LETTER https://doi.org/10.1038/s41586-018-0160-9 Molecular tuning of electroreception in sharks and skates Nicholas W. Bellono1,2,3*, Duncan B. Leitch1,3 & David Julius1* Ancient cartilaginous vertebrates, such as sharks, skates and rays, antagonist (Fig. 1b, Extended Data Fig. 2d). Instead, the voltage-gated possess specialized electrosensory organs that detect weak electric KV channel inhibitor 4-aminopyridine (4-AP) blocked outward 1–4 fields and relay this information to the central nervous system . currents from shark electrosensory cells (IKV, Fig. 1b). IKV was selective Sharks exploit this sensory modality for predation, whereas skates for K+ and exhibited a relatively high voltage-activation threshold may also use it to detect signals from conspecifics5. Here we analyse (Fig. 1c, Extended Data Fig. 2e, f). Furthermore, we observed fast shark and skate electrosensory cells to determine whether discrete activation and deactivation kinetics, whereas voltage-dependent inac- physiological properties could contribute to behaviourally relevant tivation (similar to desensitization) in response to prolonged voltage sensory tuning. We show that sharks and skates use a similar low pulses was weak and slow, which results in a K+ conductance of undi- threshold voltage-gated calcium channel to initiate cellular activity minished current amplitude even after repeated activation–deactivation but use distinct potassium channels to modulate this activity. cycles (Fig. 1d, e, Extended Data Fig. 2g). Among voltage-gated K+ Electrosensory cells from sharks express specially adapted voltage- channels, transcripts that encode the pore-forming subunit of KV1.3 gated potassium channels that support large, repetitive membrane predominated in shark ampullary organs (together with several KV voltage spikes capable of driving near-maximal vesicular release auxiliary subunits) and co-localized with CaV1.3 in electrosensory cells from elaborate ribbon synapses. By contrast, skates use a calcium- (Fig. 1f, g, Extended Data Fig. 2h). Outside of ampullary organs, only a activated potassium channel to produce small, tunable membrane truncated form of KV1.3 that lacks an essential N-terminal tetrameriza- voltage oscillations that elicit stimulus-dependent vesicular tion domain was observed in the brain (Extended Data Fig. 2i). KV1.3 release. We propose that these sensory adaptations support expression was not detected in skate ampullary organs, and only shark amplified indiscriminate signal detection in sharks compared with electrosensory cells exhibited 4-AP-sensitive voltage-gated K+ currents selective frequency detection in skates, potentially reflecting the (Extended Data Fig. 3a, b). Furthermore, both shark and skate elec- electroreceptive requirements of these elasmobranch species. Our trosensory cells expressed BK transcripts that, when heterologously findings demonstrate how sensory systems adapt to suit the lifestyle expressed, produced channels with similar properties; however, func- or environmental niche of an animal through discrete molecular and tional BK currents were observed only in the latter (Extended Data biophysical modifications. Fig. 3). As such, BK channels do not contribute appreciably to the major Electrosensory cells from the little skate (Leucoraja erinacea) express K+ conductance in shark electrosensory cells, at least not under the 2+ specialized low threshold CaV1.3 voltage-gated calcium (Ca ) channels developmental or physiological conditions examined here. In summary, + and big-conductance potassium (K , BK) channels that functionally shark electrosensory cells express a specific IKV with voltage-dependent couple to produce cellular membrane voltage oscillations6,7. In elec- properties that support repetitive stimulation. trosensory cells from the chain catshark (Scyliorhinus retifer, Fig. 1a), Shark KV1.3 is 80% identical to the human orthologue, but its voltage we similarly observed voltage-activated inward calcium currents threshold for activation was shifted to more depolarized values 2+ (ICaV) that were sensitive to L-type voltage-gated Ca -channel (CaV) compared with human KV1.3 (Fig. 2a, Extended Data Fig. 4a). modulators, had a low voltage threshold for activation, had steep voltage- Furthermore, shark KV1.3 was activated at slightly slower rates and dependence, and had a slow inactivation profile that contributed to deactivated with rapid kinetics, requiring substantially less negative a large window-current across physiological membrane voltages voltage to return to the resting state compared with the human channel (Extended Data Fig. 1a–d, f). As with skates, the pore-forming α (Fig. 2b, c, Extended Data Fig. 4b, c). Shark KV1.3 inactivation was subunit of CaV1.3 was the predominant CaV channel subtype expressed slow and only weakly voltage-dependent compared to human KV1.3 in shark electrosensory (ampullary) organs (Extended Data Fig. 1e). (Fig. 2d, Extended Data Fig. 4d). Consequently, shark KV1.3 produced