Mongeon R , Walogorsky M , Urban J , Mandel G , Ono F , Brehm P .

NS-18205 NINDS NIH HHS , Howard Hughes Medical Institute , R01 NS018205 NINDS NIH HHS , R37 NS018205 NINDS NIH HHS

	Figure 1. Comparison of decay time constants (τdecay) of spontaneous synaptic currents (mEPCs) between wild-type and acetylcholinesterase-null (zimtm205) 72–82-hpf larval zebrafish. (A) Sample mEPCs from wild-type fast (left) and wild-type slow (right) skeletal muscle. The 10–90% decay phase is fit to a single-exponential function (red) to obtain τdecay. (B) Sample mEPCs from zimtm205 fast (left) and zimtm205 slow (right) skeletal muscle, each fit in the same manner as those in A. (C) The frequency histogram of τdecay values for individual mEPCs from wild-type fast muscle (black; n = 14 recordings), zimtm205 fast muscle (red; n = 28 recordings), wild-type slow muscle (green; n = 15 recordings), and zimtm205 slow muscle (blue; n = 15 recordings). (D) The mean τdecay values for each recording (filled circles) along with the overall mean ± SD (open circle) are shown for each condition.
	Figure 2. Single-channel ACh-activated currents from fast skeletal muscle of 72-hpf larval zebrafish. (A) A sample ∼0.25-s trace showing two amplitude classes of ACh-activated channels (300 nM) recorded from a cell-attached patch at +30 mV applied potential. The open channel levels for the primary (1°) and secondary (2°) amplitude classes are indicated by the dashed lines. (B) The amplitude frequency histogram for openings recorded from a single patch at +50 mV applied potential. The mean value for each recording (filled symbols) is shown below the sample histogram for both the 1° and 2° classes, along with overall mean values ± SD (open circles). (C) The current–voltage relations for the 1° (black) and 2° (red) single-channel amplitude classes. Each symbol represents the mean amplitude ± SD values of all patches (n = 12) for each applied potential. (D) A burst-duration frequency histogram for the 1° amplitude class from a single recording fit with an exponential function. The mean burst-duration values for each recording of the 1° class (black filled symbols) and 2° class (red filled symbols) are shown along with the overall mean ± SD (open circles) below the histogram. (E) The relationship between applied potential and mean burst duration of the 1° amplitude class is exponential and corresponds to an e-fold change per 122 mV. Each symbol represents the mean ± SD (n = 8 recordings).
	Figure 3. Comparison of single-channel mean burst duration and τdecay of synaptic current for fast and slow muscle recordings. The mean values for individual recordings are indicated by filled symbols, and the overall mean ± SD is indicated as the open symbol for each condition.
	Figure 4. Single-channel ACh-activated currents from slow skeletal muscle of 72-hpf larval zebrafish. (A) A sample ∼0.25-s trace showing two amplitude classes of ACh-activated channels (300 nM) recorded from a cell-attached patch at +50 mV applied potential. The open channel levels for the primary (1°) and secondary (2°) amplitude classes are indicated by the dashed lines. (B) The amplitude frequency histogram for a single recording with two amplitude classes (1° and 2°) at +50 mV applied potential. The mean values for each class are indicated for individual recordings (filled symbols) along the overall mean ± SD (open symbols) below the histogram. (C) The current–voltage relations for the pooled 1° openings. Each symbol represents the mean amplitude ± SD values of all patches (n = 8) for each applied potential. (D) A burst-duration frequency histogram from a single recording fit to the sum of two exponential functions. Only the 1° amplitude class was included in the distribution. The mean values for the brief (filled triangles) and long (filled circles) mean burst duration for the 1° class are indicated along with the overall mean ± SD (open symbols). The 2° class was fit separately to a single exponential (not depicted), and the individual mean values for burst duration for each recording (red filled circles) are indicated along with the overall mean ± SD (red open symbol). (E) The relationship between applied potential and mean burst duration is shown for all 1° class openings fit with linear (black) and exponential (red) curves. Each symbol represents the mean ± SD (n = 10 recordings).
	Figure 5. Zebrafish AChR subunit identification and functional expression in Xenopus oocytes. (A) A phylogenetic tree of AChR subunit genes constructed by the UPGMA method. Subunit sequences of human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), puffer fish (Takifugu rubripes), and frog (Xenopus tropicalis) are included. Zebrafish genes are highlighted in red. (B) Two microelectrode recordings of membrane currents elicited by 30 µM ACh (bar) at a −80 mV holding potential. Shown are responses from oocytes expressing αβδε (red trace), αβδγ (green trace), or αβδ (blue trace) RNA. (C) The mean ± SD macroscopic current amplitude associated with the RNA subunit combinations is indicated. Xenopus αβδγ RNA is shown for comparison, and the number of oocytes tested is indicated for each RNA combination tested. The three combinations that yielded currents suitable for single-channel recordings are indicated by colored bars.
