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Molecular tuning of electroreception in sharks and skates

Naturevolume 558pages122126 (2018) | Download Citation

Abstract

Ancient cartilaginous vertebrates, such as sharks, skates and rays, possess specialized electrosensory organs that detect weak electric fields and relay this information to the central nervous system1,2,3,4. Sharks exploit this sensory modality for predation, whereas skates may also use it to detect signals from conspecifics5. Here we analyse shark and skate electrosensory cells to determine whether discrete physiological properties could contribute to behaviourally relevant sensory tuning. We show that sharks and skates use a similar low threshold voltage-gated calcium channel to initiate cellular activity but use distinct potassium channels to modulate this activity. Electrosensory cells from sharks express specially adapted voltage-gated potassium channels that support large, repetitive membrane voltage spikes capable of driving near-maximal vesicular release from elaborate ribbon synapses. By contrast, skates use a calcium-activated potassium channel to produce small, tunable membrane voltage oscillations that elicit stimulus-dependent vesicular release. We propose that these sensory adaptations support amplified indiscriminate signal detection in sharks compared with selective frequency detection in skates, potentially reflecting the electroreceptive requirements of these elasmobranch species. Our findings demonstrate how sensory systems adapt to suit the lifestyle or environmental niche of an animal through discrete molecular and biophysical modifications.

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Acknowledgements

We thank S. Bennett from the Marine Biological Laboratory for supplying animals, J. Wong from the Gladstone/University of California, San Francisco (UCSF) transmission electron microscopy core for performing electron microscopy, A. Zimmerman for help with capacitance measurements, discussion and reading of the manuscript, R. Edwards for discussion, and R. Nicoll for input on the manuscript. This work was supported by a National Institutes of Health (NIH) Institutional Research Service Award to the UCSF CVRI (T32HL007731 to N.W.B.), a Howard Hughes Medical Institute Fellowship of the Life Sciences Research Foundation (N.W.B.), a Simons Foundation Postdoctoral Fellowship to the Jane Coffin Childs Memorial Fund (D.B.L.) and grants from the NIH (1K99DC016658 to D.B.L., K99DK115879 to N.W.B., and NS055299 and NS105038 to D.J.).

Reviewer information

Nature thanks B. Carlson, C. Lingle, H. von Gersdorff and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Nicholas W. Bellono

    Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA

  1. These authors contributed equally: Nicholas W. Bellono, Duncan B. Leitch.

Affiliations

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

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

Authors

  1. Search for Nicholas W. Bellono in:

  2. Search for Duncan B. Leitch in:

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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 interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Nicholas W. Bellono or David Julius.

Extended data figures and tables

  1. Extended Data Fig. 1 Properties of shark ICaV.

    a, Top, isolated shark ampullary organs with attached canals and nerve fibres (scale bar, 100 μm); bottom, a representative electrosensory cell patch-clamp experiment (scale bar, 10 μm). b, Left, ICaV currents elicited by increasing voltage pulses from −100 mV; right, average current–voltage (IV) relationship (n = 7). c, ICaV exhibited an L-type CaV pharmacological profile: Peak currents were regulated by Bay K (agonist), Cd2+ (blocker) and nifedipine (antagonist), but not by mibefradil (T-type antagonist). n = 4. P < 0.0001 for control versus all treatments except mibefradil, one-way ANOVA with post hoc Bonferroni test. d, ICaV conductance–voltage (GV) relationship (black) with half-maximal activation voltage (Va1/2) of −54.6 ± 1.2 mV. Inactivation–voltage relationship (grey) with half-inactivation potential (Vh1/2) of −62.9 ± 1.4 mV. A large window current was observed between −70 mV and −40 mV. GV relationships were established from current measurements during voltage pulses delivered in 10-mV increments from −100 mV. Inactivation was measured at a −20 mV test pulse after a series of voltage prepulses. n = 7. e, CaV α-subunit mRNA expression in shark ampullary organs. Bars represent FPKM. f, ICaV elicited by a 2-s depolarizing voltage step to −30 mV exhibits little inactivation. Representative of n = 5. g, CaV1.3 alignment revealed that the IVS2–S3 motif that confers low voltage threshold in skate CaV1.3 is conserved in the shark orthologue. h, Expression of CaV auxiliary subunits in shark ampullary organs. Bars represent FPKM. i, Average ICaV current density and voltage-activation threshold was similar in shark and skate electrosensory cells. n = 6. All data are represented as mean ± s.e.m, n denotes cells.

