Molecular basis of ancestral vertebrate electroreception

Journal name:
Nature
Volume:
543,
Pages:
391–396
Date published:
DOI:
doi:10.1038/nature21401
Received
Accepted
Published online
Corrected online

Abstract

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.

At a glance

Figures

  1. CaV1.3 and BK channels mediate the major cation currents in electrosensory cells.
    Figure 1: CaV1.3 and BK channels mediate the major cation currents in electrosensory cells.

    a, Dorsal profile of little skate (Leucoraja erinacea). b, Alcian blue-stained ampullary organ canals on the ventral surface of a juvenile skate. c, Isolated ampullary organs with short lengths of canal and attached afferent nerve fibres (scale bar, 400 μm). d, Electrosensory cells in a representative patch-clamp experiment (scale bar, 10 μm). e, Left, representative ICav traces: green and blue traces show current elicited by −55 and −45 mV pulses, respectively. Right, average current–voltage (IV) relationship (n = 11). Scale bars, 20 pA pF−1 vertical, 100 ms horizontal. f, Pharmacological profile of ICav. Channel subtype drug selectivity is indicated above bars. Each circle depicts one experiment; bars represent mean ± s.e.m. measured at peak amplitude; P < 0.05 for L-type channel modulators, one-way ANOVA with post hoc Bonferroni test. g, Conductance–voltage (GV) relationship with half-maximal activation voltage (Va1/2) of −52 ± 0.8 mV with slope factor (Ka) = 4.8 ± 0.6 mV. Inactivation–voltage relationship with half-inactivation potential (Vh1/2) of −55.9 ± 1.7 mV with slope factor (Ki) = −6.3 ± 1.6 mV. Window current was observed between −70 and −30 mV and peaked at about −58 mV with around 40% maximal conductance. Data represented as mean ± s.e.m., n = 10. h, Representative K+ currents. Scale bars, 20 pA pF−1, 100 ms. i, Pharmacological profile of IK. Each circle depicts one experiment and bars represent mean ± s.e.m. measured at peak amplitude, P < 0.001 for control versus treatments, one-way ANOVA with post hoc Bonferroni test. j, Left, Cav α-subunit mRNA expression in skate ampullary organs, ampullary canals, skin, and liver. Right, expression of K+ channel α-subunits in ampullary organs. Bars represent fragments per kilobase of exon per million fragments mapped (FPKM). k, Co-localization of CaV1.3 (red) and BK (green) transcripts within electrosensory cells of ampullary organs visualized by in situ hybridization histochemistry. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

  2. Skate CaV has a low voltage threshold.
    Figure 2: Skate CaV has a low voltage threshold.

    a, Representative voltage-activated currents recorded in HEK293 cells expressing skate CaV1.3 (sCaV, blue) or the homologous long isoform of rat CaV1.3 (rCaV, red). Scale bars, 200 pA, 50 ms. b, Normalized IV relationship of sCaV (blue) and rCaV (red); n = 7. c, sCaV (blue) and rCaV (red) GV (n = 8) and inactivation (n = 7) curves. d, Average Va1/2 for sCaV (−42.68 ± 0.56, n = 8) compared with rCaV (−18.16 ± 0.51, n = 7, P < 0.0001). Vh1/2 was similar, n = 7. e, Ionic (left) and gating (right) currents from representative cells expressing sCaV or rCaV. Scale bars, 100 pA, 50 ms for sCaV; 200 pA, 50 ms for rCaV. Inset: enlarged ONRight, expression of Ca2-gating currents. Scale bars, 50 pA, 3 ms. f, Relationship between relative conductance (G/Gmax, y-axis) and charge movement (QON/QONmax, x-axis) for sCaV (blue, n = 7) and rCaV (red, n = 8). P < 0.0001 for difference in QON required for half-maximal conductance (dashed line). g, Maximal tail current (Itail) versus maximal gating charge (QON,max). Slopes: 2.23 ± 0.20 for sCav (blue, n = 8); 0.79 ± 0.06 for rCav (red, n = 9). Inset: representative ON-gating currents and Itail elicited by a voltage step to reversal potential (EREV) from and returning to −100 mV. Scale bars, 100 pA, 10 ms. All data represented as mean ± s.e.m. All P values from two-tailed Student’s t-test.