	Figure 6. Functional properties of ACh-activated single-channel currents from Xenopus oocytes expressing αβδε (A–E), αβδγ (F–J), and αβδ (K–O) subunit RNA. (A) Sample recording from a patch expressing αβδε at −100 mV. (B) A representative amplitude histogram for one recording, with individual mean values for all recordings (filled circles; n = 12) shown below along with the overall mean ± SD (open circle) at −100 mV. (C) The cumulative current–voltage relations for eight recordings. (D) A representative burst-duration histogram fit by the sum of two exponentials. The time constants for each exponential are indicated for each recording (filled circles; n = 7) along with the overall mean ± SD for all recordings (open circles) at −100 mV. (E) The relationship between burst duration and membrane potential for seven recordings fit to an exponential function. The voltage dependence corresponds to e-fold change per 156 mV over this range. (F) Sample recording from a patch expressing αβδγ at −100 mV. (G) A representative amplitude histogram from one recording showing a 1° (black) and 2° (red) amplitude class of openings. The individual mean values for 10 recordings (filled circles) are shown along with the overall mean ± SD (open circles) for each class at −100 mV. (H) The cumulative current–voltage relations for the primary class for eight recordings. (I) A representative burst-duration histogram fit by the sum of two exponentials. The time constants for each exponential are indicated for each recording (filled circles; n = 9) as well as the overall mean ± SD (open circles) at −100 mV. (J) The relationship between burst duration and membrane potential for four recordings fit to an exponential function. The voltage dependence corresponds to e-fold change per 141 mV over this range. (K) Sample recording from a patch expressing αβδ at −100 mV. (L) A representative amplitude frequency histogram from a single recording with the individual mean values for all recordings (filled circles; n = 13) along with the overall mean ± SD (open circle) at −100 mV. (M) The cumulative current–voltage relations for 11 recordings. (N) A representative burst-duration histogram fit by the sum of two exponentials. The individual time constants for each exponential are indicated for each recording (filled circles; n = 6) as well as the overall mean ± SD for all recordings (open circles) at −100 mV. (O) The relationship between burst duration and membrane potential for six recordings fit to an exponential function over the range of values shown yielding an e-fold change in burst duration per 769 mV.
	Figure 7. Comparisons between fast and slow muscle ACh-activated channels and individually identified receptor isoforms expressed in Xenopus oocytes. (A) Comparisons of single-channel conductance for α2βδε, α2βδγ, and α2βδ2 receptor isoforms in oocytes to both the 1° and 2° amplitude classes from muscle. The values from individual recordings (filled circles) and overall mean ± SD values (open circles) are indicated for each condition. (B) Comparisons of single-channel mean burst duration of α2βδε, α2βδγ, and α2βδ2 receptor isoforms to the mean burst duration of 1° fast and slow muscle receptors. The values from individual recordings are indicated by filled circles, and the overall mean ± SD values are indicated by the open circle for each condition.
	Figure 8. Differences in rectification between fast and slow muscle ACh-activated single-channel currents. Sample single-channel traces at holding potentials that are either positive or negative to the current reversal potential (∼0 mV) for fast muscle (A) and slow muscle (B). (C) The current–voltage relations for fast muscle synaptic currents, with each point representing the mean ± SD of seven recordings, fit by a linear curve. (D) The current–voltage relations for synaptic currents in slow muscle, with each point representing the mean ± SD for 10 recordings, fit by a second-order polynomial curve.
	Figure 9. Inward rectification of the zebrafish αβδ channel isoform. (A) Sample ACh-activated single-channel currents recorded at the indicated potentials from cell-attached patches of Xenopus oocytes injected with either αβδ (left) or αβδε (right) RNA. (B) Whole oocyte ACh-activated macroscopic currents recorded at −80 mV and subsequently ramped from −80 to +80 mV over a 2-s period. Oocytes were either injected with αβδ (red) or αβδε (black). Leak currents recorded before ACh application are indicated in blue. (C) Current–voltage relations of macroscopic currents from αβδ (red; n = 5) and αβδε (black; n = 3) RNA-injected oocytes were normalized to peak current and fit with fifth-order polynomial (red) or linear curves (black).
	Figure 10. Comparison of synaptic current properties after α, γ, and ε AChR subunit knockdown in fast and slow muscle. All recordings were performed at a −70 mV holding potential. (A) Mean amplitude of spontaneous synaptic currents recorded from fast muscle for individual cells (filled circles) and overall mean ± SD (open circles) for each condition. (B) Mean amplitude of spontaneous synaptic currents recorded from slow muscle for individual cells (filled circles) and overall mean ± SD (open circles) for each condition. (C) Mean τdecay values for individual fast muscle recordings (filled circles) and overall mean ± SD (open circles) for each condition. (D) Mean τdecay values for individual slow muscle recordings (filled circles) and overall mean ± SD (open circles) for each condition.
	Figure 11. Alignment of residues within AChR subunits of different species. The residues outlined with red form the intermediate charged ring and have been shown to be involved in channel rectification of rat neuronal α3 and tunicate BDGE3 subunit–containing receptors. The second transmembrane region (TM2) is indicated by the shaded green, and the outer and inner charged rings are indicated by the blue shade.

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