The L-type voltage-dependent Ca2+ channel EGL-19 controls body wall muscle function in Caenorhabditis elegans

Abstract

Caenorhabditis elegans is a powerful model system widely used to investigate the relationships between genes and complex behaviors like locomotion. However, physiological studies at the cellular level have been restricted by the difficulty to dissect this microscopic animal. Thus, little is known about the properties of body wall muscle cells used for locomotion. Using in situ patch clamp technique, we show that body wall muscle cells generate spontaneous spike potentials and develop graded action potentials in response to injection of positive current of increasing amplitude. In the presence of K+ channel blockers, membrane depolarization elicited Ca2+ currents inhibited by nifedipine and exhibiting Ca2+-dependent inactivation. Our results give evidence that the Ca2+ channel involved belongs to the L-type class and corresponds to EGL-19, a putative Ca2+ channel originally thought to be a member of this class on the basis of genomic data. Using Ca2+ fluorescence imaging on patch-clamped muscle cells, we demonstrate that the Ca2+ transients elicited by membrane depolarization are under the control of Ca2+ entry through L-type Ca2+ channels. In reduction of function egl-19 mutant muscle cells, Ca2+ currents displayed slower activation kinetics and provided a significantly smaller Ca2+ entry, whereas the threshold for Ca2+ transients was shifted toward positive membrane potentials.

Figure 1.

Voltage responses and ionic currents in body wall muscle cells in the presence of standard saline. (A, left) Membrane potential was recorded in a muscle cell in the current clamp mode without current injection. The arrow indicates the time at which a negative hyperpolarizing current was injected. (B) A spontaneous spike indicated by a star in the left panel is shown on an expanded scale. (C) The internal potential was held at –70 mV by passing a constant negative current, and voltage responses were obtained in response to current injection of 20 ms duration in 100 pA increments. The arrow indicates an inflection point during the depolarizing phase. (D) Membrane currents were elicited on the same cell as in C under voltage clamp conditions by applying voltage pulses of 20 ms duration in 10-mV increments from a holding potential of –70 mV.

Figure 2.

Inward currents and mean current-voltage relationships. (A) Inward currents were evoked on the same cell by voltage pulses in the presence of K⁺ channel blockers from a holding potential of –70 mV to the indicated values. (B) Mean current-voltage relationships were established at the peak of the currents (left) and at the end of the depolarization pulses (right). The curves were fitted by using Eq. 1 (see Experimental procedures) with values for G _max, V _rev, V _0.5, and k of 180 S/F, +51 mV, 0.9 mV, and 4.6 mV (left) and of 100 S/F, +60 mV, 3.6 mV, and 6.6 mV (right).

Figure 3.

Effect of Cd^{2 +} on inward currents and on regenerative responses induced by current injection in the presence of K ⁺ channel blockers. (A) Inward currents were evoked by voltage pulses in the presence of K⁺ channel blockers from a holding potential of –70 mV in 10-mV increments in control (left) and in the same cell after addition of Cd²⁺ (right). (B) Voltage responses were obtained on the same cell under current clamp conditions using the current protocols indicated below, in the presence of a control external medium (left), and in the presence of a solution containing 20 mM TEA and 3 mM 4-AP (right). The internal potential was held at –70 mV by passing a constant negative current. The star shows the voltage response obtained after addition of 500 μM Cd²⁺ in the external solution.

Figure 4.

Effect on the inward current of the dihydropyridine compound nifedipine and of a substitution of Ba^{2 +} for Ca^{2 +} . (A) Currents were obtained on the same cell in response to a voltage pulse to +10 mV from a holding potential of –70 mV. The star indicates the current evoked after addition of 1 μM nifedipine in the external solution. (B) Currents were evoked on the same cell in response to a voltage pulse to +10 mV from a holding potential of –70 mV. The star indicates the current evoked after substitution of 6 mM Ba²⁺ for 6 mM Ca²⁺ in the external solution.

Figure 5.

