Modulation of plasma membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells

Abstract

The spatial distribution of Ca(2+) signalling molecules is critical for establishing specific interactions that control Ca(2+) signal generation and transduction. In many cells, close physical coupling of Ca(2+) channels and their targets enables precise and robust activation of effector molecules through local [Ca(2+)](i) elevation in microdomains. In T cells, the plasma membrane Ca(2+)-ATPase (PMCA) is a major target of Ca(2+) influx through Ca(2+) release-activated Ca(2+) (CRAC) channels. Elevation of [Ca(2+)](i) slowly modulates pump activity to ensure the stability and enhance the dynamic nature of Ca(2+) signals. In this study we probed the functional organization of PMCA and CRAC channels in T cells by manipulating Ca(2+) microdomains near CRAC channels and measuring the resultant modulation of PMCAs. The amplitude and spatial extent of microdomains was increased by elevating the rate of Ca(2+) entry, either by raising extracellular [Ca(2+)], by increasing the activity of CRAC channels with 2-aminoethoxyborane (2-APB), or by hyperpolarizing the plasma membrane. Surprisingly, doubling the rate of Ca(2+) influx does not further increase global [Ca(2+)](i) in a substantial fraction of cells, due to a compensatory increase in PMCA activity. The enhancement of PMCA activity without changes in global [Ca(2+)](i) suggests that local [Ca(2+)](i) microdomains near CRAC channels effectively promote PMCA modulation. These results reveal an intimate functional association between CRAC channels and Ca(2+) pumps in the plasma membrane which may play an important role in governing the time course and magnitude of Ca(2+) signals in T cells.

Figure 1. Effects of extracellular [Ca²⁺] on modulation of PMCA

TG (1 μm) was present in all solutions, beginning with 0 Ca²⁺ Ringer solution, as indicated, to deplete Ca²⁺ stores and activate CRAC channels. A, measurement of Ca²⁺ clearance rates following [Ca²⁺]_i elevation induced by 0.5 or 2 mm Ca²⁺_o. The clearance rate at a common [Ca²⁺]_i following removal of Ca²⁺_o was measured by the d[Ca²⁺]_i/dt slopes over 5 s periods indicated by the thickened lines. The Ca²⁺ clearance data during these periods are overlaid on an expanded time scale to the right. Average response of 261 cells. Bars indicate s.e.m. in this and all subsequent figures. B, measurement of Ca²⁺ clearance rates following [Ca²⁺]_i elevation induced by 2 or 20 mm Ca²⁺_o. Slopes were measured by linear regression over a 5 s period immediately following removal of [Ca²⁺]_o as indicated by the thickened lines. Data are overlaid and displayed on an expanded scale to the right. Average response of 261 cells. C, clearance rate in single cells as a function of [Ca²⁺]_i following long exposures to different [Ca²⁺]_o. Data from the experiments in A and B. Each symbol represents the initial rate of Ca²⁺ clearance after removal of Ca²⁺_o plotted against the immediately preceding plateau [Ca²⁺]_i produced by 0.5 mm (black), 2 mm (blue) or 20 mm (green) Ca²⁺_o. Each cell is represented by a pair of points (0.5 and 2 mm, or 2 and 20 mm).

Figure 2. High [Ca²⁺]_o augments PMCA modulation independently of changes in global [Ca²⁺]_i

TG-pretreated cells were exposed sequentially to 2 and 20 mm Ca²⁺_o as shown in Fig. 1B. A, distribution of the plateau [Ca²⁺]_i levels generated by 20 mm Ca²⁺_o relative to 2 mm Ca²⁺_o in 265 individual cells. In 16% of the cells, 2 and 20 mm Ca²⁺_o evoked the same global [Ca²⁺]_i plateau (‘iso-[Ca²⁺]_i’ cells; shaded bar). B, average [Ca²⁺]_i responses from the iso-[Ca²⁺]_i cells identified in A. Ca²⁺ clearance rates indicated by the thickened lines were measured over the first 5 s following Ca²⁺_o removal (left). Clearance was faster following exposure to higher [Ca²⁺]_o, despite constant global [Ca²⁺]_i. An overlay of the first 5 s of Ca²⁺ clearance following the 2 and 20 mm Ca²⁺_o applications, highlights the different PMCA rates (right).

