Brv1 Is Required for Drosophila Larvae to Sense Gentle Touch Mingfeng Zhang, Xia Li, Honglan Zheng, Xiaoxu Wen, Sihan Chen, Jia Ye, Siyang Tang, Fuqiang Yao, Yuezhou Li, Zhiqiang Yan  Cell Reports  Volume 23, Issue 1, Pages 23-31 (April 2018) DOI: 10.1016/j.celrep.2018.03.041 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 23, 23-31DOI: (10.1016/j.celrep.2018.03.041) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Brv1 Is Essential for Class III da Neurons of Larvae to Sense Gentle Touch (A) Behavioral response cores of wild-type (WT Ctrl), brv1 LOF mutant (brv1STOP and brv1DEL), brv1 rescue (brv1STOP rescue and brv1DEL rescue), brv1 knockdown (class III RNAi brv1), and control (class III RNAi Ctrl) larvae in response to gentle touch (mean ± SEM, n = 20). The score is the sum of 5 tests for each sample. The scores for the response are as follows: 0, no response; 1, pause; 2, recoil; 3, single reverse contractile wave; and 4, multiple reverse contractions. (B) Percentage of each scored response in larvae of the indicated genotype. (C) Representative Ca2+ response traces of class III da neurons in WT (red), brv1 RNAi (green), brv1STOP (blue), and brv1STOP rescue (gray) larvae. The arrowhead indicates stimulus onset (40-μm displacement for 300 ms). (D) Summary of Ca2+ responses of class III da neurons with different genotypes (mean ± SEM, total of 15 cells from 5 larvae). (E) Immunostaining of Brv1 in class III da neurons labeled with class III-neuron-specific 19-12-Gal4-driven GFP. Scale bar, 30 μm. The enlarged view shows the spike-like protrusion (indicated by an arrow), which is the hallmark of class III neurons. Genotypes are as follows. WT control: w1118. brv1STOP: brv1L563 > STOP. brv1DEL: brv1112–204 > DEL. brv1STOP rescue: brv1L563 > STOP/UAS-Brv1; 19-21-GAL4/+. brv1DEL rescue: brv1112–204 > DEL/UAS-Brv1; 19-21-GAL4/+. RNAi control: w1118, UAS-brv1-RNAi. Class III brv1 RNAi: 19-12-GAL4, UAS-brv1-RNAi. Cell Reports 2018 23, 23-31DOI: (10.1016/j.celrep.2018.03.041) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Brv1 Heterologously Expressed in HEK293T Cells Forms Cation Channels (A) Whole-cell I-V relationships of spontaneously activated Brv1 channels (red triangles), Brv1 treated with 100 μM gadolinium (Gd3+, green circles) or 50 μM ruthenium red (RR, blue triangles), or Brv1 bathing in NaMES solution (brown diamonds). Vector-transfected HEK293T served as control (black squares). The currents were evoked by 500-ms pulses from −100 to 100 mV in 20-mV increments. (B) I-V relationships of Brv1 channel currents recorded after replacing Na+ in the extracellular saline solution with the indicated cation. (C) Relative permeability of Brv1 to different cations. The permeability ratio was estimated by average reversal-potential changes relative to a CsCl-based internal solution. (D) Single-channel currents of spontaneously activated Brv1 channels recorded from an outside-out excise patch at indicated membrane potentials. Dashed horizontal lines indicate the closed states. (E) I-V relationships of the average single-channel amplitude of Brv1-transfected cells (black circles) or vector-transfected cells (red triangles). The slope was used to estimate conductance. (F) Sequence alignment of the putative pore region of Brv1 and PKD homologs. Asterisks mark negatively charged residues D552 and E558 targeted for mutagenesis. (G) I-V curves of spontaneously activated WT Brv1 (red triangles), mutants D552A (blue circles), and E558A (green squares). Data are represented as mean ± SEM, n = 10. Cell Reports 2018 23, 23-31DOI: (10.1016/j.celrep.2018.03.041) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Brv1 Conducts Mechanical Currents (A) Currents of an outside-out patch (held at −80 mV) recorded from Brv1- or vector-transfected cells in response to 500-ms pulses of negative pressure from −40 to −80 mmHg. (B) Dose-dependent curves of pressure-induced