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Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

Amjad A, Hernandez-Clavijo A, Pifferi S, Maurya DK, Boccaccio A, Franzot J, Rock J, Menini A - J. Gen. Physiol. (2015)

Bottom Line: Ion substitution experiments and partial blockade by commonly used Cl(-) channel blockers indicated that Ca(2+) activates mainly anionic currents in these neurons.We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons.Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons.

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Affiliation: Neurobiology Group, SISSA, Scuola Internazionale Superiore di Studi Avanzati, 34136 Trieste, Italy.

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Lack of Ca2+-activated currents in vomeronasal sensory neurons from TMEM16A conditional knockout mice. Representative whole-cell recordings obtained with an intracellular solution containing 1.5 µM Ca2+ from vomeronasal sensory neurons dissociated from TMEM16Afl/fl (A) or TMEM16A cKO (B) mice. Insets show the enlargement of the recordings of voltage-gated inward currents activated by a step to 0 mV from the holding potential of −100 mV, as indicated in the voltage protocol at the top of the figure. (C) Mean current amplitudes measured at −100 or 100 mV with intracellular pipette solution containing nominally 0 (black bar; n = 6) or 1.5 µM free Ca2+ from WT (blue bar; n = 28) or TMEM16A cKO mice (red bar; n = 20). Error bars indicate SEM (**, P < 0.01; Dunn–Hollander–Wolfe test after Krustal–Wallis analysis).
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fig11: Lack of Ca2+-activated currents in vomeronasal sensory neurons from TMEM16A conditional knockout mice. Representative whole-cell recordings obtained with an intracellular solution containing 1.5 µM Ca2+ from vomeronasal sensory neurons dissociated from TMEM16Afl/fl (A) or TMEM16A cKO (B) mice. Insets show the enlargement of the recordings of voltage-gated inward currents activated by a step to 0 mV from the holding potential of −100 mV, as indicated in the voltage protocol at the top of the figure. (C) Mean current amplitudes measured at −100 or 100 mV with intracellular pipette solution containing nominally 0 (black bar; n = 6) or 1.5 µM free Ca2+ from WT (blue bar; n = 28) or TMEM16A cKO mice (red bar; n = 20). Error bars indicate SEM (**, P < 0.01; Dunn–Hollander–Wolfe test after Krustal–Wallis analysis).

Mentions: To determine the contribution of TMEM16A to Ca2+-activated Cl− currents in vomeronasal sensory neurons, we measured currents in isolated neurons from TMEM16A cKO mice in the whole-cell configuration with intracellular solutions containing various amounts of free [Ca2+]i. Fig. 11 (A and B) shows the comparison between representative recordings from TMEM16Afl/fl and TMEM16A cKO mice. Currents were activated by voltage steps between −100 and 100 mV from a holding potential of 0 mV, followed by a step to −100 mV and return to 0 mV. The last part of the current trace elicited by the depolarizing voltage from −100 to 0 mV was used as a control of the viability of neurons, as it allowed the measurement of voltage-gated inward currents (likely Na+ currents; insets of Fig. 11, A and B). The kinetics of currents activated by 1.5 µM Ca2+ in neurons from TMEM16Afl/fl mice resembled those measured in control WT mice (Figs. 11 A and 1 A), whereas currents in vomeronasal neurons from TMEM16A cKO mice were not significantly different from currents in the absence of Ca2+ (Figs. 11, B and C, and 1 A). The inset of Fig. 11 B shows a representative voltage-gated current (elicited by a 0-mV step from −100 mV), indicating that the neuron was viable but did not have currents activated by Ca2+. All together, we tested 40 viable neurons from 6 TMEM16A cKO mice at [Ca2+]i varying from 0.5 µM to 2 mM Ca2+ and did not find a significant difference from currents measured in 0 Ca2+. Furthermore, we measured currents in inside-out patches from dendritic knobs of vomeronasal neurons from TMEM16A cKO mice and did not find any measurable Ca2+-activated current (not depicted).


Conditional knockout of TMEM16A/anoctamin1 abolishes the calcium-activated chloride current in mouse vomeronasal sensory neurons.

