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A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus

Voltage-clamp recordings from single renal primary cilia. All recordings were made in the standard recording solutions except as noted. In A and C, dashed lines indicate the current level when the channels were closed. (A) Single-channel fluctuations were recorded while the cilium in the pipette was still attached to the cell. Pipette potential was strongly depolarizing (−140 mV). (B) Membrane current–voltage relations of two cilia after excision from the cell. The recording shown in black has an input resistance R = 10.1 GΩ. In the recording shown in blue (R = 7.4 GΩ), single-channel openings are visible at potentials more positive than +50 mV. (C) Activation by Ca2+ and by depolarization of large-conductance channels in an excised renal primary cilium. On the left are shown the membrane currents at three voltage-clamp potentials in the presence of 0.1 μM free cytoplasmic Ca2+. On the right are recordings with 3 μM free cytoplasmic Ca2+ present. (D) A macroscopic current activated by high cytoplasmic Ca2+ in an excised cilium. The current–voltage relation was measured in each of two pseudointracellular baths: a bath containing 0.1 μM Ca2+ (black); and a bath with 300 μM Ca2+ (red).
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Figure 3: Voltage-clamp recordings from single renal primary cilia. All recordings were made in the standard recording solutions except as noted. In A and C, dashed lines indicate the current level when the channels were closed. (A) Single-channel fluctuations were recorded while the cilium in the pipette was still attached to the cell. Pipette potential was strongly depolarizing (−140 mV). (B) Membrane current–voltage relations of two cilia after excision from the cell. The recording shown in black has an input resistance R = 10.1 GΩ. In the recording shown in blue (R = 7.4 GΩ), single-channel openings are visible at potentials more positive than +50 mV. (C) Activation by Ca2+ and by depolarization of large-conductance channels in an excised renal primary cilium. On the left are shown the membrane currents at three voltage-clamp potentials in the presence of 0.1 μM free cytoplasmic Ca2+. On the right are recordings with 3 μM free cytoplasmic Ca2+ present. (D) A macroscopic current activated by high cytoplasmic Ca2+ in an excised cilium. The current–voltage relation was measured in each of two pseudointracellular baths: a bath containing 0.1 μM Ca2+ (black); and a bath with 300 μM Ca2+ (red).

Mentions: Beads coated with ciliated cells were placed in a recording chamber and observed under phase-contrast microscopy. By applying positive pressure through the recording micropipette, it was often possible to rotate a bead until a cilium was positioned near the tip of the pipette. Suction was then applied through the pipette to draw the single cilium into the pipette (Figure2, Additional file 1). The cilium easily followed the suction because the bead was small and not attached to any surface. In 47% of 838 attempts, suction resulted in a membrane-pipette seal of resistance ≥  1 GΩ. With a cilium in the recording pipette and the cell still attached, bursts of single-channel activity were seen in a minority of cells, particularly at depolarizing potentials (Figure3A).


A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Voltage-clamp recordings from single renal primary cilia. All recordings were made in the standard recording solutions except as noted. In A and C, dashed lines indicate the current level when the channels were closed. (A) Single-channel fluctuations were recorded while the cilium in the pipette was still attached to the cell. Pipette potential was strongly depolarizing (−140 mV). (B) Membrane current–voltage relations of two cilia after excision from the cell. The recording shown in black has an input resistance R = 10.1 GΩ. In the recording shown in blue (R = 7.4 GΩ), single-channel openings are visible at potentials more positive than +50 mV. (C) Activation by Ca2+ and by depolarization of large-conductance channels in an excised renal primary cilium. On the left are shown the membrane currents at three voltage-clamp potentials in the presence of 0.1 μM free cytoplasmic Ca2+. On the right are recordings with 3 μM free cytoplasmic Ca2+ present. (D) A macroscopic current activated by high cytoplasmic Ca2+ in an excised cilium. The current–voltage relation was measured in each of two pseudointracellular baths: a bath containing 0.1 μM Ca2+ (black); and a bath with 300 μM Ca2+ (red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539729&req=5

Figure 3: Voltage-clamp recordings from single renal primary cilia. All recordings were made in the standard recording solutions except as noted. In A and C, dashed lines indicate the current level when the channels were closed. (A) Single-channel fluctuations were recorded while the cilium in the pipette was still attached to the cell. Pipette potential was strongly depolarizing (−140 mV). (B) Membrane current–voltage relations of two cilia after excision from the cell. The recording shown in black has an input resistance R = 10.1 GΩ. In the recording shown in blue (R = 7.4 GΩ), single-channel openings are visible at potentials more positive than +50 mV. (C) Activation by Ca2+ and by depolarization of large-conductance channels in an excised renal primary cilium. On the left are shown the membrane currents at three voltage-clamp potentials in the presence of 0.1 μM free cytoplasmic Ca2+. On the right are recordings with 3 μM free cytoplasmic Ca2+ present. (D) A macroscopic current activated by high cytoplasmic Ca2+ in an excised cilium. The current–voltage relation was measured in each of two pseudointracellular baths: a bath containing 0.1 μM Ca2+ (black); and a bath with 300 μM Ca2+ (red).
Mentions: Beads coated with ciliated cells were placed in a recording chamber and observed under phase-contrast microscopy. By applying positive pressure through the recording micropipette, it was often possible to rotate a bead until a cilium was positioned near the tip of the pipette. Suction was then applied through the pipette to draw the single cilium into the pipette (Figure2, Additional file 1). The cilium easily followed the suction because the bead was small and not attached to any surface. In 47% of 838 attempts, suction resulted in a membrane-pipette seal of resistance ≥  1 GΩ. With a cilium in the recording pipette and the cell still attached, bursts of single-channel activity were seen in a minority of cells, particularly at depolarizing potentials (Figure3A).

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus