Author response:
The following is the authors’ response to the previous reviews.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Line 127. Provide a few more words describing the voltage protocol. To the uninitiated, panels A and B will be difficult to understand. "The large negative step is used to first close all channels, then probe the activation function with a series of depolarizing steps to re-open them and obtain the max conductance from the peak tail current at -36 mV. "
We have revised the text as suggested (revision lines 127 to Line 131): “From a holding potential within the gK,L activation range (here –74 mV), the cell is hyperpolarized to –124 mV, negative to EK and the activation range, producing a large inward current through open gK,L channels that rapidly decays as the channels deactivate. We use the large transient inward current as a hallmark of gK,L. The hyperpolarization closes all channels, and then the activation function is probed with a series of depolarizing steps, obtaining the max conductance from the peak tail current at –44 mV (Fig. 1A).”
Incidentally, why does the peak tail current decay?
We added this text to the figure legend to explain this: “For steps positive to the midpoint voltage, tail currents are very large. As a result, K+ accumulation in the calyceal cleft reduces driving force on K+, causing currents to decay rapidly, as seen in A (Lim et al., 2011).”
The decay of the peak tail current is a feature of gK,L (large K+ conductance) and the large enclosed synaptic cleft (which concentrates K+ that effluxes from the HC). See Govindaraju et al. (2023) and Lim et al. (2011) for modeling and experiments around this phenomenon.
Line 217-218. For some reason, I stumbled over this wording. Perhaps rearrange as "In type II HCs absence of Kv1.8 significantly increased Rin and tauRC. There was no effect on Vrest because the conductances to which Kv1.8 contributes, gA and gDR activate positive to the resting potential. (so which K conductances establish Vrest???).
We kept our original wording because we wanted to discuss the baseline (Vrest) before describing responses to current injection.
->Vrest is presumably maintained by ATP-dependent Na/K exchangers (ATP1a1), HCN, Kir, and mechanotransduction currents. Repolarization is achieved by delayed rectifier and A-type K+ conductances in type II HCs.
Figure 4, panel C - provides absolute membrane potential for voltage responses. Presumably, these were the most 'ringy' responses. Were they obtained at similar Vm in all cells (i.e., comparisons of Q values in lines 229-230).
We added the absolute membrane potential scale. Type II HC protocols all started with 0 pA current injection at baseline, so they were at their natural Vrest, which did not differ by genotype or zone. Consistent with Q depending on expression of conductances that activate positive to Vrest, Q did not co-vary with Vrest (Pearson’s correlation coefficient = 0.08, p = 0.47, n= 85).
Lines 254. Staining is non-specific? Rather than non-selective?
Yes, thanks - Corrected (Line 264).
Figure 6. Do you have a negative control image for Kv1.4 immuno? Is it surprising that this label is all over the cell, but Kv1.8 is restricted to the synaptic pole?
We don’t have a null-animal control because this immunoreactivity was done in rat. While the cuticular plate staining was most likely nonspecific because we see that with many different antibodies, it’s harder to judge the background staining in the hair cell body layer. After feedback from the reviewers, we decided to pull the KV1.4 immunostaining from the paper because of the lack of null control, high background, and inability to reproduce these results in mouse tissue. In our hands, in mouse tissue, both mouse and rabbit anti-KV1.4 antibodies failed to localize to the hair cell membrane. Further optimization or another method could improve that, but for now the single-cell expression data (McInturff et al., 2018) remain the strongest evidence for KV1.4 expression in murine type II hair cells.
Lines 400-404. Whew, this is pretty cryptic. Expand a bit?
We simplified this paragraph (revision lines 411-413): “We speculate that gA and gDR(KV1.8) have different subunit composition: gA may include heteromers of KV1.8 with other subunits that confer rapid inactivation, while gDR(KV1.8) may comprise homomeric KV1.8 channels, given that they do not have N-type inactivation .”
Line 428. 'importantly different ion channels'. I think I understand what is meant but perhaps say a bit more.