currents recorded from outside-out patches in Brv1-transfected cells (black circles) or vector-transfected cells (red triangles). (C) Sample traces showing the short latency of negative pressure-induced currents. The arrow indicates the onset of stimulus. The activation time constant (τ activation) was obtained from a single exponential fit of the negative pressure-activated currents to reach peak amplitude. (D) Activation time constant of Brv1 is pressure dependent. (E) Adaption time constant of Brv1 is pressure dependent. τ adaptation was obtained from a single exponential fit of the decay of negative pressure-activated currents. (F) Sample traces of repetitive 0.5-s stimuli (−80 mmHg) at 1-s intervals between stimuli. (G) Amplitude of pressure-activated currents in response to repetitive stimuli. (H) Sample trace of Brv1 currents in response to a series of testing and adapting stimuli (−10 mmHg) at a 100-ms interval between the two stimuli. (I) Amplitudes of Brv1 currents in response to serial adapting stimuli. (J) Single-channel currents of spontaneously activated (0 mmHg) or negative pressure-activated (−80 mmHg) Brv1 channels. Multiple openings (O1–O3) are shown in histogram analysis. O, open; C, closed. The dashed horizontal line indicates the closed state. Solid horizontal lines indicate the open state. (K) Single-channel current amplitude of spontaneously activated (spon. curr.) and negative pressure-activated (mech. curr.) Brv1 channels. (L) Sample currents of an inside-out excised patch (held at −80 mV) recorded from Brv1- or vector-transfected cells in response to 500-ms pulses of negative pressure from −40 to −80 mmHg. (M) Dose-dependent curves of pressure-induced currents recorded from inside-out patches in Brv1-transfected cells (black circles) or vector-transfected cells (red triangles). (N) Image of Brv1-reconstituted liposomes. The arrow indicates the membrane blisters used for patch clamp. (O) Sample traces of reconstituted Brv1 currents recorded from an inside-out patch (held at −20 mV) in response to 500-ms pulses of negative pressure from −30 to −70 mmHg. (P) Dose-dependent curve of pressure-induced currents recorded in the Brv1-reconstituted liposome. (Q) I-V relationship of the average single-channel amplitude of pressure-induced Brv1 activation. Data are represented as mean ± SEM, n = 10. Cell Reports 2018 23, 23-31DOI: (10.1016/j.celrep.2018.03.041) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Co-expression of Brv1 Alters Poke-Induced NOMPC Currents (A) Whole-cell I-V relationships of recorded in HEK293T cells transfected with Brv1 (red triangles), NOMPC (green squares), and Brv1+NOMPC (blue circles). The currents were evoked by 500-ms potential pulses from −100 to 100 mV in +20-mV increments. (B) Currents of an outside-out patch (held at −80 mV) recorded from Brv1-, NOMPC-, or Brv1+NOMPC-transfected cells in response to −60 mmHg of negative pressure. (C) Dose-dependent curves of pressure-induced currents recorded from outside-out patches in Brv1-transfected cells (black circles), NOMPC-transfected cells (green squares), or Brv1+NOMPC-transfected cells (red triangles). (D) Representative traces of poke-induced currents in whole-cell recording from vector-, Brv1-, NOMPC-, or Brv1+NOMPC-transfected cells. Cells were subjected to a series of mechanical steps in 1-μm increments using a glass probe and at a holding potential of −80 mV. (E) Co-expression of Brv1 increases the amplitude of poke-induced NOMPC current. (F) Co-expression of Brv1 prolongs the adaption time constant of NOMPC. τ adaptation was obtained from a single exponential fit of the decay of poke-activated currents. (G) Co-expression of Brv1 decreases the adaption of poke-induced NOMPC current. Data are represented as mean ± SEM, n = 10. Cell Reports 2018 23, 23-31DOI: (10.1016/j.celrep.2018.03.041) Copyright © 2018 The Author(s) Terms and Conditions