  3. Positively charged motif confers skate CaV voltage threshold.
    Figure 3: Positively charged motif confers skate CaV voltage threshold.

    a, Predicted topology of CaV1.3 α1-subunit. Species alignment reveals a positively charged insert in DIVS2–S3 of the skate orthologue. Charge-neutralized skate CaV1.3 (neutral-sCaV) was generated by replacing charged residues (KKKER) of the skate motif with glutamines. b, Representative currents from HEK293 cells expressing wild-type skate CaV1.3 (WT-sCaV, blue) or neutral-sCaV (red). Scale bars, 100 pA, 50 ms. c, IV relationships for WT-sCaV (blue) and neutral-sCaV (red). n = 7 per condition. Va1/2 from WT-sCaV (−41.13 ± 0.75 mV) compared with neutral-sCaV (−25.99 ± 0.92 mV), n = 7 per condition, P < 0.0001. d, GQON relationships of neutral-sCaV (red) and WT-sCaV (blue). n = 7 per condition, P < 0.0001 for difference in QON required for half-maximal conductance (dashed line). e, Itail versus QON,max. Slopes: 1.92 ± 0.15 for WT-sCaV (blue, n = 9), 0.66 ± 0.16 for neutral-sCaV (red, n = 7). Inset: representative maximal ON-gating currents and Itail. Scale bars, 100 pA, 10 ms. f, Representative currents from HEK293 cells expressing rCaV1.3 with the charged skate motif (charged-rCaV) or rCaV1.3 with a neutralized skate motif insert (neutral-rCaV). Scale bars, 100 pA, 50 ms. g, IV relationships for charged-rCaV (blue) and neutral-rCaV (red). n = 9 per condition. Va1/2 from charged-rCaV (−37.24 ± 0.32 mV) compared with neutral-rCaV (−19.6 ± 0.32 mV), n = 9 per condition, P < 0.0001. h, GQON relationships of charged-rCaV (blue, n = 9) and neutral-rCaV (red, n = 8). P < 0.0001 for difference in QON required for half-maximal conductance (dashed line). i, Itail versus QON,max. Slopes: 2.45 ± 0.19 for charged-rCaV (blue), 1.19 ± 0.04 for neutral-rCaV (red). n = 9 per condition. Inset: representative maximal ON-gating currents and Itail. Scale bars, 100 pA, 10 ms. All data represented as mean ± s.e.m. All P values from two-tailed Student’s t-test.

  4. Skate BK has a small conductance and short open time.
    Figure 4: Skate BK has a small conductance and short open time.

    a, Representative sBK and mBK single-channel currents recorded at the indicated membrane voltages from excised patches from transfected HEK293 cells. Scale bars, 25 pA vertical, 50 ms horizontal. Average IV relationship of sBK (blue) and mBK (red). n = 10 patches per condition, P < 0.0001 for difference in amplitude at each voltage. b, Representative sBK or mBK single-channel kinetics recorded at 80 mV. Scale bars, 10 pA, 1 ms. Inset: longer traces from same experiment (scale bars, 10 pA, 50 ms). Histograms of channel open times from 60-s records fitted with a single exponential to calculate open-state dwell time constants (sBK = 1.09 ± 0.02 ms, mBK = 5.06 ± 0.07 ms, n = 5, P < 0.0001). All P values from two-tailed Student’s t-test.