Revised (Line 438): “biophysically distinct and functionally different ion channels”.
Random thought. In addition to impacting Rin and TauRC, do you think the more negative Vrest might also provide a selective advantage by increasing the driving force on K entry from endolymph?
When the calyx is perfectly intact, gK,L is predicted to make Vrest less negative than the values we report in our paper, where we have disturbed the calyx to access the hair cell (–80, Govindaraju et al., 2023, vs. –87 mV, here). By enhancing K+ accumulation in the calyceal cleft, the intact calyx shifts EK—and Vrest—positively (Lim et al., 2011), so the effect on driving force may not be as drastic as what you are thinking.
Reviewer #2 (Recommendations For The Authors):
(1) Introduction: wouldn't the small initial paragraph stating the main conclusion of the study fit better at the end of the background section, instead of at the beginning?
Thank you for this idea, we have tried that and settled on this direct approach to let people know in advance what the goals of the paper are.
(2) Pg.4: The following sentence is rather confusing "Between P5 and P10, we detected no evidence of a non-gK,L KV1.8-dependent.....". Also, Suppl. Fig 1A seems to show that between P5 and P10 hair cells can display a potassium current having either a hyperpolarised or depolarised Vhalf. Thus, I am not sure I understand the above statement.
Thank you for pointing out unclear wording. We used the more common “delayed rectifier” term in our revision (Lines 144-147): “Between P5 and P10, some type I HCs have not yet acquired the physiologically defined conductance, gK,L.. N effects of KV1.8 deletion were seen in the delayed rectifier currents of immature type I HCs (Suppl. Fig. 1B), showing that they are not immature forms of the Kv1.8-dependent gK,L channels. ”
(3) For the reduced Cm of hair cells from Kv1.8 knockout mice, could another reason be simply the immature state of the hair cells (i.e. lack of normal growth), rather than less channels in the membrane?
There were no other signs to suggest immaturity or abnormal growth in KV1.8–/– hair cells or mice. Importantly, type II HCs did not show the same Cm effect.
We further discussed the capacitance effect in lines 160-167: “Cm scales with surface area, but soma sizes were unchanged by deletion of KV1.8 (Suppl. Table 2). Instead, Cm may be higher in KV1.8+/+ cells because of gK,L for two reasons. First, highly expressed trans-membrane proteins (see discussion of gK,L channel density in Chen and Eatock, 2000) can affect membrane thickness (Mitra et al., 2004), which is inversely proportional to specific Cm. Second, gK,L could contaminate estimations of capacitive current, which is calculated from the decay time constant of transient current evoked by small voltage steps outside the operating range of any ion channels. gK,L has such a negative operating range that, even for Vm negative to –90 mV, some gK,L channels are voltage-sensitive and could add to capacitive current.”
(4) Methods: The electrophysiological part states that "For most recordings, we used .....". However, it is not clear what has been used for the other recordings.
Thanks for catching this error, a holdover from an earlier ms. version. We have deleted “For most recordings” (revision line 466).
Also, please provide the sign for the calculated 4 mV liquid junction potential.
Done (revision line 476).
Reviewer #3 (Recommendations For The Authors):
(1) Some of the data in panels in Fig. 1 are hard to match up. The voltage protocols shown in A and B show steps from hyperpolarized values to -71mV (A) and -32 mV (B). However, the value from A doesn't seem to correspond with the activation curve in C.
Thank you for catching this. We accidentally showed the control I-X curve from a different cell than that in A. We now show the G-V relation for the cell in A.
Also the Vhalf in D for -/- animals is ~-38 mV, which is similar to the most positive step shown in the protocol.
The most positive step in Figure 1B is actually –25 mV. The uneven tick labels might have been confusing, so we re-labeled them to be more conventional.
Were type I cells stepped to more positive potentials to test for the presence of voltage-activated currents at greater depolarizations? This is needed to support the statement on lines 147-148.
