Voltage-dependent conductance changes in the store-operated Ca2+ current ICRAC in rat basophilic leukaemia cells

Abstract

Tight-seal whole-cell patch-clamp experiments were carried out in order to investigate the effects of different holding potentials on the rate of development and amplitude of the Ca2+ release-activated Ca2+ current ICRAC in rat basophilic leukaemia (RBL-1) cells. ICRAC was monitored at -80 mV from fast voltage ramps, spanning 200 mV in 50 ms. At hyperpolarised potentials, the macroscopic CRAC conductance was lower than that seen at depolarised potentials. The conductance increased almost 5-fold over the voltage range -60 to +40 mV and was seen when the stores were depleted either by the combination of IP3 and thapsigargin in high Ca2+ buffer, or passively with 10 mM EGTA or BAPTA. The voltage-dependent conductance of the CRAC channels could not be fully accounted for by Ca2+-dependent fast inactivation, nor by other slower inhibitory mechanisms. It also did not seem to involve intracellular Mg2+ or the polycations spermine and spermidine. Voltage step relaxation experiments revealed that the voltage-dependent conductance changes developed and reversed slowly, with a time constant of several seconds at -60 mV. In the presence of physiological levels of intracellular Ca2+ buffers, ICRAC was barely detectable when cells were clamped at -60 mV and dialysed with IP3 and thapsigargin, but at 0 mV the current in low Ca2+ buffer was as large as that seen in high Ca2+ buffer. Our results suggest that CRAC channels exhibit slow voltage-dependent conductance changes which can triple the current amplitude over the physiological range of voltages normally encountered by these cells. The role of this conductance change and possible underlying mechanisms are discussed.

Figure 1. Conductance of CRAC channels depends on the holding potential

A, cells were clamped at the indicated holding potentials (V_h; −80 mV in i, −40 mV in ii, 0 mV in iii and +40 mV in iv). I_CRAC was activated by dialysing cells with IP₃ (30 μm), 10 mm EGTA and 2 μm thapsigargin and was monitored by applying fast voltage ramps (−100 to +100 mV in 50 ms at 0.5 Hz) superimposed on the holding potential. The amplitude of the current was always measured at −80 mV from the ramps. Upper panels plot the amplitude of I_CRAC against time for each V_h (corresponding to different cells) and the lower panels show the I-V relationship obtained from the ramps. Bi, plot of the pooled data for I_CRAC (•) from several cells for each V_h. Each point represents data from between 5 and 35 cells. The relationship between current amplitude and holding potential could be reasonably well fitted with a modified Boltzmann-type equation of the form: I_CRAC (−80 mV) = 1/(1 + exp(V–V_½)/S) where V_½ is the voltage at which the current is one-half its maximal amplitude and S is the slope factor (RT/zF), where R, T and F have their usual meanings and z is the gating valency of the voltage-dependent step. For the pooled data, V_½ was −3.1 mV and z was 1.9. This is a macroscopic empirical estimate of z and is only a very rough indication. All points were significantly different from that at 0 mV (P < 0.05). The size of the non-store-operated current as a function of different holding potentials, measured for up to 300 s is also shown (^). For these recordings, cells were dialysed with a solution lacking IP₃ and Ca²⁺ was buffered at 120 nm (see text). Note that this current is constant, irrespective of the membrane potential, and very small. It would therefore not affect the voltage dependence of I_CRAC (•). Bii, summary of the relationship between holding potential and the time constant of activation (τ-activation). There was a tendency for the current to activate more quickly as the holding potential became more negative. τ-activation at −80, −60, −40 and −20 mV was significantly different from that at 0 mV, whereas τ-activation at +20 and +40 mV was not.

Figure 2. Reduction in the extent of fast Ca²⁺-dependent inactivation does not prevent HII of I_CRAC

In A, the rate and extent of activation of I_CRAC at 0 and −60 mV holding potentials with IP₃+ EGTA + thapsigargin in the pipette are compared with those seen with IP₃+ BAPTA + thapsigargin. Both chelators were used at 10 mm. A₁ refers to IP₃+ 10 mm EGTA + thapsigargin at a V_h of 0 mV, A₂ to IP₃+ 10 mm EGTA + thapsigargin at a V_h of −60 mV, A₃ to IP₃+ 10 mm BAPTA + thapsigargin at a V_h of 0 mV and A₄ to IP₃+ 10 mm BAPTA + thapsigargin at a V_h of −60 mV. Each trace is from a different cell. Pooled data are summarised in the right-hand panel, which plots current amplitude against τ-activation. Each point represents data from 5–9 cells. In B, I_CRAC was activated passively following dialysis with 10 mm EGTA or BAPTA at the indicated holding potentials. IP₃ was not present in the patch pipette. Point B₁ here refers to 10 mm EGTA at a V_h of 0 mV, B₂ to 10 mm EGTA at a V_h of −60 mV, B₃ to 10 mm BAPTA at a V_h of 0 mV and B₄ to 10 mm BAPTA at a V_h of −60 mV. The right-hand panel plots the amplitude of I_CRAC against half-time to peak (time at which I_CRAC had reached half its steady-state amplitude) and this has been corrected for the delay before the current started to develop (typically around 70 s). In C, I_CRAC was activated by dialysing cells with IP₃ and 10 mm EGTA + thapsigargin under conditions where extracellular Ca²⁺ was reduced from 10 to 2 mm. Point C₁ refers to a V_h of 0 mV and C₂ to a V_h of −60 mV.

