Molecular basis of ancestral vertebrate electroreception.

Elasmobranch fishes, including sharks, rays, and skates, use specialized electrosensory organs called ampullae of Lorenzini to detect extremely small changes in environmental electric fields. Electrosensory cells within these ampullae can discriminate and respond to minute changes in environmental voltage gradients through an unknown mechanism. Here we show that the voltage-gated calcium channel CaV1.3 and the big conductance calcium-activated potassium (BK) channel are preferentially expressed by electrosensory cells in little skate (Leucoraja erinacea) and functionally couple to mediate electrosensory cell membrane voltage oscillations, which are important for the detection of specific, weak electrical signals. Both channels exhibit unique properties compared with their mammalian orthologues that support electrosensory functions: structural adaptations in CaV1.3 mediate a low-voltage threshold for activation, and alterations in BK support specifically tuned voltage oscillations. These findings reveal a molecular basis of electroreception and demonstrate how discrete evolutionary changes in ion channel structure facilitate sensory adaptation.

Sharks[1,2], weakly electric fishes[3], amphibians[4], and monotremes[5] can sense incredibly small electrical signals to communicate, detect prey, or navigate through the earth’s electromagnetic field. Transduction of these electrical signals occurs through specialized electrosensory organs that differ in morphology and distribution through vertebrate lineages. Ancient electrosensory systems in elasmobranch fishes[1,2] such as the little skate (Leucoraja erinacea) detect electrical stimuli as small as 5 nV/cm through dermal pores that connect through low-resistance canals to specialized electrosensory cells within Ampullae of Lorenzini[1,2,6] (). Depolarization of electrosensory cells triggers neurotransmitter release onto afferent nerve fibers that project to the central nervous system[6,7,8]. Functional properties of ampullary organs have been described[7-11], but direct recordings from electrosensory cells are limited and biophysical properties of this unique sensory system are not well studied. Here, we identify a CaV1.3 voltage-gated calcium (Ca2+) channel orthologue (sCaV1.3) as the major voltage-gated cation channel in electrosensory cells of the little skate. sCaV1.3 exhibits an unusually low voltage threshold, which is conferred by a positively charged intracellular motif in the α1 subunit. We show that sCaV1.3 works in conjunction with a skate BK channel (sBK) that is molecularly adapted to support specific, behaviorally relevant voltage oscillation frequencies and amplitude[12-14], providing a mechanism for stimulus discrimination. Furthermore, treatment of behaving skates with CaV and BK modulators substantiates roles for these channels in electroreception.

Cation currents in electrosensory cells

We obtained whole-cell patch-clamp recordings from dissociated electrosensory cells () using cesium (Cs+) to block potassium (K+) currents, thereby revealing a low-threshold voltage-activated inward current (). This current (ICav) was blocked by nonspecific CaV pore blockers (), enhanced by the L-type agonist Bay K, and partially inhibited by L-type antagonists (). ICav was not affected by inhibitors of P/Q-, N-, or T-type CaV channels, or by a NaV channel inhibitor (). The conductance-voltage (G-V) relationship was steep with a relatively negative half maximal conductance compared with other CaV channels[15]. Channel inactivation was slow, contributing to a large ‘window current’ representing sustained channel activity within a physiologically relevant voltage range (). Thus, we conclude that ICav is mediated by a low-threshold L-type Ca2+ channel with steep voltage dependence.

Previous electrophysiological recordings from little skate ampullary organs suggest that K+ channels contribute to detection of weak electrical signals and membrane voltage oscillations, which are required for stimulus selectivity[7,8,10]. We measured K+ currents directly using a K+-based intracellular solution, revealing a large outward current in response to voltage pulses () that was blocked by the K+ channel pore blocker TEA+. Furthermore, pharmacological agents that modulated ICav also regulated IK (), suggesting that a Ca2+-activated K+ channel mediates IK. Indeed, IK was blocked by selective inhibitors of BK channels, which are Ca2+-activated ().

Cav and BK in electrosensory cells

To identify ion channel subtypes mediating ICav and IK, we transcriptionally profiled little skate ampullary organs. The orthologue of cacna1d, which encodes the α1 subunit of CaV1.3, was the predominant Ca2+ channel subtype expressed in ampullae and greatly enriched (>90-fold) compared to other tissues examined (). Several CaV auxiliary subunits were also expressed (). Interestingly, mammalian CaV1.3 has a relatively low voltage threshold compared to other L-type CaV channels, and plays a critical role in auditory hair cells, which are related to electrosensory cells[16-20]. We also examined expression profiles of pore-forming α subunits of K+ channels and, consistent with our functional data, found that kcnma1 (α subunit of BK) is the most abundant K+ channel in ampullary organs, expressed at levels substantially higher (>35-fold) than other Ca2+-activated K+ channels (). At the cellular level, both CaV1.3 and BK transcripts were robustly expressed in ampullary receptor cells and absent in supporting cells and tubule structures (). Expression of other CaV and Ca2+-activated K+ channels was at or below the level of detection, but it remains possible that currents in electrosensory cells are not carried exclusively by CaV1.3 and BK.

