the main Cav3 subtype in cones and have modified our discussion of these results accordingly. These experiments did not reveal any changes in Cav3 subtype expression in G369i KI vs WT cones.

(3) Lines 95/96- onward, spend more time telling the story. When working out the biophysical and pharmacological behavior of the Ca2+ currents, you might want to initially refer to the membrane current as a membrane current, and then state how your voltage protocols, intra- and extra-cell solutions, and drugs helped you verify 1) L-type and 2) T-type Ca2+ currents.

We have modified the text with more detail.

(4) If data is in hand, add a ramp I-V to Figure S2, which shows the response of the ground squirrel cone. The steps in S2a are excellent for making your point that a transient current is missing, and the bipolar is a great control to illustrate ML218 works. However, a comparison of a squirrel cone ramp to a bipolar ramp response could complete the figure.

See Reponse to #5 below.

(5) Consider moving Supplementary Figures S2 and S3 to the main text; these are highly relevant to the story, novel, and well-executed.

Fig.S2 and S3 were added as new Figs.4,5. The new Fig.4 includes voltage ramps in ground squirrel cones (panel a) to compare with the bipolar data (panel f).

(6) The nice electron microscopy reconstructions are not elaborated on in any detail, and there is no mention of ribbon size. Is the resolution sufficient to estimate ribbon size, the number of synaptic vesicles around the ribbon and in the adjacent cytosol? The images indicate major changes in the morphology of the terminals. Is the glial envelope similar in WT and KI?

Since ribbons were quantified extensively in the confocal analyses in Fig.6, we felt it unnecessary to add this to the EM analysis which focused mainly on aspects of 3D structure (i.e., arrangement of ribbons, postsynaptic wiring, cone pedicle morphology). We added further discussion of the change in morphology of the G369i KI cone pedicle (lines 200-203): “Compared to WT, ribbons in G369i KI pedicles appeared disorganized and were often parallel rather than perpendicular to the presynaptic membrane (Fig.7a-c). Consistent with our confocal analyses (Fig.1), G369i KI cone pedicles extended telodendria in multiple directions rather than just apically (Fig. 7a).”

While we did not opt to characterize the glial envelope in WT cones, we did add an analysis of synaptic vesicles around ribbons to Table 2.

(7) Discussion line 250: "we found no evidence for a functional contribution of Cav3 in our recordings of cones in WT mice (Figures. 2,3), ground squirrels, or macaque (Supplementary Figures S2 and S3).". I would not use "functional" in this context because when comparing your work to Davison et al 2022, they defined functional as a separate response component driven by Cav3. For instance, they examined the influence of their T-type current on exocytosis (by membrane capacitance) and other features like spiking Ca2+ transients. Suggestion: substitute functional with "detectable", and say "we found no detectable Cav currents". Or if you had Ttype staining, but not T-type Ca2+ currents, then say "no functional current even though there is staining...".

We have modified the text as (lines 336-338): “However, in contrast to recordings of WT mouse cone pedicles in a previous study21, we found no evidence for Cav3-mediated currents in somatic recordings of cones in WT mice (Figs.2,3).”

We propose an alternative interpretation of the results in the Davison et al study concerning the conclusion that Cav3 channels contribute to Ca2+ spikes and exocytosis. That study used 100 µM Ni2+ to block a “T-type” contribution to spike activity in cones. In their Figs.4,5, the spikes are suppressed by 100 µM Ni2+ and 10 µM nifedipine, a Cav1 antagonist, and spared by the T-type selective drug Z944. This is problematic for several reasons. First, as shown by the authors

(their Fig.2A1,A2) and others (PMID: 15541900), 100 µM Ni2+ inhibits Cav1-type currents in photoreceptors. Second, Z944 potentiates Cav1 current in their mouse cones (their Fig.2C1,C2). Thus, both reagents are suboptimal for dissecting the contribution of either Cav subtype to spiking activity. With respect to Cav3 channels and exocytosis, these authors interpreted a reduction in exocytosis upon holding at -39 mV compared to at -69 mV as indicating a loss of a T-type driven component of release. However, Cav1 channel inactivation (PMID: 12473074) could lead to the observed reduction in exocytosis at -30 mV.