  2. Extended Data Fig. 2 Properties of shark IKV.

    a, Currents elicited by 500-ms voltage ramps in shark (red) or skate (blue) electrosensory cells in the presence of intracellular Cs+ (left) or K+ (right). The insets on the right show the inward ICaV in the presence of intracellular K+. b, Average ICaV current density elicited by voltage ramps was similar for both shark and skate cells in the presence of intracellular Cs+, but larger in shark cells in the presence of intracellular K+. Outward K+ current density was significantly larger in shark cells. n = 5, P < 0.0001 for shark versus skate ICaV or IK with intracellular K+, two-tailed Student’s t-test. c, The percentage of ICaV remaining in the presence of K+ compared with Cs+ is markedly greater in shark cells compared with those from skate. d, Inward currents elicited by increasing voltage pulses from −100 mV were not affected by IbTx or 4-AP, but were blocked by Cd2+. n = 5, P < 0.0001 for inward control currents versus Cd2+, two-way ANOVA with post hoc Tukey test. Peak inward currents were not affected by IbTx or 4-AP. e, Reversal potential for IKV in shark electrosensory cells is near the reversal potential for selective K+ permeation (EK, blue arrow on the IV plot). n = 5. Arrows indicate when currents were measured at the indicated voltages after an activating prepulse of 40 mV (also shown in expanded view). Extracellular Cd2+ was included to block ICaV for biophysical studies of IKV. f, IKV currents elicited by a voltage protocol to obtain the GV relationship. Arrows indicate when tail currents were measured at −30 mV after voltage steps that increased in 10-mV increments from −100 mV (expanded view within inset). Representative of n = 11. g, Voltage-dependent inactivation properties of IKV. The arrow indicates when inactivation was measured during 40-mV pulses after a series of prepulses that increased in 10-mV increments from −100 mV. Vh1/2 was −5.5 ± 1.7 mV and inactivation was incomplete. n = 6. h, mRNA expression of the KV channel auxiliary subunits in shark ampullary organs. Bars represent FPKM. i, mRNA expression of kcna3 isoforms. The major isoform studied is indicated in red. Other low-expression ampullary isoforms were similar. The only isoform that was appreciably expressed outside of ampullae was truncated and found in the brain (grey). All data are represented as mean ± s.e.m, n denotes cells.

  3. Extended Data Fig. 3 Properties of shark BK.

    a, mRNA expression of major K+-channel α-subunits (Kcna3, KV1.3) and (Kcnma1, BK) in shark and skate electrosensory cells. b, Average K+ current density, 4-AP-sensitive current (IKV), and IbTx-sensitive current (IBK) in shark and skate electrosensory cells. n = 5, P < 0.0001 for shark versus skate cells for all comparisons, two-tailed Student’s t-test. c, Co-localization of CaV1.3 (red) and BK (green) transcripts within shark ampullary organs. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. Representative of n = 4. d, In the presence of 4-AP, a relatively small outward current remained that was insensitive to IbTx and was slightly increased by NS11021 at very positive voltages. n = 5, P < 0.05 for control versus NS11021 at 70 or 80 mV, two-way ANOVA with post hoc Tukey test. e, BK alignment revealed that residues found to reduce conductance in skate BK are conserved in the shark orthologue. f, Heterologously expressed shark and skate BK had relatively small single-channel conductance compared with mouse BK. Shark BK = 109 ± 4 pS; skate BK = 105 ± 5 pS; mouse BK = 259 ± 12 pS. n = 5, P < 0.0001 for mouse versus shark or skate BK, two-way ANOVA with post hoc Tukey test. g, 1 μM Ca2+ increased open probability in shark BK expressed in excised inside-out patches, and 10 μM NS11021 increased open probability of channels in excised outside-out patches. NS11021 modulation was blocked by 100 nM IbTx. Holding voltage was 80 mV. NPo: basal, 0.0061 ± 0.0014; Ca2+, 0.11 ± 0.011, NS11021, 0.24 ± 0.026. n = 5, P < 0.0001 for basal versus Ca2+ or NS11021, two-tailed Student’s t-test. All data are represented as mean ± s.e.m, n denotes cells or tissue sections.