sCav has low voltage-activation threshold

The pore-forming subunit of sCaV1.3 is 78% identical to the well-characterized long isoform of rat CaV1.3 (rCaV1.3), and heterologous expression of sCaV1.3 produced voltage-gated currents with ion sensitivity and pharmacological profiles resembling those of rCaV1.3 or native electrosensory cell ICav (). However, like native ICav, the voltage threshold of sCaV1.3 was significantly decreased compared to rCaV1.3. Currents produced by sCaV1.3 were activated at more negative potentials and increased steeply to maximal amplitude with increasing voltage (). While inactivation was similar between sCaV1.3 and rCaV1.3, the G-V curve was significantly shifted in the negative direction for sCaV1.3, contributing to a substantially larger window current for the skate channel (). sCaV1.3 also exhibited reduced Ca2+-dependent inactivation compared to rCaV1.3 (). These functional properties match those of native ICav, suggesting that sCaV1.3 forms the predominant voltage-gated Ca2+ channel in electrosensory cells.

What accounts for the decreased voltage threshold of sCaV1.3? Measuring ionic and gating currents from the same cells allowed us to examine the relationship between relative conductance and voltage sensor movement (represented by ON gating charge, QON) for skate versus rat orthologues. Both gating current kinetics and QON-voltage relationships were similar (); however, the G-V relationship was shifted to more negative voltages, and the QON-G relationship was extremely steep for sCaV1.3 compared with rCaV1.3 (), suggesting that only minimal voltage sensor movement is required to elicit maximal channel opening for the skate channel (). As another index of coupling efficiency, we measured maximal QON in the absence of pore blockers by applying voltage pulses to the channel’s reversal potential (EREV) and then stepping to −100mV to induce large tail currents (Itail)[21]. Similar QON induced significantly larger Itail for sCaV1.3 compared with rCaV1.3 (, suggesting that sCaV1.3 exhibits greater channel open probability or open-state stability in response to equal voltage sensor movement. Collectively, these data indicate that the low voltage threshold of the skate channel originates from increased coupling between voltage sensors and channel opening.

Alignment of the α1 subunit of sCaV1.3 with human, rat, and zebrafish orthologues revealed a skate-specific insertion that introduces four positively charged residues (KKKER) into an intracellular loop of domain IV (DIVS2-S3) (). Remarkably, a charge-neutralized mutant (neutral-sCaV1.3; ) required significantly greater depolarization for maximal activation and exhibited decreased current density compared with WT-sCaV1.3 (). Gating current properties were not affected by charge neutralization (), but consistent with increased voltage threshold of neutral-sCaV1.3, more relative QON was required for maximal conductance compared with WT-sCaV1.3 (). Furthermore, maximal QON elicited by voltage pulses to EREV resulted in decreased Itail amplitude in neutral-sCaV1.3 versus WT-sCaV1.3 (). These results suggest that coupling between voltage sensor movement and channel opening is decreased in neutral-sCaV1.3 and that the low voltage threshold of sCaV1.3 is determined by the charged insertion in DIVS2-S3. Indeed, the charged motif from sCav1.3 (but not a neutralized control) was sufficient to confer skate-like voltage sensitivity to rCaV1.3,(). Gating current properties of ‘charged-rCaV1.3’ and ‘neutral-rCaV1.3’ were similar (), but comparatively less relative QON was required for maximal conductance of charged-rCaV1.3 and maximal QON elicited larger Itail (), indicating enhanced coupling between voltage sensor movement and pore opening.

According to recent structural models of a related mammalian CaV[22], the charged skate motif within the intracellular loop of DIVS2-S3 could be relatively close to the bottom of the charged voltage sensor (DIVS4) such that electrostatic interactions could repel DIVS4 into a partially activated or primed state to decrease voltage threshold. To test this hypothesis, we examined voltage-dependent channel activation kinetics and found that activation occurred more rapidly in cells expressing charged-rCaV1.3 compared with neutral-rCaV1.3 or WT-rCaV1.3 (). If charge interactions between the skate motif and DIVS4 position the voltage sensor in a primed state, then extremely negative voltages might force the voltage sensor into a resting state, resulting in activation kinetics similar to WT channels. Indeed, following a long negative prepulse (1 s, −170 mV), activation kinetics for charged-rCaV1.3, neutral-rCaV1.3 and WT-rCaV1.3 were identical. As we increased the prepulse voltage to more positive values, the charged-rCaV1.3 activation rate increased, while neutral-rCaV1.3 and WT-rCaV1.3 rates did not change (). These Cole-Moore shifts[23] demonstrate that an additional voltage-dependent step in channel activation occurs at very negative potentials in the presence of the charged skate motif, supporting our hypothesis that charge repulsion regulates the domain IV voltage sensor to decrease voltage threshold and enhance open-state stability at physiological membrane potentials (). Gating currents measure the movement of all voltage sensors (domains I–IV) irrespective of heterogeneity, thus a small difference, such as a partially activated voltage sensor, could be missed. While Cole-Moore effects support our model, further structural insights are required to confirm this hypothesis.

sBK has small conductance

We wondered if skate BK (sBK) is also specially adapted for electrosensation. Single-channel recordings from HEK293 cells expressing the α subunit (kcnma1) of skate or mouse BK showed that sBK had drastically reduced current amplitude at all voltages compared with mBK, resulting in a markedly decreased slope conductance (). Both channels were similarly sensitive to intracellular Ca2+ (), but sBK single-channel currents were of smaller amplitude and had shorter open-state dwell time () such that sBK passes significantly less current than mBK.