(8) Additional literature related to your Intro and Discussion. Regarding CSNB2, related mutations of active zone proteins, and what happens to Ca2+ currents when ribbons are deleted, you might want to consider the following studies that measure Ca2+ currents from rods: conditional KO of RIM1/2 (Grabner et al 2015 JN), KO of ELKS1/2 (Hagiwara et al, 2018 JCB), and KO of Ribeye (Grabner and Moser eLife 2021). In these studies, the Cav currents were absent in rods of the ELKS1/2 DKO, strongly reduced (80%) in the RIM1/2DKO, but altered in more subtle ways (activation-inactivation) without significantly changing steady-state Ca2+ current in the Ribeye KO. This does not seem to support some of the arguments you have made in the Introduction and Discussion regarding ribbon size and Ca2+ currents, yet the suggested literature is related to the topic at hand.

A description of these synaptic proteins as potential mediators of the effect of Cav1.4 on ribbon morphogenesis was added to the Discussion, lines 325-327.

(9) Line 129: "Along with the major constituents of the ribbon, CtBP2, and RIBEYE", for clarity Ribeye has two domains, one that is identical to CtBP2 (B-domain) and the unique Ribeye domain (A-domain) that is only expressed at ribbon synapses. And, Piccolino is also embedded in the ribbon (Brandstaetter lab, Wichmann/Moser labs). In other words, Ribeye and Piccolino are the major constituents of the ribbon.

To avoid confusion, we simply mention Ctbp2 and RIBEYE in the context of the corresponding antibodies that were used to label ribbons.

(10) Abstract: consider to rephrase "Ca2+-independent role of Cav1.4" by "Ca2+-permeationindependent role of Cav1.4" or alike

Sentence changed to: “In CSNB2, we propose that Cav3 channels maintain cone synaptic output provided that the nonconducting role of Cav1.4 in cone synaptogenesis remains intact.”

Reviewer #3 (Recommendations For The Authors):

Cav1.4 voltage-gated calcium channels play an important role in neurotransmission at mammalian photoreceptor synapses. Mutations in the CACNA1f gene lead to congenital stationary night blindness that particularly affects the rod pathway. Mouse Cav1.4 knockout and Cav1.4 knockin models suggest that Cav1.4 is also important for the cone pathway. Deletion of Cav1.4 in the knockout models leads to signaling malfunctions and to abundant morphological re-arrangements of the synapse suggesting that the channel not only has a role in the influx of Ca2+ but also in the morphological organization of the photoreceptor synapse. Of note, also additional Cav-channels have been previously detected in cone synapses by different groups, including L-type Cav1.3 (Wu et al., 2007; pmid; Kersten et al., 2020; pmid), and also T-type Cav3.2 (Davison et al., 2021; pmid 35803735).

In order to study a conductivity-independent role of Cav1.4 in the morphological organization of photoreceptor synapses, the authors generated the knockin (KI) mouse Cav1.4 G369i in a previous study (Maddox et al., eLife 2020; pmid 32940604). The Cav1.4 G369i KI channel no longer works as a Ca2+-conducting channel due to the insertion of a glycine in the pore-forming unit (Madox et al. elife 2020; pmid 32940604). In this previous study (Madox et al. elife 2020; pmid 32940604), the authors analyzed Cav1.4 G369i in rod photoreceptor synapses. In the present study, the authors analyzed cone synapses in this KI mouse.

For this purpose, the authors performed a comprehensive set of experimental methods

including immunohistochemistry with antibodies (also with quantitative analyses), electrophysiological measurements of presynaptic Ca2+ currents from cone photoreceptors in the presence/absence of inhibitors of L-type- and T-type- calcium channels, electron microscopy (FIB-SEM), ERG recordings and visual behavior tests of the Cav G369i KI in comparison to the Cav1.4 knockout and wild-type control mice.

