Electrophysiological characteristics of enteric neurons isolated from the immortomouse

Abstract

Background: Recently, two enteric neuronal cell lines, one fetal and the other post-natal (IM-PEN), have been developed from the H-2K(b)-tsA58 transgenic mouse (immortomouse). However, their electrophysiological properties are not known. The goal of this study was to determine the electrical excitability and ionic conductance of the immortalized postnatal enteric neuronal (IM-PEN) cell line.

Methods: Whole cell patch clamp studies, immunohistochemistry and RT-PCR were performed on differentiated IM-PEN cells following propagation at 33 °C and differentiation at 37 °C.

Results: Differentiated IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5. The mRNA for several ion channels expressed in enteric neurons were detected by RT-PCR. In current clamp, the resting membrane potential was -24.6 ± 2.1 mV (n = 6) for IM-FEN and -29.8 ± 0.9 mV (n = 30) for IM-PEN. Current injections from Vh -80 mV resulted in passive responses but not action potentials. Depolarizing pulses in the whole cell voltage clamp configuration from Vh -80 mV elicited small nifedipine-sensitive inward currents. Additionally, outward currents with slow deactivating tail currents were blocked by niflumic acid and low chloride solution. A volume-regulated anion current was elicited by hypo-osmotic solution and inhibited by 10 μM DCPIB. Growth with rabbit gastrointestinal smooth muscle did not yield significant differences in the active properties of the IM-PEN cell line. Transient expression of L-type Ca(2+) channels produced large inward currents demonstrating a working mechanism for protein folding and transport.

Conclusion: The electrophysiological characteristics of IM-PEN cells suggest that chloride channels in IM-PEN cells play an important role in their resting state, and membrane trafficking of some of the ion channels may preclude their electrical excitability.

Figure 1. IM-PEN exhibits positive immunoreactivity for the neuronal marker PGP 9.5 and βIII tubulin (Tuj1)

A) PGP9.5 immunoreactivity (green) 20X B) βIII-tubulin immunoreactivity (red) 20X C) higher magnification of βIII-tubulin (red) 60X D) control in the absence of primary antibody 20X. Each is shown with its corresponding brightfield image.

Figure 2. Expression of neuronal markers and channels in IM-FEN cultured at 39°C (3 days) compared to cells cultured at 33°C

(A) RNA was isolated from IM-FEN cultured at 33°C or 39°C (3 days) and amplified for cRet, Kcnn4, NaV_1.3, TASK1, Clca1, and GAPDH (loading control), n=3. The two lanes/gene correspond to RNA isolated from cells grown at 33°C and 39°C. (B) Histogram for IM-FEN mRNA assessed by Real Time PCR showing the relative level of expression of various channels and neuronal markers, Peripherin and Ret after normalization to GAPDH expression. Results are mean ± S.E., n=3. **P < 0.01, ***P < 0.001.

Figure 3. Whole cell current clamp recording of IM-PEN cell

A) Depolarizing current pulses elicited passive responses and large electrotonic potential with hyperpolarizing pulses. A hyperpolarizing current was applied to maintain a holding potential of −80 mV.

Figure 4. Voltage gated Na⁺ and Ca²⁺ currents

A) A current-voltage relationship of peak currents in control (closed circles, n=11), following replacement of Na⁺ with NMDG (open circles) (n=3) and in the presence of TTX (2 μM) (n=4) (closed triangles). B) Raw tracings of inward currents at the peak potential of −20 mV from same cell, treated with NMDG (blue) or TTX (red). C) Current-voltage relationship, control (closed circles) and inhibition with 1μM nifedipine (open circles)(n=4). D) Raw trace shows inhibition of the inward current with 1μM nifedipine.

Figure 5. IM-PEN cells contain Cl⁻ currents

A) Voltage clamp experiment in the whole cell configuration with K⁺ channels blocked by 140mM CsCl in the internal solution and 144mM Cl⁻ in the external solution E_Cl = −38mV. B) Same cell recorded with 40mM Cl⁻ in the external solution produces smaller currents. C) Cl⁻ sensitive current obtained by subtraction. D) IV curve of chloride currents (n=6). E) Niflumic acid (1μM NA) inhibited both the outward currents and tail currents. F) Expanded traces of tail currents from test pulse of +30 mV in the absence and presence of 1μM NA. G) IV relationship of amplitude of end of pulse current in the absence and presence of 1μM NA.

Figure 6. A hypoosmotically activated chloride current

A) Whole cell voltage clamp recording at Vh= −60mV initiated an inward current upon the perfusion of hypoosmotic solution (230mOsm). This current was inhibited and remained inhibited after treatment with 10μM DCPIB. B) IV- relationship showing swelling activated current and inhibition by DCPIB. C) Whole cell recording of IM-PEN with Vh = +30mV. Addition of hypoosmotic solution initiated an outward current which was inhibited when Cl⁻ was replaced in the external solution with gluconate. The current returned when Cl⁻ was returned to the external solution.

Figure 7. TRPV1 channel

A) TRPV1 positive immuno-reactivity of IM-PEN after 7 days at 37°C (20X). Side panel shows immuno-reactivity in intracellular vesicles at higher magnification (60X). B) Whole cell voltage clamp recording from IM-PEN cell held at −60 mV and treated with 10μM capsaicin. No currents were elicited in the presence of capsaicin.

Figure 8. IM-PEN grown on rabbit smooth muscle

A) Representative voltage clamp recording from GFP transfected IM-PEN grown on rabbit smooth muscle. B) Current clamp recording from the same cell shows no action potentials to depolarizing pulses with a Vh −80mV. C) Image of GFP transfected IM-PEN (white) plated on cultured rabbit smooth muscle cells. D) Current voltage relationship of IM-PEN grown in 3 different conditions with smooth muscle cells.

Figure 9. Transfection of IM-PEN with the voltage gated Ca²⁺ channel (Ca_v1.2b)

A) Raw trace of whole cell voltage clamp recording with internal CsCl (140mM) in the presence of 10mM external Ba²⁺ of a GFP transfected cell. B) Raw trace of GFP and Ca_v1.2b transfected cell displayed a peak inward current of −8.7 ± 0.72 pA pF⁻¹ at 20mV (Vh = −60mV). C) Current-voltage relationship of GFP transfected cells vs. those transfected with GFP and Ca_v1.2b.