Considering its unique conductance profile, we aligned the pore region of sBK with that of mouse, rat, human, and zebrafish orthologues to reveal high conservation (~87% identical to mBK), with a few notable alterations within an intracellular region near the pore that affects channel conductance through electrostatic interactions with K+ ()[24-28]. To determine if the altered amino acids affect sBK properties, we converted arginine and/or alanine of sBK to match cognate residues of mBK. sR340S significantly affected both conductance and open-state dwell time, while the effect of sA347E was less pronounced (). Remarkably, substitution of both amino acids (sBK-SE) produced a single-channel conductance nearly identical to that of mBK, with open-dwell time akin to the mouse channel (). Conversely, these two amino acids from sBK were sufficient to convert mBK conductance and open-time to that of sBK (mBK-RA, ).

We next asked if altered K+ concentration near the pore accounts for the reduced conductance of sBK. When patches expressing wild-type and mutant BK channels were exposed to various concentrations of intracellular K+ (140mM, 640mM, or saturating 3.14M), single-channel amplitude increased for all BK channels with increasing K+ concentration, and sBK and mBK-RA exhibited the smallest current amplitude at 140mM and 640mM (). Current amplitude was the same for all channels when exposed to a saturating K+ concentration of (3.14M), indicating that the pore is intrinsically capable of passing the same current (). Notably, in the presence of 640mM, sBK channels passed nearly as much current as sBK-SE or WT mBK in 140 mM (). Thus, adaptations in sBK alter intracellular electrostatics near the pore to decrease the apparent conductance by reducing local K+ concentration by >500mM.

Voltage oscillations in electroreception

Membrane voltage (Vm) oscillations, previously described in ampullary epithelial current-clamp experiments, control neurotransmitter release from electrosensory cells onto postsynaptic nerve fibers[7,8,29]. Under our conditions, electrosensory cells had a resting Vm of ~-55 mV and exhibited small, low frequency voltage oscillations. Injecting current to bring the Vm to various potentials modulated oscillatory behavior (). Because oscillations occur over voltages where sCaV1.3-mediated ICav is activated, we plotted oscillation amplitude versus membrane voltage and overlaid the normalized window current of ICav (. Interestingly, average oscillation amplitude increased with window current, suggesting that tonic ICav activity underlies the depolarization phase of electrosensory cell Vm oscillations. In the presence of TEA+, current injection elicited prolonged depolarization (), suggesting that sBK-mediated IK contributes to Vm oscillations, potentially by restoring cells to a hyperpolarized state after the initial depolarization. Furthermore, spontaneous oscillatory behavior was significantly reduced by TEA+ or nifedipine (). Taken together, our data suggest that ICav and IK couple to mediate Vm oscillations.

How might sBK properties affect functional coupling of the two channels? As expected from reduced sBK conductance and open time, intracellular Ca2+ elicited smaller whole-cell currents from HEK293 cells expressing sBK compared to sBK-SE or mBK (). Voltage pulses in cells coexpressing CaV1.3 and sBK also elicited smaller K+ currents and decreased K+ permeability compared with sBK-SE or mBK. Thus, sBK allows for relatively more CaV-mediated Ca2+ current, while sBK-SE- and mBK-mediated K+ currents quickly occlude measurable Ca2+ current ().

To determine if sBK-specific properties are important in native electrosensory cells, we used the selective BK agonist NS11021 (NS)[30] to pharmacologically increase Po and open-state dwell time, producing a BK channel that more closely resembles mBK (). In recordings from cells coexpressing CaV1.3 and sBK, or from native electrosensory cells, NS increased outward current amplitude and shifted reversal potentials in the negative direction, indicating increased BK activity and K+ permeability (). In current-clamp experiments from electrosensory cells, treatment with NS dramatically reduced voltage oscillation amplitude and increased frequency (). The addition of iberiotoxin blocked oscillations, consistent with a requirement for BK channels in spontaneous electrosensory cell Vm oscillations (). Thus, evolutionary tuning of BK decreases conductance and its activity controls Vm oscillation amplitude and frequency. We hypothesize that because CaV-mediated Ca2+ influx immediately activates a BK current to limit the CaV-mediated depolarization, a smaller BK current will more slowly return Vm to rest, thus supporting large amplitude, low frequency oscillation events.