  4. Extended Data Fig. 4 Properties of shark KV.

    a, Voltage-activated currents recorded in HEK293 cells expressing shark (red) or human (black) KV1.3. Arrows indicate when currents were measured at −30 mV after voltage pulses that increased in 10-mV increments from −100 mV. The inset shows expansions of measured currents following arrows. b, Left, normalized currents elicited by a 40-mV voltage pulse demonstrated that expressed shark KV channels open more slowly compared with human orthologues. Right, average activation kinetics were slower for shark KV compared with human KV in response to voltages from 20 mV to 50 mV. n = 6, P < 0.0001 for contribution of orthologue identity to series variance, two-way ANOVA with post hoc Bonferroni test. c, Deactivation kinetics of normalized currents from shark and human KV channels at various repolarizing voltages after an activating prepulse of 40 mV. The arrow indicates when current properties were measured during the voltage protocol. d, Inactivation properties (left) and average inactivation–voltage relationships (right) of shark (red, Vh1/2 = 0.1 ± 2.8 mV) and human (black, Vh1/2 = −30.6 ± 0.9 mV) KV1.3 channels. The arrow indicates when inactivation was measured during 40-mV pulses after a series of prepulses that increased in 10-mV increments from −100 mV. n = 9, P < 0.0001 for Vh1/2, two-tailed Student’s t-test. e, IKV was reduced by 4-AP or quinidine in native shark electrosensory cells or heterologously expressed shark KV1.3. Currents were elicited by increasing voltage pulses from −90 mV (native) or −100 mV (heterologous). f, Pharmacological profile of shark electrosensory cell IKV and heterologously expressed shark KV1.3. Currents measured at peak amplitude were reduced by 4-AP or quinidine, but not by other treatments. Pharmacological modulation of native IKV and shark KV1.3 was similar. g, The short human isoform of KV1.3 (short N-terminal truncation) was used to study gating currents because of enhanced expression, but channel properties are identical15. Similarly, we found that activation threshold and GV relationship, voltage-dependent inactivation, and deactivation were similar between long and short isoforms. n = 6. All data are represented as mean ± s.e.m, n denotes cells.

  5. Extended Data Fig. 5 Shark KV gating currents.

    a, Gating currents recorded in HEK293 cells expressing non-conductive shark (red) or human (black) KV1.3 elicited by increasing voltage pulses in 10-mV increments from a −100-mV holding potential. b, Average charge (Q)–V relationships. Shark KV1.3 Va1/2 was −33.4 ± 0.5 mV, Ka = 6.1 ± 0.5 mV; and human KV1.3 Va1/2 was −54.25 ± 0.7 mV, Ka = 5.2 ± 0.6 mV. n = 9, P < 0.0001 for Va1/2 two-tailed Student’s t-test. Dotted lines indicate associated GV relationships. c, QON kinetics after voltage sensor activation from a holding potential of −100 mV were significantly slower in shark (red) compared with human (black). n = 7, P < 0.0001 for contribution of orthologue identity to series variance, two-way ANOVA with post hoc Bonferroni test. d, Representative OFF gating current (QOFF) kinetics during repolarization in 10-mV increments after a 40-mV prepulse. The arrow indicates when deactivation rates were measured and purple traces show deactivation at −50 mV. e, QOFF kinetics were significantly faster in shark (red) compared with human (black). n = 11, P < 0.0001 for contribution of orthologue identity to series variance, two-way ANOVA with post hoc Bonferroni test. f, Gating currents from shark (red) or human (black) KV1.3 after decreasing voltage pulses in increments of 10 mV from a holding potential of 0 mV. Scale bars, 500 pA, 25 ms. g, Average charge (Q)–V relationships of downward voltage sensor movement (QOFF) in response to decreasing voltage pulses from a holding potential of 0 mV. Shark KV1.3 Va1/2 was −61.6 ± 1.9 mV and human KV1.3 Va1/2 was −110.9 ± 1.01 mV. n = 7, P < 0.0001 for contribution of orthologue identity to series variance, two-way ANOVA with post hoc Bonferroni test. h, QOFF kinetics of voltage sensor deactivation from a holding potential of 0 mV were significantly faster in shark (red) compared with human (black). n = 7, P < 0.0001 for contribution of orthologue identity to series variance, two-way ANOVA with post hoc Bonferroni test. All data are represented as mean ± s.e.m. i, Ion tail currents (indicating channel closure) deactivated faster in shark (red) compared with human (black) KV1.3. Tail currents were measured at −100 mV after a series of activating voltage pulses that increased in 10-mV increments. Inset, arrows indicate when tail currents were measured after activating voltage pulses. Representative of n = 10. τ for deactivation from 60-mV pulse: shark = 1.5 ± 0.1 ms; human = 5.5 ± 0.4 ms. P < 0.0001, two-tailed Student’s t-test. All data are represented as mean ± s.e.m, n denotes cells.