Inactivation properties of inward currents. (A) Currents were elicited by the voltage protocol indicated next to each current trace; a first step of 20 ms duration and various amplitude was followed by a 200-ms pulse to +10 mV with a short interpulse hyperpolarization of 2-ms duration to –70 mV. (B) Relative peak current (□) and steady state current (▴) amplitudes are plotted as a function of the 20-ms prepulse potential.

Figure 6.

Ca^{2 +} transients and Ca^{2 +} currents in voltage-clamped cells. (A) The light micrograph in the left panel shows a muscle cell with a recording pipette sealed. The two other panels show the fluorescence images of the same field before (middle) and during a voltage pulse given to +10 mV (right). (B) The same cell was depolarized by 500 ms duration pulses from a holding potential of –70 mV to the indicated potentials. The upper and lower traces correspond to membrane currents and Ca²⁺ transients, respectively, on different time scales. Fluorescence was sampled at 50 Hz. Inset shows the membrane current and the Ca²⁺ transient obtained in response to a voltage pulse given to +10 mV on the same time scale. Bars below the fluorescence traces indicate the time during which depolarizing pulses were applied. (C) The running integral of the Ca²⁺ currents and the corresponding Ca²⁺ transients were superimposed for pulses to the indicated potentials. For each pulse, the integral of the current was normalized to the peak value of the Ca²⁺ transient to allow comparison of kinetics. For pulses to –10 and +10 mV, the integrals of current merge into one. (D) The amplitude of the Ca²⁺ transients were plotted against the integral of the corresponding Ca²⁺ currents for two different cells (⋄ and ♦, respectively) depolarized by 500 ms duration pulses from a holding potential of –70 mV up to +30 mV. A linear function was fitted to data points for each cell.

Figure 7.

Spontaneous spikes and inward current properties in egl-19 mutant muscle cells. (A) Spontaneous spikes recorded in the current clamp mode without current injection in a wild-type and in an egl-19 mutant muscle cell are superimposed. Voltage responses have been shifted on voltage axe to allow comparison. (B) Inward currents were elicited from a holding potential of –70 mV to the indicated voltages. (C) The time constant of activation of the inward current was plotted as a function of the membrane potential in egl-19 (○) and wild-type (•) muscles. Inset shows Ca²⁺ currents obtained at +10 mV in a wild-type and egl-19 muscle cell superimposed on an expanded scale. (D) The mean current-voltage relationship of the steady state inward current in wild-type and egl-19 muscle are presented. The curve for egl-19 muscle was fitted by using Eq. 1 (see Experimental procedures) with values for G _max, V _rev, V _0.5, and k of 86 S/F, +59 mV, 13.6 mV, and 8.4 mV. (E) The mean normalized conductance was plotted as a function of voltage in egl-19 (○) and wild-type (•) muscles. The curves were fitted by using a Boltzman equation with V _0.5 = 5.6 and k = 7.5 in wild-type and V _0.5 = 14.5 and k = 9.4 in egl-19 muscles, respectively.

Figure 8.

Ca^{2 +} transients and Ca^{2 +} currents in egl-19 mutant muscle cells. (A) The cell was depolarized by 500-ms duration pulses from a holding potential of –70 mV to the indicated potentials. The upper and lower traces correspond to membrane currents and Ca²⁺ transients, respectively, on different time scales. Fluorescence was sampled at 50 Hz. Inset shows the membrane current and the Ca²⁺ transient obtained in response to a voltage pulse given to +10 mV on the same time scale. Bars below the fluorescence traces indicate the time during which depolarizing pulses were applied. (B) The mean normalized amplitude of the Ca²⁺ transients and the mean normalized value of the integral of the Ca²⁺ currents in wild-type and egl-19 muscle cells were plotted as a function of membrane potential. Data points correspond to values averaged from five series of voltage pulses applied in three egl-19 cells and four series of voltage pulses applied in two wild-type cells, respectively. Data points were normalized to the maximal mean value obtained in wild-type and egl-19 muscle cells, respectively. The curves were drawn by eyes.