Electrosensory cells likely contain a mechanism to dampen BK-mediated hyperpolarization, thus maintaining a membrane voltage where CaV could initiate another oscillation event. Indeed, the highest expressed transcript in ampullary organs is parvalbumin 8, a Ca2+-binding protein implicated in Vm oscillations[31] that could chelate CaV-mediated Ca2+ influx to produce only brief BK activation (). Consistent with this hypothesis, a plasma membrane Ca2+-ATPase is also highly enriched in ampullary organs, presumably to support persistent oscillations ().

To examine the relative contributions of sCaV1.3 and sBK at an organismal level, we preincubated behaving skates with vehicle or with nifedipine to inhibit Cav1.3, NS to stimulate BK, or mibefradil, a T-type CaV antagonist that does not affect ICav (). We then asked if they favored a zone in which a submerged dipole electrical stimulus was buried under a sand-covered surface ( For each treatment condition, a startle response was subsequently measured to confirm that the drug did not generally affect mobility (). While both untreated and mibefradil-treated skates spent a majority of their time in the vicinity of the hidden electrical stimulus, skates treated with nifedipine or NS spent significantly less time near the active electrode (. These results are consistent with the notion that CaV1.3 and low-level BK activity are important for electroreception-related behaviors.

Discussion

Electroreception is an ancient sensory modality that has independently evolved multiple times to facilitate the detection of environmental electrical signals for predation, navigation, or communication[6]. Electrosensory systems in elasmobranch fishes are among the most sensitive, and we have therefore exploited this system to gain molecular insights into mechanisms underlying this unique sensory modality. Our results demonstrate that low-threshold sCaV1.3 couples to sBK to produce electrosensory cell Vm oscillations. This is reminiscent of electrical resonance in evolutionarily-related auditory hair cells, which also contain ribbon synapses and use CaV and BK orthologues to produce Vm oscillations that regulate vesicle release dynamics, allowing for the coding of stimulus strength and frequency[32-37]. In some animals, auditory hair cell electrical resonance tuning similarly contributes to frequency selectivity for incoming auditory signals[38,39]. Considering the simplicity and flexibility of this tuning mechanism, physiological state-dependent posttranslational modifications in electrosensory cell transducers may provide a means to tune Vm oscillations for selective electrical frequency detection of salient signals from the environment according to developmental maturation, reproductive state, or nutritional condition. Oscillating tuberous electrosensory organs in weakly electric fishes (e.g., Gymnotiformes and Mormyriformes) are functionally tuned to electromagnetic fields related to self-generated electric organ discharges[6]; whether these systems use similar molecular mechanisms remains to be determined.

Methods

Animals and cells

Male and female little skates (Leucoraja erinacea) were provided by the Marine Biological Laboratory (Woods Hole, MA) and their use was approved by the UCSF Animal Care and Use Committee. Animals used for cellular physiology experiments were euthanized with tricaine methanesulfonate (MS222, 1g/L). Hyoid capsules were removed on ice, and individual ampullae were dissected by cutting the canals and afferent nerve fibers. Ampullae were treated with papain for 2-3 mins and then electrosensory cells were mechanically dissociated over the recording chamber. Isolated electrosensory cells were identified by the presence of their large single kinocilium. HEK293T cells (ATCC) were grown in DMEM, 10% fetal calf serum, and 1% penicillin/streptomycin at 37°C, 5% CO2. Cells were transfected using Lipofectamine 2000 (Invitrogen/Life Technologies) according to manufacturer’s protocol. 1 μg of skate or rat cacna1d was co-expressed with 1 μg rat cacnb3, 1 μg rat cacna2d1, and 0.3 μg GFP. Mock transfection experiments (1 μg rat cacnb3, 1 μg rat cacna2d1, and 0.3 μg GFP, but no cacna1d) were performed as controls, in which no voltage-activated inward currents were observed. For BK experiments, 1 μg of skate or mouse kcnma1 was co-expressed with 0.3 μg GFP. Mock transfection experiments with 0.3 μg GFP were performed as controls. To enhance expression of wild-type and charge-neutralized skate CaV1.3, cells were transfected for 6 – 8 hrs and then incubated at 28°C for 3 – 4 days, plated on poly-L-lysine-coated coverslips, incubated for an additional 3 – 4 days at 28°C, and then used for experiments[40].

Molecular biology

Cacna1d and kcnma1 from little skate ampullary organs were synthesized by Genscript (Piscataway, NJ). Rat cacna1d, cacnb3, and cacna2d1 were gifts from Diane Lipscombe (Addgene plasmids 49332, 26574, 26575) and mouse kcnma1 was from Larry Salkoff (Addgene plasmid 16195). Cacna1d mutagenesis was carried out and verified by Genscript (Piscataway, NJ). BK point mutations were induced using QuikChange Lightning site-directed mutagenesis kit (Agilent Genomics).

