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Membrane currents in cultured human intestinal smooth muscle cells

A. V. Zholos, C. J. Fenech, S. A. Prestwich and T. B. Bolton

1. Using whole-cell patch-clamp recording techniques, we have examined voltage-gated ion currents in a cultured human intestinal smooth muscle cell line (HISM). Experiments were performed at room temperature on cells after passages 16 and 17.

2. Two major components of the whole-cell current were a tetraethylammonium-sensitive (IC₅₀ = 9 mM), iberiotoxin-resistant, delayed rectifier K⁺ current and a Na⁺ current inhibited by tetrodotoxin (IC50 Å 100 nM). No measurable inward current via voltage-gated Ca²⁺ channels could be detected in these cells even with 10 mM Ca²⁺ or Ba²⁺ in the external solution. No current attributable to calcium-activated K⁺ channels was found and no cationic current in response to muscarinic receptor activation was present.

3. In divalent cation-free external solution two additional currents were activated: an inwardly rectifying hyperpolarization-activated current, I_HA, and a depolarization-activated current, I_DA.

4. I_HA and I_DA could be carried by several monovalent cations; the sizes of currents in descending order were: K⁺ > Cs⁺ > Na⁺ for I_HA and Na⁺ > K⁺ >> Cs⁺ for I_DA. I_HA was activated and deactivated instantaneously and showed no inactivation whereas I_DA was activated, inactivated and deactivated within tens of milliseconds. These currents were inhibited by external calcium with an IC₅₀ of 0.3 M for I_DA and an IC₅₀ of 20 M for I_HA.

5. Cyclopiazonic acid (CPA) induced an outward, but not an inward current. SK&F 96365, a blocker of store-operated Ca²⁺ channels, suppressed I_DA with a half-maximal inhibitory concentration of 9 M but was ineffective in inhibiting I_HA at concentrations up to 100 M. Gd³⁺ and La³⁺ strongly suppressed I_DA at 1 and 10 M, respectively and were less effective in blocking I_HA (complete inhibition required a concentration of 100 M for both). Carbachol at 10-100 M evoked about a 3-fold increase in I_HA amplitude and completely abolished I_DA.

6. We conclude that I_HA and I_DA are Ca²⁺-blockable cationic currents with different ion selectivity profiles that are carried by different channels. I_DA shows novel voltage-dependent properties for a cationic current.