  5. Intracellular electrostatic adaptations in the pore of skate BK.
    Figure 5: Intracellular electrostatic adaptations in the pore of skate BK.

    a, Left, species alignment of skate BK α-subunit (kcnma1) reveals differences in charged residues. Right, net charge in this region determines local K+ concentration and conductance of BK. b, Representative single-channel records from patches at 120 mV expressing the indicated BK channels. Scale bars, 25 pA, 20 ms. c, Average IV relationships. Slope conductances: Wild-type sBK (blue) = 104 ± 5.4 pS, sR340S (purple) = 200 ± 9.9 pS, sA347E (teal) = 115 ± 5.4 pS, sBK-SE (green) = 287 ± 14 pS, mBK (red) = 263 ± 15 pS, mBK-RA (orange) = 97 ± 3.6 pS. n = 5. d, Wild-type sBK and mBK-RA have significantly shorter open-state dwell times than all other BK channels tested (at 80 mV). P < 0.001, one-way ANOVA with post hoc Tukey’s test, n = 7. e, Average single-channel amplitudes in response to changes in intracellular K+ concentration. n = 5. Dashed lines indicate single-channel current amplitude for sBK at 140 mM (bottom), 640 mM (middle), and 3.14 M (top). All data represented as mean ± s.e.m.

  6. ICav and IK tune voltage oscillations and electroreceptive behaviours.
    Figure 6: ICav and IK tune voltage oscillations and electroreceptive behaviours.

    a, Representative traces showing membrane voltage-dependent oscillations at indicated membrane potentials (Vm). Scale bars, 5 mV, 500 ms. b, Normalized amplitude of Vm oscillations (from rest, averaged over 500 ms) at indicated Vm (n = 4). Overlaid normalized ICav window current (black trace). c, Current injection (10 pA, 5 ms at arrow) at −65 mV elicited oscillations or a sustained depolarization in the presence of TEA+ (representative of n = 3). Scale bars, 5 mV, 100 ms. d, Representative Vm oscillations were inhibited by TEA+ or nifedipine. Scale bars, 5 mV, 100 ms. e, Average oscillation amplitude and frequency. Each circle depicts one experiment; P < 0.001, one-way ANOVA with post hoc Tukey’s test. f, Vm oscillations in response to the BK agonist NS11021 (NS) or NS + iberotoxin (IbTx). Scale bars, 10 mV, 250 ms. g, Average Vm oscillation amplitude and frequency. n = 4, P < 0.01 for amplitude, P < 0.05 for frequency, paired two-tailed Student’s t-test. h, Top-down view of control (left) and NS-treated (right) skates orienting towards a submerged electrical stimulus. Bold line depicts movement from start time to end during 30-min trials. Scale bar, 5 cm. i, Normalized percent time spent in area of submerged electrode for control and nifedipine-, NS-, or mibefradil-treated skates during basal conditions or in the presence of an electrical stimulus. n = 10 trials for all conditions, P < 0.001 for control stimulus versus basal, P < 0.01 for control stimulus versus all treatments except mibefradil, two-way ANOVA with post hoc Tukey’s test. All data represented as mean ± s.e.m.

  7. CaV and K+ channel expression in little skate.
    Extended Data Fig. 1: 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.

  8. Skate CaV ion selectivity and Ca2+-dependent inactivation.
    Extended Data Fig. 2: Skate CaV ion selectivity and Ca2+-dependent inactivation.

    a–c, Representative currents measured from electrosensory cells (native ICaV, a), HEK293 cells expressing skate CaV1.3 (sCaV, b), or HEK293 cells expressing rat CaV1.3 (rCaV, c) 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 cells expressing rCaV1.3 had only about 20% of current remaining. In electrosensory cells or cells expressing heterologous sCaV1.3 or rCaV1.3, the percentage of remaining current was significantly increased by replacing extracellular Ca2+ with 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 pulse that elicited maximal currents (± s.e.m., n = 5 per condition).

  9. Skate CaV pharmacology.
    Extended Data Fig. 3: Skate CaV pharmacology.

    a, b, Pharmacology of skate CaV1.3 (sCav). Representative currents recorded in response 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 (Fig. 1e). Dose–response relationships of current amplitudes measured at voltages that elicited maximal currents. Data are represented as mean ± s.e.m., 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.