Electrophysiology

Recordings were carried out at room temperature using a MultiClamp 700B amplifier (Axon Instruments) and digitized using a Digidata 1322A (Axon Instruments) interface and pClamp software (Axon Instruments). Whole-cell recording data were filtered at 1 kHz and sampled at 10 kHz. Data were leak subtracted online using a P/4 protocol, and membrane potentials were corrected for liquid junction potentials. Single-channel data were filtered at 5 kHz and sampled at 50 kHz. Electrosensory cell recordings were made using borosilicate glass pipettes polished to 8 – 10 MΩ. The extracellular solution was a modified “elasmobranch Ringer’s solution” containing (mM): 250 NaCl, 6 KCl, 4 CaCl2, 1 MgCl2, 10 glucose, 5 HEPES, 360 urea, pH 7.6. Two intracellular solutions were used: for recording ICav (mM): 250 CsMeSO4, 1 MgCl2, 11 Cs-EGTA, 10 HEPES, 30 sucrose, 360 urea, pH 7.6. For recording IK or membrane potential (mM): 250 K-gluconate, 1 MgCl2, 11 K-EGTA, 10 HEPES, 30 sucrose, 360 urea, pH 7.6. For heterologous expression experiments in HEK293, recordings were made using pipettes polished to 3 – 4 MΩ. For CaV1.3 recordings, Intracellular solution contained (mM): 150 NMDGMeSO4, 1 MgCl2, 10 Cs-EGTA, 10 HEPES, 10 sucrose, pH 7.3. Extracellular solution for measuring ionic current contained (mM): 150 choline chloride, 5 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.3. For measuring gating currents, CaCl2 was replaced with MgCl2 and pore blockers (500 μM Cd2+ and 200 μM La3+) were added to the extracellular solution. During ion substitution experiments, Ca2+ was substituted for an equal concentration of Ba2+ or Sr2+. For BK single-channel recordings, intracellular solution contained (mM): 136 K-gluc, 4 KCl, 1 K-EGTA, 1 HEDTA, 10 HEPES, 10 glucose, pH 7.3. Extracellular solution contained (mM): 136 K-gluc, 4 KCl, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.3. Heterologous BK whole-cell recordings used an intracellular solution containing (mM): 140 K-gluc, 1 MgCl2, 0.1 K-EGTA, 10 HEPES, 10 sucrose, pH 7.2. Extracellular solution contained (mM): 140 NaCl, 5 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 10 glucose, pH 7.4. Calculated concentrations of buffered Ca2+ added to intracellular solution were made using MaxChelator (C. Patton, Stanford University).

The pharmacological inhibitors or agonists Bay K (Tocris), nifedipine (Tocris), nimodipine (Tocris), ω-agatoxin (Tocris), ω-conotoxin (Tocris), TTX (Tocris), charybdotoxin (Alamone Labs), iberiotoxin (Alamone Labs), and NS11021 (Tocris) were dissolved in <1% vehicle (DMSO or water), which was used for a control. Ionic pore blockers stocks were made in standard extracellular solution and diluted before use. Unless stated otherwise, the following concentrations were used: 2 mM Co2+, 100 μM Cd2+, 1 μM Bay K, 10 μM nifedipine, 10 μM nimodipine, 300 nM ω-agatoxin, 1 μM ω-conotoxin, 5 μM mibefradil, 50 μM nickel (low concentration to block T-type CaV), 1 μM tetrodotoxin, 1 μM charybdotoxin, 100 nM iberiotoxin, 10 mM TEA+ 10 μM NS11021. Pharmacological effects were quantified by differences in normalized current from the same cell following bath application of the drug (Itreatment / Icontrol).

Unless stated otherwise, currents were measured in response to 200 ms voltage pulses in 10 mV increments from a −115 mV holding potential. G-V relationships were derived from I-V curves by calculating G: G = ICa / (Vm-Erev) and fit with a Boltzman equation. Voltage-dependent inactivation was measured during −20 mV voltage pulses following a series of 1 s prepulses ranging from −115 to 65 mV in 10 mV increments. Voltage-dependent inactivation was quantified as I / Imax, with Imax occurring at the voltage pulse following a −115 mV prepulse. QON represents the integral of nonlinear ON-gating current measured during voltage pulses from a holding potential of −110 mV. QON was only quantified from cells with no ionic current. QON – Itail relationships were examined by applying short pulses to predetermined Erev for each cell from −100 mV and stepping back to −100 mV to induce large Itail.

Transcriptome sequencing and analysis

Poly A+ RNA was extracted from the ampullae, ampullary tubules/canals, non-electroreceptor covered skin, and liver of an adult L. erinacea then was reverse transcribed using the SuperScript III kit (Invitrogen/Life Sciences). Sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit according to the manufacturer’s instructions. Libraries were sequenced on the Illumina Hi-Seq 4000 (V. C. Genomics Sequencing Lab, University of California, Berkeley) using 150 cycles of paired end reads, producing 20 to 30 million inserts for each sample.

Transcriptomes for each sample were assembled de novo using the Trinity suite (version 2.1.0). Sequences were aligned to the zebrafish protein database (NCBI assembly GRCz10) using the blastx tool from NCBI blast (version 2.2.31) using a maximum E value of 1 ×10−5. Reciprocal blastx alignments (using zebrafish protein sequences that aligned to L. erinacea sequences) were performed to the human protein database. Estimates of relative abundance for differential expression comparisons were performed using the RSEM software package within Trinity. These values are reported as fragments per kilobase of exon per million fragments mapped (FPKM).

