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Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system.

Phelan P, Goulding LA, Tam JL, Allen MJ, Dawber RJ, Davies JA, Bacon JP - Curr. Biol. (2008)

Bottom Line: A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons.When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification.These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Kent, Canterbury, UK. p.phelan@kent.ac.uk

ABSTRACT
Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

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ShakB(N+16) and ShakB(L) Form Heterotypic Channels that Are Asymmetrically Gated by Voltage, Whereas Homotypic Channels Exhibit Symmetrical Voltage ResponsesXenopus oocytes injected with shakB RNAs were paired and recorded by dual voltage clamp. Both cells of a pair were clamped at a holding potential of −80 mV. Transjunctional voltage steps (Vj, mV) were then applied to one cell while the current (Ij, nA) required to maintain the other cell at the holding potential was recorded. Junctional conductance (Gj, μS) is Ij/Vj.(A–E) Heterotypic cell pairs.(A–C) Representative traces from cell pairs injected with 0.25 ng RNA. Junctional currents (A and C) were elicited by application of Vj steps (B) to (A) the ShakB(N+16)-expressing cell or (C) the ShakB(L)-expressing cell. Gj is shown for depolarizing (below baseline) and hyperpolarizing (above baseline) 10 mV steps. Mean values are provided in Table S3.(D–E) Gj/Vj plots. Initial (open symbols) and steady-state (closed symbols) Gjs recorded in response to application of Vj steps to (D) the ShakB(N+16)-expressing cell or (E) the ShakB(L)-expressing cell. Gjs, normalized to their values at −10 mV (D) and 10 mV (E), are mean ± SD for n = 4 pairs injected with 0.1–0.25 ng RNA. Steady-state data are fitted to a Boltzmann equation (parameters in Table S3). Heterotypic channels respond asymmetrically to applied voltage.(F–J) Homotypic cell pairs.(F–H) Typical recordings and Gj/Vj plots (I and J) for oocyte pairs in which both cells expressed (F and I) ShakB(N+16) (0.5–2 ng RNA) or (H and J) ShakB(L) (0.05–0.25 ng RNA). (F and H) Gj is shown for depolarizing and hyperpolarizing 10 mV steps. (I and J) Initial (open symbols) and steady-state (closed symbols) Gjs normalized to their values at Vj = ±10 mV are mean ± SD for n = 8 (I) and n = 3 (J) pairs. Curves in (J) are Boltzmann fits of the data. ShakB(N+16) channels show no significant voltage sensitivity. ShakB(L) channels exhibit a symmetrical response to applied voltage. Mean Gjs and Boltzmann parameters are in Table S3.
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fig4: ShakB(N+16) and ShakB(L) Form Heterotypic Channels that Are Asymmetrically Gated by Voltage, Whereas Homotypic Channels Exhibit Symmetrical Voltage ResponsesXenopus oocytes injected with shakB RNAs were paired and recorded by dual voltage clamp. Both cells of a pair were clamped at a holding potential of −80 mV. Transjunctional voltage steps (Vj, mV) were then applied to one cell while the current (Ij, nA) required to maintain the other cell at the holding potential was recorded. Junctional conductance (Gj, μS) is Ij/Vj.(A–E) Heterotypic cell pairs.(A–C) Representative traces from cell pairs injected with 0.25 ng RNA. Junctional currents (A and C) were elicited by application of Vj steps (B) to (A) the ShakB(N+16)-expressing cell or (C) the ShakB(L)-expressing cell. Gj is shown for depolarizing (below baseline) and hyperpolarizing (above baseline) 10 mV steps. Mean values are provided in Table S3.(D–E) Gj/Vj plots. Initial (open symbols) and steady-state (closed symbols) Gjs recorded in response to application of Vj steps to (D) the ShakB(N+16)-expressing cell or (E) the ShakB(L)-expressing cell. Gjs, normalized to their values at −10 mV (D) and 10 mV (E), are mean ± SD for n = 4 pairs injected with 0.1–0.25 ng RNA. Steady-state data are fitted to a Boltzmann equation (parameters in Table S3). Heterotypic channels respond asymmetrically to applied voltage.(F–J) Homotypic cell pairs.(F–H) Typical recordings and Gj/Vj plots (I and J) for oocyte pairs in which both cells expressed (F and I) ShakB(N+16) (0.5–2 ng RNA) or (H and J) ShakB(L) (0.05–0.25 ng RNA). (F and H) Gj is shown for depolarizing and hyperpolarizing 10 mV steps. (I and J) Initial (open symbols) and steady-state (closed symbols) Gjs normalized to their values at Vj = ±10 mV are mean ± SD for n = 8 (I) and n = 3 (J) pairs. Curves in (J) are Boltzmann fits of the data. ShakB(N+16) channels show no significant voltage sensitivity. ShakB(L) channels exhibit a symmetrical response to applied voltage. Mean Gjs and Boltzmann parameters are in Table S3.

Mentions: shakB(n+16) and shakB(l) RNAs were transcribed in vitro and microinjected into connexin-depleted Xenopus oocytes [6, 27]. Figure S2 confirms that both RNAs are efficiently translated by oocytes. The ability of the expressed proteins to form channels was assessed by dual voltage clamp electrophysiology [28] of cell pairs in which one cell expressed ShakB(N+16) and the other ShakB(L) (heterotypic) or both cells of a pair expressed the same protein (homotypic). In heterotypic configuration, channels were reliably induced at RNA levels of 0.1–0.5 ng; the voltage sensitivity of these channels differed significantly from that of homotypic channels composed of either protein (Figure 4 and Table S3).


Molecular mechanism of rectification at identified electrical synapses in the Drosophila giant fiber system.