Furthermore, both skate and shark orthologues contain a charged motif a conductance that could be repetitively stimulated with undiminished within the S2–S3 region of the IV repeat domain that confers low volt- amplitude, whereas human KV1.3 quickly inactivated with repetitive 6 age threshold to skate CaV1.3 (Extended Data Fig. 1g). Shark ampul- voltage pulses (Fig. 2e). These biophysical properties resemble those of lary organs also expressed several CaV auxiliary subunits, and the ICaV native shark IKV, which also exhibited a comparable pharmacological current density and activation threshold exhibited in shark elec- profile (Extended Data Fig. 4e, f). One notable difference is that the trosensory cells was similar to that of skates (Extended Data Fig. 1h, i). deactivation kinetics of native IKV were faster than those of the cloned Our results therefore suggest that CaV1.3 mediates the major depolar- channel, particularly at positive voltages. As such, although KV1.3 izing current in both skate and shark electrosensory cells. forms the predominant K+ conductance in shark electrosensory cells, In skate electrosensory cells, Ca2+ influx activated outward K+ cur- additional regulatory mechanisms may be provided by auxiliary subu- rents at relatively negative potentials to occlude inward Ca2+ currents6 nits, signalling cascades or structural proteins to further enhance rapid (Extended Data Fig. 2a–c). In shark cells, however, we observed large K+ deactivation. currents that were activated at more positive voltages and did not affect The voltage-dependent properties of shark KV1.3 probably derive the amplitude of inward current, which suggests reduced functional from altered voltage-sensor domain movements, which we verified by interaction between Ca2+ and K+ currents (Extended Data Fig. 2a– comparing gating currents from modified non-conductive shark and + c). Indeed, K currents were not affected by an ICaV blocker or a BK human KV1.3. For these experiments, we analysed a human isoform that 1Department of Physiology, University of California, San Francisco, San Francisco, CA, USA. 2Present address: Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, 3 USA. These authors contributed equally: Nicholas W. Bellono, Duncan B. Leitch. *e-mail: [email protected]; [email protected] NATURE| www.nature.com/nature © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. RESEARCH LETTER a b c a Activation b Deactivation c K+ 200 pA Control 1,000 1.0 1.0 100 100 ms Cd2+ 600 IbTx 10 max 0.5 4-AP I/I 0.5 Shark (ms) pA/pF 200 W Human 1 0.0 Normalized I –80 80 0.0 0.1 –200 –100 –50 0 50 Control Cd2+ 4-AP Voltage (mV) –100 –50 050 –100–50 050 Voltage (mV) 50 ms Voltage (mV) Voltage (mV) d Deact. Act. e f g 30 d 1.0 e 100 Inactivation Shark 20 0.8 10 Cav1.3 peak 0.6 I 10 s 10 / (ms) 0.4 10s W 1 Cav1.3 I pression (FPKM) pression 0 Kv1.3 0.2 Human 0.1 Ex 1 Kv1.3 DAPI 0.0 5 ms –100 0 100 5 ms kcna3akcnqkcnb1kcnh5akcnh2a n Voltage (mV) Shark 1 s Huma Fig. 1 | Major K+ current in shark electrosensory cells. a, Profile of chain Fig. 2 | Properties of shark KV. a, Average G–V relationships from catshark (Scyliorhinus retifer). b, Representative KV currents elicited by currents measured at −30 mV after activating pulses at the indicated increasing voltage pulses from −100 mV were inhibited by the KV blocker + = − ± = ± 4-AP but not by the I blocker Cd2 . Average I–V relationship from voltage. For shark KV1.3: Va1/2 5.4 0.4 mV, Ka 7.6 0.4 mV; CaV = − ± = ± = peak currents. n = 5, P < 0.0001 for outward control currents versus 4-AP, for human KV1.3: Va1/2 30.7 0.5 mV, Ka 4.7 0.5 mV. n 10, < two-way analysis of variance (ANOVA) with post hoc Tukey test. The P 0.0001 for Va1/2, two-tailed Student’s t-test. b, Deactivation kinetics − BK channel antagonist IbTx did not affect currents. c, G–V relationships of KV currents at 30 mV normalized to maximal amplitude elicited at 40 mV. Arrows indicate when deactivation rates were measured. exhibited a half-maximal activation voltage (Va1/2) of −4.1 ± 0.8 mV Right, expanded view. c, Deactivation properties of shark (red) and with a slope factor (Ka) of 9.5 ± 0.7 mV. n = 11. d, IKV activation and deactivation kinetics. Values of the time constant τ are obtained from human (black) KV1.3 channels. τ values from single exponential fits of single exponential fits of activation in response to the indicated voltage deactivation at the indicated voltages after an activating prepulse of 40 mV. = < pulses (10-mV increments from −100 mV) or deactivation at the indicated n 6, P 0.0001 for comparison of orthologues across voltage pulses, two-way ANOVA with post hoc Bonferroni test. d, Normalized currents voltages after an activating prepulse of 40 mV. n = 6. e, IKV inactivation over a 10-s, 40-mV pulse (top, 57 ± 4% current remained) and cumulative showing inactivation in response to a 10-s, 40-mV pulse. Average current = < inactivation properties in response to repetitive 40-mV pulses delivered remaining at the end of a 10-s step.