Figure 3. High [Ca²⁺]_o increases PMCA modulation independently of global [Ca²⁺]_i in the majority of cells treated with La³⁺ to reduce Ca²⁺ influx

TG-treated cells were exposed to 2 mm Ca²⁺_o or 20 mm Ca²⁺_o + 7 nm La³⁺ as indicated in B. A, distribution of the plateau [Ca²⁺]_i levels generated by 20 mm Ca²⁺_o + 7 nm La³⁺ relative to 2 mm Ca²⁺_o in 373 cells. Iso-[Ca²⁺]_i cells (68% of the population) are indicated by the shaded bars. B, average [Ca²⁺]_i responses from the iso-[Ca²⁺]_i cells identified in A. Ca²⁺ clearance rates measured over a 5 s period are indicated by the thickened lines (left) and are plotted on an expanded scale (right).

Figure 4. 2-APB enhances PMCA modulation independently of changes in [Ca²⁺]_i and [Ca²⁺]_o

A, 2-APB increases the rate of Ca²⁺ clearance. Following store depletion with 1 μm TG, cells were exposed to 2 mm Ca²⁺_o for 300 s and following an 8 min recovery period, with 2 mm Ca²⁺_o + 5 μm 2-APB. Slopes during the first 5 s after Ca²⁺_o removal are indicated as thickened lines (left) and on an expanded scale (right). Average response of 112 cells. B, 2-APB does not affect pump activity directly. 2-APB (5 μm) was added during the washout of Ca²⁺_o. Clearance rates were unaffected, as shown by the equal slopes (thickened lines to the left, expanded scale to the right). C, distribution of the plateau [Ca²⁺]_i levels generated by 2 mm Ca²⁺_o+ 2-APB relative to 2 mm Ca²⁺_o alone from the experiment in A. Iso-[Ca²⁺]_i cells are indicated by the shaded bars. D, average response of the iso-[Ca²⁺]_i cells identified in C (left). 2-APB enhanced the clearance rate 1.7-fold despite the absence of any change in global [Ca²⁺]_i. An overlay of the first 5 s of Ca²⁺ clearance following applications of 2 mm Ca²⁺ ± 2-APB highlights the different PMCA rates (right).

Figure 5. Increasing the electrical driving force for Ca²⁺ enhances PMCA modulation

After establishing the perforated-patch voltage-clamp configuration, single indo-1-loaded cells were treated with 1 μm TG for 10 min to activate CRAC channels at a holding potential of +30 mV to maintain resting [Ca²⁺]_i levels. Antimycin (2 μm) + oligomycin (2 μm) were present to block mitochondrial Ca²⁺ uptake. A, PMCA activity is voltage independent. Hyperpolarization from +30 mV to −70 mV evoked a [Ca²⁺]_i rise to 1.4 μm in ∼10 s. Subsequent removal of Ca²⁺_o caused [Ca²⁺]_i to decay back to baseline; the dotted line indicates a single-exponential fit to the recovery phase. Changing the holding potential from −70 to +30 mV did not significantly alter the rate of Ca²⁺ clearance. B, hyperpolarization increases PMCA activity with little change in global [Ca²⁺]_i. Hyperpolarization of the holding potential (V_HOLD, bottom) evoked an increase in I_CRAC (middle, measured at the holding potential) and [Ca²⁺]_i (top). Leak-subtracted ramp current at −80 to +50 mV (inset) was collected during the period at −50 mV V_HOLD. The ramps display the inward rectification and positive reversal potential characteristic of I_CRAC. Increasing V_HOLD from −50 to −75 mV almost doubled I_CRAC with only a slight elevation of [Ca²⁺]_i, implying an increase of PMCA activity.

Figure 6. Local [Ca²⁺]_i gradients near CRAC channels and possible modes of coupling to PMCAs

A, estimate of the [Ca²⁺]_i gradient in a microdomain around a single CRAC channel using eqn (1) (see text). The 2 mm Ca²⁺_o condition was simulated using a unitary CRAC current of −1.5 fA (Zweifach & Lewis, 1993), and this value was scaled by 1.8 to simulate 20 mm; Ca²⁺_o conditions, based on a K_d for conduction of 2 mm (Premack et al. 1994). Global [Ca²⁺]_i = 1 μm; Ca²⁺ diffusion coefficient, D_Ca = 300 μm² s⁻¹. B, possible models for local control of PMCA modulation by Ca²⁺ influx through CRAC channels. Close physical coupling of PMCAs and CRAC channels may expose PMCAs directly to local [Ca²⁺]_i microdomains which enhance pump modulation. Alternatively, a Ca²⁺ sensor may receive the local Ca²⁺ signal, and via diffusion may modulate PMCAs located at more distant sites.