Whole mount preparations

L. erinacea embryos were removed from egg cases, euthanized with an overdose of MS-222 in artificial seawater, and fixed in 4% paraformaldehyde for at least 24 hours. The cartilage matrix and electroreceptor tubules were stained using Alcian Blue (20 mg Alcian Blue 8GX in 30 mL glacial acetic acid and 70 mL 100% ethanol) following previously published methods[41].

In situ hybridization histochemistry

Adult skates were euthanized with an overdose of MS-222 in artificial seawater and trans-cardially perfused with PBS followed by 4% PFA. The hyoid capsule, which contained the aveolae of the ampullary organs, was dissected and cryo-protected in 30% sucrose in PBS overnight. Cryostat sections (15 μm thick) were probed with digoxigenin-labeled cRNA for skate CaV1.3 and fluorescein-labeled cRNA for skate BK receptors. Probes were generated by T7/T3 in vitro transcription reactions using a 510 nucleotide fragment of CaV1.3 cDNA (nucleotides 4501 to 5011) and a 510 nucleotide fragment of BK cDNA (nucleotides 2934 to 3444). Hybridization was developed using anti-digoxigenin and anti-fluorescein Fab fragments, followed by incubation with Fast Red and streptavidin conjugated Dylight 488 (to probe for BK) according to published methods[42]. Following hybridization and detection, sections were coverslipped and co-stained with DAPI as a nuclear marker (Prolong Gold Antifade Mountant with DAPI; Invitrogen).

Behavioral analysis

In an isolated location and under normal lighting conditions, juvenile skates were placed in 250 mL of seawater or seawater with 5 μM nifedipine, 10 μM NS −11021, or 5 μM mibefradil for 30 minutes. Following incubation, skates were allowed to habituate for 10 minutes in an acrylic cylindrical tank (diameter = 28 cm) and were surrounded by a barrier blocking external visual cues. A DC dipole stimulus (18 μA over 5 mm), generated by threading positive and negative ends of tin-plated copper wire (300 VH, 22 gauge, NTE Electronics, Inc.) into seawater filled Tygon tubing, was randomly positioned and obscured by the sand substrate in one of four circles (diameter = 5.5 cm), all equally spaced from the center of the tank (see Extended Data Fig. 10A). All skates were exposed to a plume of Mysis shrimp odorant originating in the center of arena in order to elicit predatory/feeding behavior. A digital video camera (Sony Handicam) positioned above the tank was used to record skate activity for 30 minutes. Trials in which the skate executed >3 large movements and remained visible above the sand substrate for the majority of the time were quantified. Time spent with the majority of the pectoral disc within the outlined circle containing the electrical stimulus was compared to time spent in all other outlined circular areas. Following 30 minutes of undisturbed observation, tactile startle responses were observed from skates in response to gentle taps of the lateral pectoral fins to verify normal movement capabilities. Startle responses were quantified as the distance moved following a straight line from the dorsal side center between the eyes in still frames captured before and after the elicited startle response.

Extended Data Figure 10

Behavioral paradigm for pharmacologically-treated skates and startle response-related control

a. Schematic drawing of electrical stimulus. A 9V battery was used to generate a dipole DC stimulus through two independent leads placed into Tygon rubber tubing filled with seawater (left). The ends of these tubes were threaded through an acrylic plate to 4 different equally spaced locations on the base of the behavioral observation tank which were then obscured by sand (right).

b. Following 30 minutes of free exploration, control and pharmacologically-treated skates were gently tapped upon the pectoral fin. The average distance moved during the startle response is represented as mean ± sem; n=10. Differences were not significant according to a two-way ANOVA with post-hoc Tukey’s test.

c. Schematic drawing traced from typical example of skate startle response following pectoral fin stimulation (red arrow). The distance covered during the startle response was measured from the initial location (left) to the final location where the body axis became straight again (right), and the distance from the center between the eyes from each respective position was recorded (dotted yellow line).

Statistical analysis

Data were analyzed with Clampfit (Axon Instruments) or Prism (Graphpad). Data are represented as mean ± sem and n represents the number of cells. Data were considered significant if p < 0.05 using paired or unpaired two-tailed Student’s t-tests or one- or two-way ANOVAs. All significance tests were justified considering the experimental design and we assumed normal distribution and variance, as is common for similar experiments. Sample sizes were chosen based on the number of independent experiments required for statistical significance and technical feasibility.

Data Availability Statement

Deep sequencing data that support the findings of this study have been archived in the Gene Expression Omnibus (GEO) database repository with accession code GSE93582. GenBank accession numbers for skate CaV1.3 α subunit and skate BK α subunit are KY355736 and KY355737, respectively.

CaV and K+ channel expression in little skate

a. CaV auxiliary subunit mRNA expression in skate ampullary organs, ampullary canals, skin, and liver. Bars represent fragments per kilobase of exon per million fragments mapped (FPKM).

b. Ten most highly expressed K+ channel α subunit transcripts in ampullary organs.

