A TRP-ASSOCIATED NON-SELECTIVE CATION CHANNEL IN INTERSTITIAL CELLS OF CAJAL

C Golden, X-Y Wang, JD Huizinga

Intestinal Disease Research Program, Department of Medicine, McMaster University, Hamilton, Ontario

BACKGROUND: To understand the electrophysiological basis of gut pacemaker activity, it is important to characterize ion channels that are active at the resting membrane potential of gut pacemaking cells. The ion channels present in ICC will determine the cell’s resting membrane potential, excitability and ability to initiate gut pacemaker activity. Our previous studies provide indirect evidence for a non-selective cation channel being essential for intestinal pacemaker activity. We hypothesised the presence of a TRP-associated non-selective cation channel that is active at the resting membrane potential of ICC.

METHODS: We have employed the perforated patch technique to record whole cell currents in ICC isolated from murine small intestine. In addition, we performed immunohistochemistry to identify Kit and TRP proteins. A ramp protocol was developed to examine the current-voltage relationship over a range of –100 to +50mV. The kinetics of currents active around the resting membrane potential was observed using a voltage-step protocol from –90 to –30mV.

RESULTS: Isolated ICC were obtained after three to seven days of culture, recognized by morphology, spontaneous activity and Kit immunohistochemistry. The voltage protocols revealed constitutively active currents plus voltage activated outward currents. After blockade of outward currents by intracellular Cs, only the linear constitutively active currents remained. A low concentration of gadolinium (1mM) inhibited the current by approximately 65% at -60mV membrane potential. The reversal potential of the current was 0 mV. Furthermore, replacing NaCl with NMDG-Cl eliminated the gadolinium sensitive component of the current profile. This is all consistent with this current being mediated by non-selective cation channels. Immunohistochemistry on Kit positive ICC revealed the presence of TRP-6 protein, indicating that a TRP related cation channel may generate inward current at the resting membrane potential of ICC.

CONCLUSION: A non-selective cation channel (possibly from the TRP family) is constitutively active at the resting membrane potential and more negative membrane potentials of ICC. This channel will be important in determining cell excitability, will likely be one of the pacemaker channels creating rhythmic depolarization in these cells and may become a target for pharmacological action in ICC .

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