The authors found that the non-conducting Cav channel is properly localized in cone synapses and demonstrated that there are no gross morphological alterations (e.g., sprouting of postsynaptic components that are typically observed in the Cav1.4 knockout). These findings demonstrate that cone synaptogenesis relies on the presence of Cav1.4 protein but not on its Ca2+ conductivity. This result, obtained at cone synapses in the present study, is similar to the previously reported results observed for rod synapses (Maddox et al., eLife 2020, pmid 32940604). No further mechanistic insights or molecular mechanisms were provided that demonstrated how the presence of the Cav channels could orchestrate the building of the cone synapse.

We respectfully disagree regarding the mechanistic advance of our study. As indicated by Reviewer 2, a major advance of our study is in providing a mechanism that can explain the longstanding conundrum that congenital stationary night blindness type 2 mutations that would be expected to severely compromise Cav1.4 function do not produce complete blindness. Our study provides an important contrast to the Maddox et al 2020 study in showing that rods and cones respond differentially to loss of Cav1.4 function, which is also relevant to the visual phenotypes of CSNB2. How the presence of Cav1.4 orchestrates cone synaptogenesis is an important topic that is outside the scope of our present study.

In the present study, the authors also propose a homeostatic switch from L-type to (newly occurring) T-type calcium channels in the Cav1.4 G369i KI mouse as a consequence of the deficient calcium channel conductivity in the Cav1.4 G369i Cav1.4 KI mouse. In cones of the Cav1.4 G369i, the high-voltage activated, L-type Ca2+-entry was abolished, in agreement with their previous paper (Maddox et al., eLife 2020, pmid 32940604). The authors found a lowvoltage activated Ca2+ current instead that they assigned to T-type Ca2+-currents based on pharmacological inhibitor experiments. T-type Ca2+-currents/channels were already previously identified in other studies by independent groups and independent techniques

(electrophysiology, RT-PCR, single-cell sequencing) in cones of wild-type mice (Davison et al.,

2021, pmid 35803735; Macosko et al., 2015, pmid 26000488; Williams et al., 2022, pmid 35650675). In the present manuscript (Figures 3a/b), the authors also observed a low-voltage activated, T-type like current in cones of wild-type mice, that is isradipine-resistant and affected by the T-type inhibitor ML218. This finding appears compatible with a T-type-like current in wildtype cones and is consistent with the published data mentioned above, although the authors interpret this data in a different way in the discussion.

Due to the noise inherent in whole cell voltage clamp measurements and some crossover effects in the pharmacology, we cannot completely exclude the presence of a T-type current in WT mouse cones. However, our results very clearly support a conclusion opposite to that stated by the reviewer. Namely, if WT mouse cones have T-type Ca currents, then they are far smaller than those in the Cav1.4 G369i KI and KO cones. In particular, while we identified message for Cav3.2 in WT mouse cones, we were unable to identify a functional T-type current by either voltage clamp measurements or pharmacology. See below for a detailed rebuttal.

This proposal of a homeostatic switch is not convincingly supported in this reviewer's opinion

(for further details, please see below). Furthermore, no data on possible molecular mechanisms were provided that would support such a proposal of a homeostatic switch of calcium channels. No mechanistic/molecular insights were provided for a proposed homeostatic switch between Ltype to T-type channels that the authors propose to occur between wild-type and Cav1.4 G369i as a consequence of conduction-deficient Cav1.4 G369i channels. Is this e.g. based on posttranslational modifications that switch on T-type channels or regulation at the transcriptional level inducing expression of T-type calcium channel or on other mechanisms? The authors remain descriptive with their central hypotheses. No molecular mechanisms/signaling pathways were provided that would support the idea of such a homeostatic switch.

Homeostatic plasticity refers to the maintenance of neuronal function in response to some perturbation in neuronal activity and can result from changes in the expression of ion channel genes (PMID: 36377048, 32747440, 19778903) or regulatory pathways that modulate ion channels (PMID: 15051886, 32492405). We present multiple lines of evidence showing that Cav3 currents appear in cones upon genetically induced Cav1.4 loss of function and can support cone synaptic responses and visual behavior if cone synapse structure is maintained. Our new transcriptomic studies show no difference between levels of Cav3 channel transcripts in WT and G369i KI cones, suggesting that the appearance of the Cav3 currents in G369i KI cones does not result from an increase in Cav3 gene expression. We are currently investigating our transcriptomic dataset to determine if Cav3 regulatory pathways are upregulated in G369i KI cones and will present this in a follow-up study.

