Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig)

Abstract

• 1. Single smooth muscle cells isolated from the urinary bladder of the guinea-pig were studied at 35°C in a solution composed of 150 mM NaCl, 3.6 mM CaCl₂, 1.2 mM MgCl₂, 5.4 mM KCl, 20 mM TEA-Cl, 5 mM glucose, 10 mM HEPES/NaOH (pH 7.4). Whole cells were clamped with a single patch electrode. The clamp settled a step from −65 to −5 mV within 260 μs, and afterwards the voltage inhomogeneities were less than 2 mV (measured at the cell edge with a second electrode). The calcium inward currenti _Ca was dissected from net currents by blocking potassium outward currents by means of patch electrodes filled with 130 mM CsCl (Klöckner and Isenberg 1985 a). Pyruvate, succinate and oxalacetate in the patch electrode stabilizedi _Ca and prevented its “run down”.

• 2. 140 ms long clamp steps from −65 to −5 mV evoked a net inward current which could be reversibly blocked by 5 mM NiCl₂. The “Ni-sensitive” difference currenti _Ca peaked within 2–4 ms to about 1 nA per cell. Afterwards it completely inactivated; the inactivation could be fitted with three exponentials (time constants of 4, 30, and 250 ms, respectively). The half decay time of 16 ms suggests that most of the inactivation resulted from the fast exponential process.

• 3. The reference current in the presence of Ni was nearly time independent and almost zero; therefore,i _Ca could be approximated from the net inward current using the zero current as a reference line. Thei _Ca obtained by this “visual estimate” had nearly the same properties as the Ni-sensitive current. Data from 10 cells were averaged to 0.95±0.06 nA per cell or a density of 20±3 μA/cm² (mean±SE for steps from −65 t0 −5 mV).

• 4. Peaki _Ca plotted over the range of clamp step potentials resulted in ani-v curve which had a threshold at −40 mV, bent with negative slope to a relative minimum at −5 mV and ascended again to a reversal potential of +50 mV. Between −40 and +20 mV thei-v curve could be described by multiplying a conductance with a driving force, where the conductance followed a steady state activation curve with a slope of −6 mV and a potential of half maximal activation of −14 mV. At more positive potentials, the currents rectified as predicted by the Hess and Tsien model (1984).

• 5. Clamp steps to more and more positive potentials monotonously shortened the fast inactivation time constant which was about 7 ms close the threshold and 4 ms at +25 mV. The second inactivation process revealed an U-shaped time constant curve. Steady state inactivation was analyzed with 5 s long conditioning presteps; the S-shaped curve had a potential of half maximal inactivation of −43 mV and a slope of 6 mV. At the resting potential of −50 mV, about 75% of the calcium channels were calculated to be available.

• 6. Sodium-free media did not significantly changei _Ca, the amplitude, the time course of inactivation and the reversal potential remained constant. This data suggests that sodium ions do not carry a significant part ofi _Ca. We estimate that during a 30 ms long action potential,i _Ca transports an amount of calcium that increases the total [Ca]_i by 20 μM, in a cell volume of 6 pl. Consequences for excitation and contraction of the cell are discussed.