  10. Skate CaV gating current properties.
    Extended Data Fig. 4: 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), and peak width at 50% of maximal gating current (width) for skate CaV1.3 (sCaV) versus rat CaV1.3 (rCaV, a), wild-type skate CaV1.3 (WT) versus charge-neutralized skate CaV1.3 (neutral, b), and rat CaV1.3 with charged skate motif (charged) versus rat CaV1.3 with neutralized skate motif (neutral, c). All values were similar except for peak I for sCaV versus rCaV, which is likely to represent increased expression of rCaV compared with sCaV. Data are presented as mean ± s.e.m., n listed above bars. d, Wild-type skate CaV1.3 (sCaV, blue, n = 7) and wild-type rat CaV (rCaV, red, n = 8) relative GV and QONV relationships. Data represented as mean ± s.e.m. e. GV and QONV relationships for wild-type sCaV1.3 (WT, blue) and charge-neutralized sCaV1.3 (neutral, red). Data represented as mean ± s.e.m., n = 7 per condition. f, GV and QONV relationships for rCaV1.3 with charged skate motif (charged, blue) and rCaV1.3 with neutral skate motif (neutral, red). Data represented as mean ± s.e.m., n = 8 per condition.

  11. Charged skate motif modulates voltage-dependent activation kinetics.
    Extended Data Fig. 5: Charged skate motif modulates voltage-dependent activation kinetics.

    a, Activation kinetics were faster in charged-rCaV (blue, n = 6) than in wild-type rCaV1.3 (WT-rCaV, grey, n = 7) or neutral-rCaV (red, n = 8). Data represent mean ± s.e.m., 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 mV 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 mV (green), were observed in currents recorded from charged-rCaV, but not neutral-rCaV. Scale bars, 50 pA, 10 ms. c, Cole–Moore effects quantified as the time to reach half-maximal current (t1/2). Charged-rCaV (blue, n = 9) reached maximal current amplitude faster with increasing prepulse voltage, whereas WT-rCaV (grey, n = 6) and neutral-rCaV (red, n = 8) were unchanged. All data represented as mean ± s.e.m., 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, pushing it into a partially activated or primed state (gold oval) before full activation (green ovals). Because sCaV1.3 is primed for activation, channel activation requires a smaller increase in voltage compared with rCaV1.3.

  12. Skate BK properties.
    Extended Data Fig. 6: Skate BK properties.

    a, Currents measured in response to 0, 1, or 10 μM intracellular Ca2+ at 80 mV from inside-out patches expressing sBK or mBK. Scale bars, 10 pA, 50 ms. Po for sBK compared with mBK was similar for all concentrations tested. Data represented as mean ± s.e.m., n = 5. b, Representative single-channel records at various voltages from patches expressing indicated BK channels. Scale bars, 25 pA, 20 ms. c, Representative currents recorded at 80 mV from patches expressing indicated BK channels. The same patch was exposed to local intracellular 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 (maroon). Scale bars, 50 pA, 20 ms.

  13. Adaptations in skate BK promote increased relative ICaV current during channel coupling.
    Extended Data Fig. 7: Adaptations in skate BK promote increased relative ICaV current during channel coupling.

    a, Whole-cell currents in response to 200-ms voltage pulses from −80 mV to +80 mV from HEK293 cells expressing sBK, sBK-SE, or mBK in the presence of 0 or 20 μM intracellular Ca2+. Scale bars, 5 nA, 50 ms. b, Average IV relationships for sBK (blue), sBK-SE (green) and mBK (red) in the presence of 0 or 20 μM intracellular Ca2+. n = 7. c, Whole-cell currents from HEK293 cells expressing charged-rCaV1.3 coexpressed with sBK, sBK-SE, or mBK. Scale bars, 500 pA, 50 ms. t, transient current evoked by voltage pulse; s, sustained current. In the presence of CaV1.3, average transient and sustained IV 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 bars, 100 pA, 5 ms). Transient EREV: sBK = 32.96 ± 2.17, mBK = 8.43 ± 2.76, sBK-s.e. = 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 ± s.e.m. and P values from two-tailed Student’s t-test.