Amjad A, Hernandez-Clavijo A, Pifferi S, Maurya DK, Boccaccio A, Franzot J, Rock J, Menini A - J. Gen. Physiol. (2015)

Lack of Ca2+-activated currents in vomeronasal sensory neurons from TMEM16A conditional knockout mice. Representative whole-cell recordings obtained with an intracellular solution containing 1.5 µM Ca2+ from vomeronasal sensory neurons dissociated from TMEM16Afl/fl (A) or TMEM16A cKO (B) mice. Insets show the enlargement of the recordings of voltage-gated inward currents activated by a step to 0 mV from the holding potential of −100 mV, as indicated in the voltage protocol at the top of the figure. (C) Mean current amplitudes measured at −100 or 100 mV with intracellular pipette solution containing nominally 0 (black bar; n = 6) or 1.5 µM free Ca2+ from WT (blue bar; n = 28) or TMEM16A cKO mice (red bar; n = 20). Error bars indicate SEM (**, P < 0.01; Dunn–Hollander–Wolfe test after Krustal–Wallis analysis).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig11: Lack of Ca2+-activated currents in vomeronasal sensory neurons from TMEM16A conditional knockout mice. Representative whole-cell recordings obtained with an intracellular solution containing 1.5 µM Ca2+ from vomeronasal sensory neurons dissociated from TMEM16Afl/fl (A) or TMEM16A cKO (B) mice. Insets show the enlargement of the recordings of voltage-gated inward currents activated by a step to 0 mV from the holding potential of −100 mV, as indicated in the voltage protocol at the top of the figure. (C) Mean current amplitudes measured at −100 or 100 mV with intracellular pipette solution containing nominally 0 (black bar; n = 6) or 1.5 µM free Ca2+ from WT (blue bar; n = 28) or TMEM16A cKO mice (red bar; n = 20). Error bars indicate SEM (**, P < 0.01; Dunn–Hollander–Wolfe test after Krustal–Wallis analysis).
Mentions: To determine the contribution of TMEM16A to Ca2+-activated Cl− currents in vomeronasal sensory neurons, we measured currents in isolated neurons from TMEM16A cKO mice in the whole-cell configuration with intracellular solutions containing various amounts of free [Ca2+]i. Fig. 11 (A and B) shows the comparison between representative recordings from TMEM16Afl/fl and TMEM16A cKO mice. Currents were activated by voltage steps between −100 and 100 mV from a holding potential of 0 mV, followed by a step to −100 mV and return to 0 mV. The last part of the current trace elicited by the depolarizing voltage from −100 to 0 mV was used as a control of the viability of neurons, as it allowed the measurement of voltage-gated inward currents (likely Na+ currents; insets of Fig. 11, A and B). The kinetics of currents activated by 1.5 µM Ca2+ in neurons from TMEM16Afl/fl mice resembled those measured in control WT mice (Figs. 11 A and 1 A), whereas currents in vomeronasal neurons from TMEM16A cKO mice were not significantly different from currents in the absence of Ca2+ (Figs. 11, B and C, and 1 A). The inset of Fig. 11 B shows a representative voltage-gated current (elicited by a 0-mV step from −100 mV), indicating that the neuron was viable but did not have currents activated by Ca2+. All together, we tested 40 viable neurons from 6 TMEM16A cKO mice at [Ca2+]i varying from 0.5 µM to 2 mM Ca2+ and did not find a significant difference from currents measured in 0 Ca2+. Furthermore, we measured currents in inside-out patches from dendritic knobs of vomeronasal neurons from TMEM16A cKO mice and did not find any measurable Ca2+-activated current (not depicted).

Bottom Line: Ion substitution experiments and partial blockade by commonly used Cl(-) channel blockers indicated that Ca(2+) activates mainly anionic currents in these neurons.We used the Cre-loxP system to selectively knock out TMEM16A in cells expressing the olfactory marker protein, which is found in mature vomeronasal sensory neurons.Immunohistochemistry confirmed the specific ablation of TMEM16A in vomeronasal neurons.

View Article: PubMed Central - HTML - PubMed

Affiliation: Neurobiology Group, SISSA, Scuola Internazionale Superiore di Studi Avanzati, 34136 Trieste, Italy.

Show MeSH
Related in: MedlinePlus