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Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon
Sok Han Kang1, Pieter Vanden Berghe, Terence K Smith
1Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA.
Abstract:
Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs(+) to remove K(+) currents. Depolarization elicited a slowly activating time-dependent outward current (I(tdo)), whereas repolarization was followed by a slowly deactivating tail current (I(tail)). I(tdo) and I(tail) were present in approximately 70% of neurons. We identified these currents as Cl(-) currents (I(Cl)), because changing the transmembrane Cl(-) gradient altered the measured reversal potential (E(rev)) of both I(tdo) and I(tail) with that for I(tail) shifted close to the calculated Cl(-) equilibrium potential (E(Cl)). I(Cl) are Ca(2+)-activated Cl(-) current [I(Cl(Ca))] because they were Ca(2+) dependent. E(Cl), which was measured from the E(rev) of I(Cl(Ca)) using a gramicidin perforated patch, was -33 mV. This value is more positive than the resting membrane potential (-56.3 +/- 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl(-). omega-Conotoxin GIVA [0.3 microM; N-type Ca(2+) channel blocker] and niflumic acid [10 microM; known I(Cl(Ca)) blocker], decreased the I(Cl(Ca)). In conclusion, these neurons have I(Cl(Ca)) that are activated by Ca(2+) entry through N-type Ca(2+) channels. These currents likely regulate postspike frequency adaptation.
Insights
Myenteric neurons in the colon possess calcium-activated chloride currents (ICl(Ca)) that are regulated by N-type calcium channels. These currents influence neuronal firing patterns.
Area of Science:
- Neuroscience
- Gastroenterology
- Ion Channel Physiology
Background:
- Myenteric neurons in the murine proximal colon control gastrointestinal motility.
- Understanding ion channel function is crucial for elucidating neuronal signaling in the gut.
Purpose of the Study:
- To identify and characterize calcium-activated chloride currents (ICl(Ca)) in myenteric neurons.
- To investigate the role of these currents in neuronal excitability and regulation.
Main Methods:
- Whole-cell patch-clamp recordings from cultured murine myenteric neurons.
- Utilized cesium-containing solutions to block potassium currents.
- Manipulated chloride gradients and used specific channel blockers (omega-conotoxin GIVA, niflumic acid).
Main Results:
- Identified slowly activating time-dependent outward currents (Itdo) and slowly deactivating tail currents (Itail) in ~70% of neurons.
- These currents were characterized as chloride currents (ICl) due to their dependence on the chloride gradient.
- Demonstrated that ICl are calcium-dependent (ICl(Ca)) and are activated by calcium influx through N-type calcium channels.
Conclusions:
- Murine myenteric neurons express functional ICl(Ca).
- These currents are activated by calcium entry via N-type calcium channels.
- ICl(Ca) likely play a role in regulating postspike frequency adaptation in myenteric neurons.