  14. BK agonist NS11021 modulates skate BK channels.
    Extended Data Fig. 8: 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 bars, 5 pA, 100 ms. 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: basal, 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 IV relationships from HEK293 cells expressing charged-rCaV1.3 and sBK (scale bars, 500 pA, 50 ms). Transient and sustained IV 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 (scale bars, 100 pA, 50 ms). d, Transient and sustained IV relationships from normalizing currents in the presence of NS to basal currents. IV 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 ± s.e.m. and P values from two-tailed Student’s t-test.

  15. Ca2+-handling proteins are enriched in ampullae of Lorenzini.
    Extended Data Fig. 9: Ca2+-handling proteins are enriched in ampullae of Lorenzini.

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

  16. Behavioural paradigm for pharmacologically treated skates and startle response-related control.
    Extended Data Fig. 10: Behavioural paradigm for pharmacologically treated skates and startle response-related control.

    a, Schematic drawing of electrical stimulus. A 9-V 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 four different equally spaced locations on the base of the behavioural observation tank, which were then obscured by sand (right). b, Following 30 min 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 ± s.e.m.; 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 centre between the eyes from each respective position was recorded (dotted yellow line).

Accession codes

Primary accessions

Gene Expression Omnibus

NCBI Reference Sequence

Change history

Corrected online 12 March 2017
A minor change was made to the legend to Fig. 1, panel j.

References

  1. Kalmijn, A. J. The electric sense of sharks and rays. J. Exp. Biol. 55, 371383 (1971)
  2. Kalmijn, A. J. Electric and magnetic field detection in elasmobranch fishes. Science 218, 916918 (1982)
  3. Lissmann, H. W. & Machin, K. E. The mechanism of object location in Gymnarchus niloticus and similar fish. J. Exp. Biol. 35, 451486 (1958)
  4. Munz, H., Claas, B. & Fritzsch, B. Electroreceptive and mechanoreceptive units in the lateral line of the axolotl Ambystoma mexicanum. J. Comp. Physiol. 154, 3344 (1984)
  5. Scheich, H., Langner, G., Tidemann, C., Coles, R. B. & Guppy, A. Electroreception and electrolocation in platypus. Nature 319, 401402 (1986)
  6. Bullock, T. H. Electroreception. Annu. Rev. Neurosci. 5, 121170 (1982)
  7. Clusin, W. T. & Bennett, M. V. The ionic basis of oscillatory responses of skate electroreceptors. J. Gen. Physiol. 73, 703723 (1979)
  8. Clusin, W. T. & Bennett, M. V. The oscillatory responses of skate electroreceptors to small voltage stimuli. J. Gen. Physiol. 73, 685702 (1979)
  9. Peters, R. C., Zwart, R., Loos, W. J. G. & Bretschneider, F. Transduction at electroreceptor cells manipulated by exposure of apical membranes to ionic channel blockers. Comp Biochem Phys C 94, 663669 (1989)
  10. Araneda, R. C. & Bennett, M. Electrical properties of electroreceptor cells isolated from skate ampulla of Lorenzini. Biol. Bull. 185, 310311 (1993)
  11. Lu, J. & Fishman, H. M. Ion channels and transporters in the electroreceptive ampullary epithelium from skates. Biophys. J. 69, 24672475 (1995)
  12. Sisneros, J. A., Tricas, T. C. & Luer, C. A. Response properties and biological function of the skate electrosensory system during ontogeny. J. Comp. Physiol. A 183, 8799 (1998)
  13. Tricas, T. C., Michael, S. W. & Sisneros, J. A. Electrosensory optimization to conspecific phasic signals for mating. Neurosci. Lett. 202, 129132 (1995)
  14. Baker, C. A., Huck, K. R. & Carlson, B. A. Peripheral sensory coding through oscillatory synchrony in weakly electric fish. eLife 4, e08163 (2015)
  15. Catterall, W. A., Perez-Reyes, E., Snutch, T. P. & Striessnig, J. International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels. Pharmacol. Rev. 57, 411425 (2005)
  16. Platzer, J. et al. Congenital deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2+ channels. Cell 102, 8997 (2000)
  17. Koschak, A. et al. a1D (Cav1.3) subunits can form L-type Ca2+ channels activating at negative voltages. J. Biol. Chem. 276, 2210022106 (2001)
  18. Xu, W. & Lipscombe, D. Neuronal CaV1.3α1 L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J. Neurosci. 21, 59445951 (2001)
  19. Brandt, A., Striessnig, J. & Moser, T. CaV1.3 channels are essential for development and presynaptic activity of cochlear inner hair cells. J. Neurosci. 23, 1083210840 (2003)
  20. Modrell, M. S., Bemis, W. E., Northcutt, R. G., Davis, M. C. & Baker, C. V. H. Electrosensory ampullary organs are derived from lateral line placodes in bony fishes. Nat. Commun. 2, 496 (2011)
  21. Hulme, J. T., Yarov-Yarovoy, V., Lin, T. W., Scheuer, T. & Catterall, W. A. Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain. J. Physiol. (Lond.) 576, 87102 (2006)
  22. Wu, J. et al. Structure of the voltage-gated calcium channel CaV1.1 complex. Science 350, aad2395 (2015)
  23. Cole, K. S. & Moore, J. W. Potassium ion current in the squid giant axon: dynamic characteristic. Biophys. J. 1, 114 (1960)
  24. Fodor, A. A. & Aldrich, R. W. Convergent evolution of alternative splices at domain boundaries of the BK channel. Annu. Rev. Physiol. 71, 1936 (2009)
  25. Soom, M., Gessner, G., Heuer, H., Hoshi, T. & Heinemann, S. H. A mutually exclusive alternative exon of slo1 codes for a neuronal BK channel with altered function. Channels 2, 278282 (2008)
  26. King, B. L., Shi, L. F., Kao, P. & Clusin, W. T. Calcium activated K+ channels in the electroreceptor of the skate confirmed by cloning. Details of subunits and splicing. Gene 578, 6373 (2016)
  27. Brelidze, T. I., Niu, X. & Magleby, K. L. A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification. Proc. Natl Acad. Sci. USA 100, 90179022 (2003)
  28. Nimigean, C. M., Chappie, J. S. & Miller, C. Electrostatic tuning of ion conductance in potassium channels. Biochemistry 42, 92639268 (2003)
  29. Teeter, J. H. & Bennett, M. V. L. Synaptic transmission in the ampullary electroreceptor of the transparent catfish, Kryptopterus. J. Comp. Physiol. 142, 371377 (1981)
  30. Bentzen, B. H. et al. The small molecule NS11021 is a potent and specific activator of Ca2+-activated big-conductance K+ channels. Mol. Pharmacol. 72, 10331044 (2007)
  31. Schwaller, B. et al. Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice. Am. J. Physiol. 276, C395C403 (1999)
  32. Sejnowski, T. J. & Yodlowski, M. L. A freeze-fracture study of the skate electroreceptor. J. Neurocytol. 11, 897912 (1982)
  33. Zanazzi, G. & Matthews, G. The molecular architecture of ribbon presynaptic terminals. Mol. Neurobiol. 39, 130148 (2009)
  34. Lewis, R. S. & Hudspeth, A. J. Voltage- and ion-dependent conductances in solitary vertebrate hair cells. Nature 304, 538541 (1983)
  35. Hudspeth, A. J. & Lewis, R. S. A model for electrical resonance and frequency tuning in saccular hair cells of the bull-frog, Rana catesbeiana. J. Physiol. (Lond.) 400, 275297 (1988)
  36. Miranda-Rottmann, S., Kozlov, A. S. & Hudspeth, A. J. Highly specific alternative splicing of transcripts encoding BK channels in the chicken’s cochlea is a minor determinant of the tonotopic gradient. Mol. Cell. Biol. 30, 36463660 (2010)
  37. Rosenblatt, K. P., Sun, Z. P., Heller, S. & Hudspeth, A. J. Distribution of Ca2+-activated K+ channel isoforms along the tonotopic gradient of the chicken’s cochlea. Neuron 19, 10611075 (1997)
  38. Fettiplace, R. & Fuchs, P. A. Mechanisms of hair cell tuning. Annu. Rev. Physiol. 61, 809834 (1999)
  39. Ramakrishnan, N. A. et al. Voltage-gated Ca2+ channel CaV1.3 subunit expressed in the hair cell epithelium of the sacculus of the trout Oncorhynchus mykiss: cloning and comparison across vertebrate classes. Brain Res. Mol. Brain Res. 109, 6383 (2002)
  40. Senatore, A., Boone, A. N. & Spafford, J. D. Optimized transfection strategy for expression and electrophysiological recording of recombinant voltage-gated ion channels in HEK-293T cells. J. Vis. Exp. 47, 2314 (2011)
  41. Gillis, J. A., Dahn, R. D. & Shubin, N. H. Chondrogenesis and homology of the visceral skeleton in the little skate, Leucoraja erinacea (Chondrichthyes: Batoidea). J. Morphol. 270, 628643 (2009)
  42. Ishii, T., Omura, M. & Mombaerts, P. Protocols for two- and three-color fluorescent RNA in situ hybridization of the main and accessory olfactory epithelia in mouse. J. Neurocytol. 33, 657669 (2004)