Skate CaV ion selectivity and Ca2+-dependent inactivation

a – c. Representative currents measured from electrosensory cells (native ICaV, top), HEK293 expressing skate CaV1.3 (sCaV, middle), or HEK293 expressing rat CaV1.3 (rCaV, bottom) in the presence of 5 mM extracellular Ca2+, Ba2+, or Sr2+. At the end of a 200 ms voltage pulse eliciting maximal current, approximately 50% of current remained in native electrosensory cell ICav or HEK293 cells heterologously expressing sCaV1.3, whereas rCaV1.3 had only ~20% current remaining. In electrosensory cells, heterologous sCaV1.3, or rCaV1.3, the percentage of remaining current was significantly increased by substituting extracellular Ca2+ for Ba2+ or Sr2+ (p < 0.05, one-way ANOVA with post-hoc Bonferroni test). Data represented as mean relative current remaining at the end of the 200 ms voltage pulses that elicited maximal currents (± sem, n = 5 per condition).

Skate CaV pharmacology

a – b. Pharmacology of skate CaV1.3 (sCav). Representative currents recorded in responses to voltage pulses in the presence of vehicle (control, <0.1% DMSO) or 10 μM nifedipine or nimodipine. Currents were incompletely inhibited similar to native electrosensory cell ICav (). Dose response relationships of current amplitudes measured at voltages that elicited maximal currents. Data are represented as mean ± sem, n = 6 per treatment.

c – d. Pharmacology of rat CaV1.3 (rCaV). Representative currents in the presence of vehicle or 10 μM nifedipine or nimodipine and associated dose-response relationships. n = 6 per treatment.

Skate CaV gating current properties

a – c. Gating current properties including peak amplitude (peak I), time-to-peak (TTP), exponential decay time constant (τ decay), peak width at 50% of maximal gating current (width) for skate CaV1.3 (sCaV) versus rat CaV1.3 (rCaV, a, top), wild-type skate CaV1.3 (WT) versus charge-neutralized skate CaV1.3 (neutral, b, middle), and rat CaV1.3 with charged skate motif (charged) versus rat CaV1.3 with neutralized skate motif (neutral, c, bottom). All values were similar except for peak I for sCaV versus rCaV, likely representing increased expression for rCaV compared with sCaV. Data are presented as mean ± sem, n listed above bars.

d. Wild-type skate CaV1.3 (sCaV, blue, n = 7) and wild-type rat CaV (rCaV, red, n = 8) relative conductance (G)-voltage (V) and ON-gating charge movement (QON)-V relationships. Data represented as mean ± sem.

e. G-V and QON-V relationships for wild-type sCaV1.3 (WT, blue) and charge-neutralized sCaV1.3 (neutral, red) relative conductance (G)-voltage (V) and ON-gating charge movement (QON)-V relationships. Data represented as mean ± sem, n = 7 per condition.

f. G-V and QON-V relationships for rCaV1.3 with charged skate motif (charged, blue) and rCaV1.3 with neutral skate motif (neutral, red). Data represented as mean ± sem, n = 8 per condition.

Charged skate motif modulates voltage-dependent activation kinetics

a. Activation kinetics were faster in charged-rCaV (blue, n = 6) compared with wild-type rCaV1.3 (WT-rCaV, grey, n = 7) or neutral-rCaV (red, n = 8). Data represent mean ± sem, p < 0.05 at all voltages for charged-rCaV versus WT-rCaV1.3 or neutral-rCaV, two-way ANOVA with post-hoc Bonferroni test.

b. Representative currents recorded in response to 1 s voltage pulses between −170 and −90 followed by a pulse to −10 mV for 20 ms. Cole-Moore effects, indicated by increased current activation rate at −90 mV (purple) versus −170 (green), were observed in currents recorded from charged-rCaV, but not in neutral-rCaV motif. Scale bar: 50 pA, 10 ms.

c. Cole-Moore effects quantified as the time to reach half maximal current (t1/2). With increasing voltage during prepulses, charged-rCaV (blue, n = 9) reached maximal current amplitude faster while WT-rCaV (grey, n = 6) and neutral-rCaV (red, n = 8) were unchanged. All data represented as mean ± sem, n ≥ 7, p < 0.05 for charged-rCaV t1/2 comparing −170 with −130, −110, or −90 mV, two-way ANOVA with post-hoc Bonferroni test).

d. Hypothetical model depicting the intracellular charged motif in the domain IV voltage sensor of sCaV1.3 destabilizing the inactive state of the channel by electrostatic repulsion, pushing it into a partially activated or primed state (gold oval) prior to full activation (green ovals). Because sCaV1.3 is primed for activation, channel activation requires a smaller increase in voltage compared with rCaV1.3.