We added “no additional K+ conductance activated up to +40 mV” (revision line 149-150). Our standard voltage-clamp protocol iterates up to ~+40 mV in KV1.8–/– hair cells, but in Figure 1 we only showed steps up to –25 mV because K+ accumulation in the synaptic cleft with the calyx distorts the current waveform even for the small residual conductances of the knockouts. KV1.8–/– hair cells have a main KV conductance with a Vhalf of ~–38 mV, as shown in Figure 1, and we did not see an additional KV conductance that activated with a more positive Vhalf up to +40 mV.
(2) Line 151 states "While the cells of Kv1.8-/- appeared healthy..." how were epithelia assessed for health? Hair cells arise from support cells and it would be interesting to know if Kv1.8 absence influences supporting cells or neurons.
We added our criteria for cell health to lines 477-479: “KV1.8–/– hair cells appeared healthy in that cells had resting potentials negative to –50 mV, cells lasted a long time (20-30 minutes) in ruptured patch recordings, membranes were not fragile, and extensive blebbing was not seen.”
Supporting cells were not routinely investigated. We characterized calyx electrical activity (passive membrane properties, voltage-gated currents, firing pattern) and didn’t detect differences between +/+, +/–, and –/– recordings (data not shown). KV1.8 was not detected in neural tissue (Lee et al., 2013).
(3) Several different K+ channel subtypes were found to contribute to inner hair cell K+ conductances (Dierich et al. 2020) but few additional K+ channel subtypes are considered here in vestibular hair cells. Further comments on calcium-activated conductances (lines 310-317) would be helpful since apamin-sensitive SK conductances are reported in type II hair cells (Poppi et al. 2018) and large iberiotoxin-sensitive BK conductances in type I hair cells (Contini et al. 2020). Were iberiotoxin effects studied at a range of voltages and might calcium-dependent conductances contribute to the enhanced resonance responses shown in Fig. 4?
We refer you to lines 310-317 in the original ms (lines 322-329 in the revised ms), where we explain possible reasons for not observing IK(Ca) in this study.
(4) Similar to GK,L erg (Kv11) channels show significant Cs+-permeability. Were experiments using Cs+ and/or Kv11 antagonists performed to test for Kv11?
No. Hurley et al. (2006) used Kv11 antagonists to reveal Kv11 currents in rat utricular type I hair cells with perforated patch, which were also detected in rats with single-cell RT-PCR (Hurley et al. 2006) and in mice with single-cell RNAseq (McInturff et al., 2018). They likely contribute to hair cell currents, alongside Kv7, Kv1.8, HCN1, and Kir.
(5) Mechanosensitive ("MET") channels in hair cells are mentioned on lines 234 and 472 (towards the end of the Discussion), but a sentence or two describing the sensory function of hair cells in terms of MET channels and K+ fluxes would help in the Introduction too.
Following this suggestion we have expanded the introduction with the following lines (78-87): “Hair cells are known for their large outwardly rectifying K+ conductances, which repolarize membrane voltage following a mechanically evoked perturbation and in some cases contribute to sharp electrical tuning of the hair cell membrane. Because gK,L is unusually large and unusually negatively activated, it strongly attenuates and speeds up the receptor potentials of type I HCs (Correia et al., 1996; Rüsch and Eatock, 1996b). In addition, gK,L augments a novel non-quantal transmission from type I hair cell to afferent calyx by providing open channels for K+ flow into the synaptic cleft (Contini et al., 2012, 2017, 2020; Govindaraju et al., 2023), increasing the speed and linearity of the transmitted signal (Songer and Eatock, 2013).”
(6) Lines 258-260 state that GKL does not inactivate, but previous literature has documented a slow type of inactivation in mouse crista and utricle type I hair cells (Lim et al. 2011, Rusch and Eatock 1996) which should be considered.