The authors show residual photopic signaling in the non-conducting Cav1.4 G369i KI mouse as judged by the recording of postsynaptic currents, ERG recordings and visual behavior tests though in a reduced manner. The residual cone-based signaling could be based on the nonaffected T-type Ca2+ channel conductivity in cone synapses. Given that the L-type current through Cav1.4 is gone in the Cav1.4 G369i KI as previously shown (Maddox et al., 2020, pmid 32940604), the T-type calcium current will remain. However as discussed above, this does not necessarily support the idea of a homeostatic switch.

A major point which we highlighted with new results is that despite the expression of Cav3 transcripts in WT mouse cones, Cav3 channels do not contribute to the cone Ca2+ current. This is at odds with the Davison et al study (PMID: 35803735, see our response to Reviewer 2, pt 7 for caveats of this study), but our results convincingly show that the Cav3 current appears only when Cav1.4 is genetically inactivated. Pharmacological or electrophysiological methods that should reveal the presence of Cav3 currents do not change the properties of the Ca2+ current in cones of WT mice, ground squirrel, or macaque:

• Figs.2-4: Voltage steps to -40 mV (Fig 2e) that activate a sizeable T-current in G369i KI mouse cones produce a negligible transient at pulse onset in WT mouse cones. Similarly, transient currents that are obvious in G369i KI mouse cones during the final step to -30 mV are absent in WT cones. When we block Cav1.4 with isradipine either in cones of WT mice or ground squirrel, the current that remains does not resemble a Cav3 current but rather a scaled down version of the L-type current. ML218, which readily blocks Cav3 channels in HEK293T cells and in G369i KI cones, has only minor effects in cones of WT mice and ground squirrel; these effects of ML218 can be attributed to non-specific actions on Cav1.4 (new Supp.Fig.S2). New Fig.4 (moved from the supplementary data to the main article) clearly shows that the ML218-sensitive current in ground squirrel cones exhibits properties of Cav1.4 not Cav3 channels.

• Figs.2,5: Holding voltages that inactivate Cav3 channels have no effect on the Ca2+ current in cones of WT mice or macaque (recordings of macaque cones were moved from the supplement to the main article as new Fig.5).

In Figure 4 the authors measured an increase in the size of the active zone (as judged by the size of the bassoon cluster) and of the synaptic ribbons in the Cav1.4 G369i. A mechanistic explanation for this phenomenon was not provided and the underlying molecular mechanisms were not unraveled.

The FIB-SEM data uncover some ultrastructural alteration/misalignments of the synaptic ribbons and misalignments of the regular arrangement of the postsynaptic dendrites in the G369i KI mice. Also concerning this observation, the study remains descriptive and does not reveal the underlying mechanisms as it would be expected for eLife.

We respectfully disagree on the descriptive nature of our study and the need for a full characterization of the molecular mechanism underlying the cone synaptic defects in the G369i KI mouse.

An important study in the field (Zanetti et al., Sci. Rep. 2021; pmid 33526839) should be also cited that used a gain-of-function mutation of Cav1.4 to analyze its functional and structural role in the cone pathway.

We have added citation of this paper to the Discussion (lines 354-356).

In conclusion, the study has been expertly performed but remains descriptive without deciphering the underlying molecular mechanisms of the observed phenomena, including the proposed homeostatic switch of synaptic calcium channels. Furthermore, a relevant part of the data in the present paper (presence of T-type calcium channels in cone photoreceptors) has already been identified/presented by previous studies of different groups (Macosko et al., 2015; pmid 26000488; Davison et al., 2021; pmid 35803735; Williams et al., 2022; pmid 35650675). The degree of novelty of the present paper thus appears limited. I think that the study might be better suited in a more specialized journal than eLife.

We thank the reviewer for acknowledging the rigor of our study but disagree with their evaluation regarding the novelty of our work as outlined in our responses above.