  6. Extended Data Fig. 6 Shark KV voltage sensor domain relaxation.

    a, QOFF kinetics after either 10-ms or 1-ms activating prepulses of 40 mV. Purple traces indicate deactivation at −50 mV. Arrow indicates when current properties were measured during the voltage protocol. b, Average QOFF kinetics were faster in shark compared with human during deactivation after 40-mV prepulses of 25, 10, or 5 ms duration, but rates were similar after 1-ms activating prepulses. Kinetics were measured at voltages that decreased in 10-mV increments from 40 mV. n = 9, P < 0.0001 for contribution of orthologue identity to series variance at 25, 10, or 5 ms, but no significant difference was observed at 1 ms, two-way ANOVA with post hoc Bonferroni test. c, Shark KV1.3 QOFF kinetics were relatively unaffected by the duration of the activating voltage pulse, whereas human KV1.3 entered a proposed ‘relaxed’ state that resulted in the slowing of QOFF with increasing pulse length. Deactivation was measured at −100 mV after a series of 40-mV voltage pulses of varying duration from 0.5 ms to 30 ms. d, Average QOFF kinetics in response to indicated voltage pulse lengths. n = 6, P < 0.0001 for comparison of orthologue QOFF kinetics after a 30-ms voltage pulse. e, Hypothetical model of shark and human KV1.3. Compared with its human orthologue, shark KV1.3 exhibits reduced voltage sensor domain relaxation, which stabilizes pore opening in human KV1.3. Reduced voltage sensor relaxation is indicated by dotted lines to suggest that this state (or states) may occur to a lesser extent in the shark orthologue. Thus, compared with human KV1.3, the shark orthologue requires relatively less repolarizing voltage to more quickly return to a resting/closed state. All data are represented as mean ± s.e.m, n denotes cells.

  7. Extended Data Fig. 7 Shark–human KV chimaeric analyses.

    a, Chimaeric shark–human KV1.3 channels reveal that shark KV S1–S6 confers differences in activation voltage threshold, deactivation kinetics and inactivation. Top, the chimaera constructs analysed (shark in red, human in black). Middle, the arrow indicates when voltage-activated currents were measured at −30 mV after a series of voltage pulses that increased in 10-mV increments from −100 mV. Bottom, the arrow indicates when inactivation was measured during 40-mV pulses after a series of prepulses that increased in 10-mV increments from −100 mV. b, Compared with wild-type human KV, average GV relationships for wild-type shark, shark (s)S1–S6, and sS1–S4 channels were similarly shifted to positive voltages with more gradual slopes and deactivation kinetics were faster. Va1/2 (mV) for wild-type human = −30.7 ± 0.5 mV, slope factor (Ka) = 4.7 ± 0.5 mV; wild-type shark Va1/2 = −5.4 ± 0.5 mV, Ka = 7.6 ± 0.4 mV; sS1–S6 Va1/2 = −9.7 ± 0.7 mV, Ka = 8.1 ± 0.6 mV; sS1–S4 Va1/2 = −6.5 ± 0.7 mV, Ka = 10.7 ± 1.2 mV. Average deactivation kinetics of wild-type shark, sS1–S6, and sS1–S4 KV channels were faster than those of wild-type human channels. Substitution of human (h)S1–S6 or hS1–S4 into the shark KV channel also partially shifted the activation threshold and deactivation kinetics. Channels containing hS5–S6 exhibited the strongest voltage-dependent inactivation—nearly as efficient as wild-type human KV—whereas channels containing sS5–S6 displayed weaker inactivation. sS1–S4 had a smaller effect on inactivation. Vh1/2 for wild-type human = −34.6 ± 0.9 mV, wild-type shark = 0.1 ± 2.8 mV, hS1–S6 = −18.3 ± 0.4 mV, hS1–S4 = −12.3 ± 1.2 mV, sS1–S6 = −9.2 ± 0.8 mV, sS1–S4 = −16.0 ± 0.9 mV. n = 9 for each of wild-type shark, wild-type human, hS1–S6, hS1–S4 and sS1–S6, and 7 for sS1–S4. c, Currents elicited from the indicated S5–S6 chimaeric channels in response to voltage protocols to access voltage-dependence for activation and inactivation. d, sS5–S6 reduces voltage-dependent inactivation and hS5–S6 partially confers strong voltage-dependent inactivation in shark channels. n = 6. P < 0.0001 for hS5–S6 versus sS5–S6 or wild-type shark, sS5–S6 versus hS5–S6 or wild-type human, two-way ANOVA with post hoc Tukey test. e, Top, S5–S6 substitution did not greatly affect voltage-dependent activation. n = 6 for hS5–S6, 7 for sS5–S6. Bottom, S5–S6 substitution did not affect deactivation kinetics. n = 7 for hS5–S6, 9 for sS5–S6. All data are represented as mean ± s.e.m, n denotes cells.