Figure 4. Voltage step relaxation studies reveal that the development of, and recovery from, HII is slow

A, a cell was dialysed with IP₃+ 10 mm EGTA + thapsigargin and held at 0 mV. Once I_CRAC had reached steady state (•), the potential was changed to −60 mV. I_CRAC then decayed slowly to reach a new steady-state (^). Stepping back to 0 mV resulted in a mono-exponential increase in the current (•) which almost reached the level it had prior to pulsing to −60 mV. B, similar experiment to A in a different cell which was initially held at −60 mV (^) and then stepped to 0 mV (•). In C, the amplitude of I_CRAC is plotted against the activation time constant corresponding to each change in membrane potential. A₁ represents the size of the current and τ-activation when cells were held at 0 mV upon break-in. A₂ refers to the change in A₁ upon stepping to −60 mV. A₃ reflects the amplitude of I_CRAC and τ-activation when cells were held at −60 mV upon break-in and A₄ is the subsequent change when these cells were then stepped to 0 mV. Note that A₁ and A₄ are virtually identical, and A₂ and A₃ are not significantly different, indicating that the changes in I_CRAC following each voltage relaxation do not seem to depend on the preceding state. Voltage relaxation experiments in the absence of store depletion (dialysis with an internal solution without IP₃ and in which Ca²⁺ was strongly buffered at 120 nm to prevent passive store emptying) failed to generate slowly developing currents on stepping from 0 to −60 mV and vice versa. Instead, there was an instantaneous change in the current amplitude (< 10 pA). It is unlikely therefore that there was a major contribution from other conductances, including Na⁺-Ca²⁺ exchange, to the voltage relaxation experiments.

Figure 3. HII of CRAC channels is not mediated by cytoplasmic Mg²⁺ or polycations

A, cells were dialysed with IP₃+ 10 mm EGTA + thapsigargin and the indicated concentrations of Mg²⁺. 0 Mg²⁺ refers to a pipette solution in which Mg²⁺ was omitted and 2 mm EDTA was added together with 10 mm EGTA. The left-hand panel shows examples of recordings from cells dialysed with 0 or 4 mm Mg²⁺ at a holding potential of either 0 mV (filled symbols) or −60 mV (corresponding open symbols). The middle and right-hand panels show how the amplitude of I_CRAC and activation time constant are related to internal Mg²⁺ concentration. B shows the effects of the different Mg²⁺ concentrations on the properties of I_CRAC when the latter was evoked by passive depletion of stores (10 mm EGTA). The left-hand panel depicts data from 3 cells dialysed with 0, 1 (control) or 4 mm Mg²⁺ at a holding potential of 0 mV and the middle panel shows the relationship between Mg²⁺ concentration and current size following passive store depletion. Each point represents data from 3–6 cells. The right-hand panel shows that fast Ca²⁺-dependent inactivation is not affected by changes in intracellular Mg²⁺ levels. Cells were held at 0 mV and then stepped to −80 mV for 250 s. Scale bars refer to 50 ms and −0.5 pA pF⁻¹. In C, I_CRAC was activated following dialysis with 10 mm EGTA and 2 mm spermine or spermidine and cells were clamped at either 0 or −60 mV. The left-hand panel depicts examples of the development of I_CRAC in the presence of spermine (at either 0 (A₁) or −60 mV (A₂)) and spermidine (A₃ at 0 and A₄ at −60 mV). Data from 2 different cells are shown for each voltage. The middle graph plots the amplitude of I_CRAC against half-time (time at which I_CRAC had reached half its steady-state amplitude) for the different conditions. Filled circles represent the control (no polycation) at 0 mV and open circles correspond to the control at −60 mV. The right-hand panel shows that fast inactivation of I_CRAC is largely unaffected by the polycations compared with control upon pulsing to −80 mV. Scale bars as in B.

Figure 5. I_CRAC in low intracellular Ca²⁺ buffer is very small at a holding potential of −60 mV

A shows the time course of I_CRAC for 2 cells held at 0 mV (filled symbols) and for 2 different cells held at −60 mV (open symbols). Internal solution contained IP₃+ 0.1 mm EGTA + 2 μm thapsigargin. Note that one of the cells held at −60 mV did not generate I_CRAC at all. In B, the amplitude histogram compares the size of I_CRAC in low Ca²⁺ buffer for the 2 different holding potentials. At a V_h of 0 mV, I_CRAC was around 5-fold larger than at a V_h of −60 mV.

Scheme 1