Phelan P, Goulding LA, Tam JL, Allen MJ, Dawber RJ, Davies JA, Bacon JP - Curr. Biol. (2008)

ShakB(N+16) and ShakB(L) Form Heterotypic Channels that Are Asymmetrically Gated by Voltage, Whereas Homotypic Channels Exhibit Symmetrical Voltage ResponsesXenopus oocytes injected with shakB RNAs were paired and recorded by dual voltage clamp. Both cells of a pair were clamped at a holding potential of −80 mV. Transjunctional voltage steps (Vj, mV) were then applied to one cell while the current (Ij, nA) required to maintain the other cell at the holding potential was recorded. Junctional conductance (Gj, μS) is Ij/Vj.(A–E) Heterotypic cell pairs.(A–C) Representative traces from cell pairs injected with 0.25 ng RNA. Junctional currents (A and C) were elicited by application of Vj steps (B) to (A) the ShakB(N+16)-expressing cell or (C) the ShakB(L)-expressing cell. Gj is shown for depolarizing (below baseline) and hyperpolarizing (above baseline) 10 mV steps. Mean values are provided in Table S3.(D–E) Gj/Vj plots. Initial (open symbols) and steady-state (closed symbols) Gjs recorded in response to application of Vj steps to (D) the ShakB(N+16)-expressing cell or (E) the ShakB(L)-expressing cell. Gjs, normalized to their values at −10 mV (D) and 10 mV (E), are mean ± SD for n = 4 pairs injected with 0.1–0.25 ng RNA. Steady-state data are fitted to a Boltzmann equation (parameters in Table S3). Heterotypic channels respond asymmetrically to applied voltage.(F–J) Homotypic cell pairs.(F–H) Typical recordings and Gj/Vj plots (I and J) for oocyte pairs in which both cells expressed (F and I) ShakB(N+16) (0.5–2 ng RNA) or (H and J) ShakB(L) (0.05–0.25 ng RNA). (F and H) Gj is shown for depolarizing and hyperpolarizing 10 mV steps. (I and J) Initial (open symbols) and steady-state (closed symbols) Gjs normalized to their values at Vj = ±10 mV are mean ± SD for n = 8 (I) and n = 3 (J) pairs. Curves in (J) are Boltzmann fits of the data. ShakB(N+16) channels show no significant voltage sensitivity. ShakB(L) channels exhibit a symmetrical response to applied voltage. Mean Gjs and Boltzmann parameters are in Table S3.
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fig4: ShakB(N+16) and ShakB(L) Form Heterotypic Channels that Are Asymmetrically Gated by Voltage, Whereas Homotypic Channels Exhibit Symmetrical Voltage ResponsesXenopus oocytes injected with shakB RNAs were paired and recorded by dual voltage clamp. Both cells of a pair were clamped at a holding potential of −80 mV. Transjunctional voltage steps (Vj, mV) were then applied to one cell while the current (Ij, nA) required to maintain the other cell at the holding potential was recorded. Junctional conductance (Gj, μS) is Ij/Vj.(A–E) Heterotypic cell pairs.(A–C) Representative traces from cell pairs injected with 0.25 ng RNA. Junctional currents (A and C) were elicited by application of Vj steps (B) to (A) the ShakB(N+16)-expressing cell or (C) the ShakB(L)-expressing cell. Gj is shown for depolarizing (below baseline) and hyperpolarizing (above baseline) 10 mV steps. Mean values are provided in Table S3.(D–E) Gj/Vj plots. Initial (open symbols) and steady-state (closed symbols) Gjs recorded in response to application of Vj steps to (D) the ShakB(N+16)-expressing cell or (E) the ShakB(L)-expressing cell. Gjs, normalized to their values at −10 mV (D) and 10 mV (E), are mean ± SD for n = 4 pairs injected with 0.1–0.25 ng RNA. Steady-state data are fitted to a Boltzmann equation (parameters in Table S3). Heterotypic channels respond asymmetrically to applied voltage.(F–J) Homotypic cell pairs.(F–H) Typical recordings and Gj/Vj plots (I and J) for oocyte pairs in which both cells expressed (F and I) ShakB(N+16) (0.5–2 ng RNA) or (H and J) ShakB(L) (0.05–0.25 ng RNA). (F and H) Gj is shown for depolarizing and hyperpolarizing 10 mV steps. (I and J) Initial (open symbols) and steady-state (closed symbols) Gjs normalized to their values at Vj = ±10 mV are mean ± SD for n = 8 (I) and n = 3 (J) pairs. Curves in (J) are Boltzmann fits of the data. ShakB(N+16) channels show no significant voltage sensitivity. ShakB(L) channels exhibit a symmetrical response to applied voltage. Mean Gjs and Boltzmann parameters are in Table S3.
Mentions: shakB(n+16) and shakB(l) RNAs were transcribed in vitro and microinjected into connexin-depleted Xenopus oocytes [6, 27]. Figure S2 confirms that both RNAs are efficiently translated by oocytes. The ability of the expressed proteins to form channels was assessed by dual voltage clamp electrophysiology [28] of cell pairs in which one cell expressed ShakB(N+16) and the other ShakB(L) (heterotypic) or both cells of a pair expressed the same protein (homotypic). In heterotypic configuration, channels were reliably induced at RNA levels of 0.1–0.5 ng; the voltage sensitivity of these channels differed significantly from that of homotypic channels composed of either protein (Figure 4 and Table S3).

Bottom Line: A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons.When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification.These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, University of Kent, Canterbury, UK. p.phelan@kent.ac.uk

ABSTRACT
Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates; innexins/pannexins encode gap-junction proteins in prechordates and chordates. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely. The phenomenon of rectification was first described five decades ago, but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal). When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

Show MeSH
Related in: MedlinePlus