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Author information

  1. These authors contributed equally to this work.

    • Nicholas W. Bellono &
    • Duncan B. Leitch

Affiliations

  1. Department of Physiology, University of California, San Francisco, California 94143, USA

    • Nicholas W. Bellono,
    • Duncan B. Leitch &
    • David Julius

Contributions

N.W.B. designed and performed electrophysiological studies, D.B.L. designed and performed gene expression, anatomical, and behavioural studies, and N.W.B., D.B.L. and D.J. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Reviewer Information Nature thanks B. Carlson, R. Krahe, N. Marrion and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: CaV and K+ channel expression in little skate. (108 KB)

    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.

  2. Extended Data Figure 2: Skate CaV ion selectivity and Ca2+-dependent inactivation. (216 KB)

    a–c, Representative currents measured from electrosensory cells (native ICaV, a), HEK293 cells expressing skate CaV1.3 (sCaV, b), or HEK293 cells expressing rat CaV1.3 (rCaV, c) 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 cells expressing rCaV1.3 had only about 20% of current remaining. In electrosensory cells or cells expressing heterologous sCaV1.3 or rCaV1.3, the percentage of remaining current was significantly increased by replacing extracellular Ca2+ with 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 pulse that elicited maximal currents (± s.e.m., n = 5 per condition).

  3. Extended Data Figure 3: Skate CaV pharmacology. (138 KB)

    a, b, Pharmacology of skate CaV1.3 (sCav). Representative currents recorded in response 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 (Fig. 1e). Dose–response relationships of current amplitudes measured at voltages that elicited maximal currents. Data are represented as mean ± s.e.m., 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.

  4. Extended Data Figure 4: Skate CaV gating current properties. (343 KB)

    a–c, Gating current properties including peak amplitude (peak I), time-to-peak (TTP), exponential decay time constant (τ decay), and peak width at 50% of maximal gating current (width) for skate CaV1.3 (sCaV) versus rat CaV1.3 (rCaV, a), wild-type skate CaV1.3 (WT) versus charge-neutralized skate CaV1.3 (neutral, b), and rat CaV1.3 with charged skate motif (charged) versus rat CaV1.3 with neutralized skate motif (neutral, c). All values were similar except for peak I for sCaV versus rCaV, which is likely to represent increased expression of rCaV compared with sCaV. Data are presented as mean ± s.e.m., n listed above bars. d, Wild-type skate CaV1.3 (sCaV, blue, n = 7) and wild-type rat CaV (rCaV, red, n = 8) relative GV and QONV relationships. Data represented as mean ± s.e.m. e. GV and QONV relationships for wild-type sCaV1.3 (WT, blue) and charge-neutralized sCaV1.3 (neutral, red). Data represented as mean ± s.e.m., n = 7 per condition. f, GV and QONV relationships for rCaV1.3 with charged skate motif (charged, blue) and rCaV1.3 with neutral skate motif (neutral, red). Data represented as mean ± s.e.m., n = 8 per condition.