Skate BK properties

a. Currents measured in response to 0, 1, or 10 μM intracellular Ca2+ at 80 mV from patches expressing sBK or mBK. Scale bar: 10pA, 50ms. Average open probability (Po) for sBK compared with mBK was similar for all concentrations tested. Data represented as mean ± sem, n = 5.

b. Representative single-channel records at various voltages from patches expressing indicated BK channels. Scale bar: 25pA, 20ms.

c. Representative currents recorded at 80 mV from patches expressing indicated BK channels. The same patch was exposed to local K+ concentrations of 140 mM, 640 mM, or 3.14 M. Dashed lines indicate single-channel current amplitude for sBK at 140 mM (green), 640 mM (orange), or 3.14 M (red). Scale bar: 50pA, 20ms.

Adaptations in skate BK promote increased relative ICaV current during channel coupling

a. Whole-cell currents in response to 200 ms voltage pulses from −80mV to +80mV from HEK293 expressing sBK, sBK-SE, or mBK in the presence of 0 or 20 μM intracellular Ca2+. Scale bar: 5nA, 50ms

b. Average I-V relationships for sBK (blue), sBK-SE (green) or mBK (red) in the presence of 0 or 20 μM intracellular Ca2+. n = 7.

c. Whole-cell currents from HEK293 expressing charged-rCaV1.3 coexpressed with sBK, sBK-SE, or mBK. Scale bar: 500pA, 50ms. t = transient current evoked by voltage pulse, s = sustained current. In the presence of CaV1.3, average transient and sustained current-voltage relationships showed a negative shifted reversal potential (EREV) for sBK-SE (green) or mBK (red) compared with sBK (blue), indicating increased relative K+ permeability.

d. Reversal potentials for transient and sustained currents evoked in cells coexpressing charged-rCaV1.3 and BK were affected by BK identity. Inset: transient currents mediated by coupling of CaV1.3 and BK (scale bar: 100pA, 5ms). Transient EREV: sBK = 32.96 ± 2.17, mBK = 8.43 ± 2.76, sBK-SE = 3.42 ± 2.38, p < 0.0001 for sBK versus mBK or sBK-SE. Sustained EREV: sBK = −17.00 ± 2.48, mBK = −50.95 ± 4.16, sBK-SE = −45.13 ± 4.59, p < 0.0001. n = 10. All data represented as mean ± sem and p values from two-tailed Student’s t-test.

BK agonist NS11021 modulates skate BK channels

a. In representative records from outside-out patches expressing sBK the BK agonist NS11021 (NS, 10 μM) increased the Po and open-state dwell time of sBK channels and this effect was blocked by iberotoxin (IbTx, 100 nM). Scale bar: 5pA, 100ms. Associated all-points histograms demonstrate the increase in open time. Po: basal = 0.0024 ± 0.00068, NS: 0.16 ± 0.041, NS + IbTx = 0.00036 ± 0.00025, p < 0.0001 for NS versus basal or NS + IbTx. Open dwell time: 0.62 ± 0.32, NS: 4.59 ± 0.34, NS + IbTx = 0.30 ± 0.010, p < 0.0001. n = 5.

b. Whole-cell currents and average transient and sustained current-voltage relationships from HEK293 expressing charged-rCaV1.3 and sBK (scale bar: 500pA, 50ms). Transient and sustained current-voltage relationships made from normalizing currents in the presence of NS to basal currents show an increase in CaV1.3-activated sBK current amplitude and negative-shifted EREV in response to 10 μM NS. Transient EREV: basal = 20.71 ± 3.46, +NS = −0.72 ± 0.94, p < 0.01. Sustained EREV: basal = −24.62 ± 0.61, NS = −47.21 ± 5.37, p < 0.05. n = 5.

c. Representative currents recorded from an electrosensory cell show that 10 μM NS increases ICav-activated IK amplitude resulting in a decrease in relative ICav current (scale bars: 100pA, 50ms).

d. Transient and sustained current-voltage relationships from normalizing currents in the presence of NS to basal currents. I-V relationships demonstrate an NS-mediated negative shift in EREV, indicating increased K+ permeability. Transient EREV: basal = −6.15 ± 5.95, +NS = −24.9 ± 8.23, p < 0.01. Sustained EREV: basal = −7.59 ± 6.02, NS = −26.65 ± 1.06, p < 0.05. n = 4. All data represented as mean ± sem and p values from two-tailed Student’s t-test.

Ca2+-handling proteins are enriched in Ampullae of Lorenzini

a. Top 4 highest expressed transcripts in ampullae. The Ca2+-binding protein (CBP) parvalbumin 8 is the highest expressed and is enriched in ampullae compared with other examined tissues. Bars represent fragments per kilobase of exon per million fragments mapped (FPKM).

b. Top 4 highest expressed ATPase transcripts in ampullae. Notably, the plasma membrane Ca2+ ATPase 1a is highly expressed and is enriched in ampullae.

c. Proposed mechanism for electrosensory cell Vm oscillations. sCaV1.3 is activated by low threshold electrical signals to depolarize the cell and mediate Ca2+ influx. Ca2+ stimulates sBK-mediated K+ current to hyperpolarize the cell. Ca2+-binding proteins (CBP) bind incoming Ca2+ to inhibit BK-mediated hyperpolarization and continue sCaV1.3-driven oscillations.