  8. Extended Data Fig. 8 Electrosensory cell ribbon synapse characteristics.

    a, Left, five highest expressed transcripts in shark ampullae. The Ca2+-binding protein parvalbumin 8 is the most highly expressed and is enriched in ampullae compared with other examined tissues. Right, five highest expressed ATPase transcripts in shark ampullae. A plasma membrane Ca2+ ATPase is highly expressed and enriched in ampullae. Bars represent FPKM. b, Expression of transcripts associated with ribbon synapses in shark and skate ampullae. Expression of vGluT3 and EAAT1 suggests that the synapse could be glutamatergic. c, Transmission electron micrograph of skate ribbon synapse with arrows indicating electrosensory cell, synaptic ribbon and afferent nerve terminal. Distinct vesicular pools are coloured: blue, attached to ribbon; green, refilling; yellow, readily releasable. An orange dotted line indicates the 150-nm region in which the readily releasable pool was quantified. Scale bar, 500 nm. d, Quantification of attached vesicles, ribbon vesicle density, ribbon shape variation and vesicle diameter was similar between shark and skate electrosensory cells. The readily releasable pool, quantified by number of vesicles 150 nm from the synapse, was significantly larger in shark versus skate electrosensory cells. n = 18, P < 0.0001, two-tailed Student’s t-test. The refilling pool density, quantified as detached cytosolic vesicles, was significantly larger in skate electrosensory cells. n = 20 shark and 21 skate, P < 0.0001, two-tailed Mann–Whitney test. Shark ribbons were more parallel to the plasma membrane in comparison to skate ribbons that were often more perpendicular. For angle quantification n = 20 shark and 21 skate ribbons, P < 0.0001, two-tailed Mann–Whitney test. All data are represented as mean ± s.e.m, n denotes counted structures.

  9. Extended Data Fig. 9 Shark electrosensory cell vesicular release characteristics.

    a, Top, currents and capacitance changes in response to a 10 ms, −20 mV voltage pulse in shark and skate electrosensory cells. Scale bars, 50 pA, 200 ms. Bottom, representative capacitance changes in response to the indicated durations of a voltage stimulus of −20 mV. Scale bars, 25 fF, 200 ms. b, −20 mV voltage pulses of various durations induced similar integrated ICaV ( Q Ca 2 + ) in shark or skate electrosensory cells. n = 6. c, ICaV elicited by simulated voltage spikes in shark electrosensory cells and smaller voltage oscillations in skate cells.. K+ currents were blocked by intracellular Cs+, extracellular 4-AP and IbTx. d, Voltage-clamp protocols developed to simulate shark electrosensory cell spiking induced the same amount of Q Ca 2 + in shark or skate cells. Similarly, voltage protocols that simulated smaller skate electrosensory cell voltage oscillations induced the same amount of Q Ca 2 + in shark or skate cells. Q Ca 2 + elicited by simulated voltage spikes was larger than Q Ca 2 + elicited by simulated oscillations. n = 10 for shark and 5 for skate. All data are represented as mean ± s.e.m, n denotes cells.

  10. Extended Data Fig. 10 Schematic representation of ampullae of Lorenzini distribution in two elasmobranch species.

    a, The dorsal surface of the chain catshark (S. retifer), with black dots corresponding to individual ampullary pores and blue lines representing canal structures. The buccal and supraorbital clusters from which electrosensory cells were obtained are indicated with arrowheads. This schematic was prepared from photographs of four individual fish. b, The ventral surface of the catshark. c, Photograph of ampullary pores, visible on the ventral rostrum of an adult catshark. d, The dorsal surface of the little skate (L. erinacea). The hyoid capsules from which electrosensory cells were obtained are indicated with arrowheads. This schematic was prepared from photographs of four individual fish. e, The ventral surface of the skate. f, Photograph of a cleared Alcian blue-stained skate, revealing the ampullary canals from the ventral surface of the skate.

Supplementary information

  1. Reporting Summary

  2. Video 1: Shark ventilation behaviour

    A juvenile catshark (S. retifer) in the behavioural observation tank, showing distinct rhythmic movement of gill slits. The frequency of these movements was recorded in responses to environmental electrical and food odorant stimuli.

  3. Video 2: Skate ventilation behaviour

    A juvenile little skate (L. erinacea) in the behavioural observation tank, showing distinct movement of spiracle, posterior to the eye. The frequency of these movements was recorded in responses to environmental electrical and food odorant stimuli.

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https://doi.org/10.1038/s41586-018-0160-9

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