  5. Extended Data Figure 5: Charged skate motif modulates voltage-dependent activation kinetics. (217 KB)

    a, Activation kinetics were faster in charged-rCaV (blue, n = 6) than in wild-type rCaV1.3 (WT-rCaV, grey, n = 7) or neutral-rCaV (red, n = 8). Data represent mean ± s.e.m., 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 mV 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 mV (green), were observed in currents recorded from charged-rCaV, but not neutral-rCaV. Scale bars, 50 pA, 10 ms. c, Cole–Moore effects quantified as the time to reach half-maximal current (t1/2). Charged-rCaV (blue, n = 9) reached maximal current amplitude faster with increasing prepulse voltage, whereas WT-rCaV (grey, n = 6) and neutral-rCaV (red, n = 8) were unchanged. All data represented as mean ± s.e.m., 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, pushing it into a partially activated or primed state (gold oval) before full activation (green ovals). Because sCaV1.3 is primed for activation, channel activation requires a smaller increase in voltage compared with rCaV1.3.

  6. Extended Data Figure 6: Skate BK properties. (232 KB)

    a, Currents measured in response to 0, 1, or 10 μM intracellular Ca2+ at 80 mV from inside-out patches expressing sBK or mBK. Scale bars, 10 pA, 50 ms. Po for sBK compared with mBK was similar for all concentrations tested. Data represented as mean ± s.e.m., n = 5. b, Representative single-channel records at various voltages from patches expressing indicated BK channels. Scale bars, 25 pA, 20 ms. c, Representative currents recorded at 80 mV from patches expressing indicated BK channels. The same patch was exposed to local intracellular 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 (maroon). Scale bars, 50 pA, 20 ms.

  7. Extended Data Figure 7: Adaptations in skate BK promote increased relative ICaV current during channel coupling. (225 KB)

    a, Whole-cell currents in response to 200-ms voltage pulses from −80 mV to +80 mV from HEK293 cells expressing sBK, sBK-SE, or mBK in the presence of 0 or 20 μM intracellular Ca2+. Scale bars, 5 nA, 50 ms. b, Average IV relationships for sBK (blue), sBK-SE (green) and mBK (red) in the presence of 0 or 20 μM intracellular Ca2+. n = 7. c, Whole-cell currents from HEK293 cells expressing charged-rCaV1.3 coexpressed with sBK, sBK-SE, or mBK. Scale bars, 500 pA, 50 ms. t, transient current evoked by voltage pulse; s, sustained current. In the presence of CaV1.3, average transient and sustained IV 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 bars, 100 pA, 5 ms). Transient EREV: sBK = 32.96 ± 2.17, mBK = 8.43 ± 2.76, sBK-s.e. = 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 ± s.e.m. and P values from two-tailed Student’s t-test.

  8. Extended Data Figure 8: BK agonist NS11021 modulates skate BK channels. (224 KB)

    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 bars, 5 pA, 100 ms. 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: basal, 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 IV relationships from HEK293 cells expressing charged-rCaV1.3 and sBK (scale bars, 500 pA, 50 ms). Transient and sustained IV 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 (scale bars, 100 pA, 50 ms). d, Transient and sustained IV relationships from normalizing currents in the presence of NS to basal currents. IV 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 ± s.e.m. and P values from two-tailed Student’s t-test.

  9. Extended Data Figure 9: Ca2+-handling proteins are enriched in ampullae of Lorenzini. (106 KB)

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

  10. Extended Data Figure 10: Behavioural paradigm for pharmacologically treated skates and startle response-related control. (139 KB)

    a, Schematic drawing of electrical stimulus. A 9-V 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 four different equally spaced locations on the base of the behavioural observation tank, which were then obscured by sand (right). b, Following 30 min 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 ± s.e.m.; 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 centre between the eyes from each respective position was recorded (dotted yellow line).

Additional data