Lim et al. (2011) concluded that K+ accumulation in the synaptic cleft can explain much of the apparent inactivation of gK,L. In our paper, we were referring to fast, N-type inactivation. We changed that line to be more specific; new revision lines 269-271: “KV1.8, like most KV1 subunits, does not show fast inactivation as a heterologously expressed homomer (Lang et al., 2000; Ranjan et al., 2019; Dierich et al., 2020), nor do the KV1.8-dependent channels in type I HCs, as we show, and in cochlear inner hair cells (Dierich et al., 2020).”
(7) Lines 320-321 Zonal differences in inward rectifier conductances were reported previously in bird hair cells (Masetto and Correia 1997) and should be referenced here.
Zonal differences were reported by Masetto and Correia for type II but not type I avian hair cells, which is why we emphasize that we found a zonal difference in I-H in type I hair cells. We added two citations to direct readers to type II hair cell results (lines 333-334): “The gK,L knockout allowed identification of zonal differences in IH and IKir in type I HCs, previously examined in type II HCs (Masetto and Correia, 1997; Levin and Holt, 2012).”
Also, Horwitz et al. (2011) showed HCN channels in utricles are needed for normal balance function, so please include this reference (see line 171).
Done (line 184).
(8) Fig 6A. Shows Kv1.4 staining in rat utricle but procedures for rat experiments are not described. These should be added. Also, indicate striola or extrastriola regions (if known).
We removed KV1.4 immunostaining from the paper, see above.
(9) Table 6, ZD7288 is listed -was this reagent used in experiments to block Gh? If not please omit.
ZD7288 was used to block gH to produce a clean h-infinity curve in Figure 6, which is described in the legend.
(10) In supplementary Fig. 5A make clear if the currents are from XE991 subtraction. Also, is the G-V data for single cell or multiple cells in B? It appears to be from 1 cell but ages P11-505 are given in legend.
The G-V curve in B is from XE991 subtraction, and average parameters in the figure caption are for all the KV1.8–/– striolar type I hair cells where we observed this double Boltzmann tail G-V curve. I added detail to the figure caption to explain this better.
(11) Supplementary Fig. 6A claims a fast activation of inward rectifier K+ channels in type II but not type I cells-not clear what exactly is measured here.
We use “fast inward rectifier” to indicate the inward current that increases within the first 20 ms after hyperpolarization from rest (IKir, characterized in Levin & Holt, 2012) in contrast to HCN channels, which open over ~100 ms. We added panel C to show that the activation of IKir is visible in type II hair cells but not in the knockout type I hair cells that lack gK,L. IKir was a reliable cue to distinguish type I and type II hair cells in the knockout.
For our actual measurements in Fig 6B, we quantified the current flowing after 250 ms at –124 mV because we did not pharmacologically separate IKir and IH.
Could the XE991-sensitive current be activated and contributing?
The XE991-sensitive current could decay (rapidly) at the onset of the hyperpolarizing step, but was not contributing to our measurement of IKir and IH, made after 250 ms at –124 mV, at which point any low-voltage-activated (LVA) outward rectifiers have deactivated. Additionally, the LVA XE991-sensitive currents were rare (only detected in some striolar type I hair cells) and when present did not compete with fast IKir, which is only found in type II hair cells.
Also, did the inward rectifier conductances sustain any outward conductance at more depolarized voltage steps?
For the KV1.8-null mice specifically, we cannot answer the question because we did not use specific blocking agents for inward rectifiers. However, we expect that there would only be sustained outward IR currents at voltages between EK and ~-60 mV: the foot of IKir’s I-V relation according to published data from mouse utricular hair cells – e.g., Holt and Eatock 1995, Rusch and Eatock 1996, Rusch et al. 1998, Horwitz et al., 2011, etc. Thus, any such current would be unlikely to contaminate the residual outward rectifiers in Kv1.8-null animals, which activate positive to ~-60 mV.
(I-HCN is also not a problem, because it could only be outward positive to its reversal potential at ~-40 mV, which is significantly positive to its voltage activation range.)
