A novel chloride conductance activated by extracellular ATP in mouse parotid acinar cells

Abstract

Salivary gland fluid secretion is driven by transepithelial Cl- movement involving an apical Cl- channel whose molecular identity remains unknown. Extracellular ATP (ATP(o)) has been shown to activate a Cl- conductance (I(ATPCl)) in secretory epithelia; to gain further insight into I(ATPCl) in mouse parotid acinar cells, we investigated the effects of ATP(o) using the whole-cell patch-clamp technique. ATP(o) and 2'- and 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (Bz-ATP) produced concentration-dependent, time-independent Cl- currents with an EC50 of 160 and 15 microM, respectively. I(ATPCl) displayed a selectivity sequence of SCN- > I- = NO3- > Cl- > glutamate, similar to the Cl- channels activated by Ca2+, cAMP and cell swelling in acinar cells. In contrast, I(ATPCl) was insensitive to pharmacological agents that are known to inhibit these latter Cl- channels, was independent of Ca2+ and was not regulated by cell volume. Moreover, the I(ATPCl) magnitude from wild-type animals was comparable to that from mice with null mutations in the Cftr, Clcn3 and Clcn2 Cl- channel genes. Taken together, our results demonstrate that I(ATPCl) is distinct from the channels described previously in acinar cells. The activation of I(ATPCl) by Bz-ATP suggests that P2 nucleotide receptors are involved. However, inhibition of G-protein activation with GDP-beta-S failed to block I(ATPCl), and Cibacron Blue 3GA and 4,4'-diisothyocyanostilbene-2,2'-disulphonic disodium salt selectively inhibited the Na+ currents (presumably through P2X receptors) without altering I(ATPCl), suggesting that neither P2Y nor P2X receptors are likely to be involved in I(ATPCl) activation. We conclude that I(ATPCl) is not associated with Cl- channels previously characterized in mouse parotid acinar cells, nor is it dependent on P2 nucleotide receptor stimulation. I(ATPCl) expressed in acinar cells reflects the activation of a novel ATP-gated Cl- channel that may play an important physiological role in salivary gland fluid secretion.

Figure 2. Voltage dependence of ATP-activated currents

A, currents obtained in the absence (left) and presence of 5 mm Tris-ATP (right). The membrane potential (V_m) was changed from −80 to +100 mV in 20 mV increments from a holding potential of 0 mV. B, current-voltage relationship of the ATP-activated current. Current amplitudes were measured at the end of the 400 ms pulse. Control currents in the absence of ATP were subtracted to obtain the ATP-activated current amplitude (n = 9).

Figure 1. Cl⁻ channels in mouse parotid acinar cells

A, Ca²⁺-dependent Cl⁻ channels. B, volume-sensitive Cl⁻ channels. C, inward-rectifier Cl⁻ channels. The data are representative of currents collected from three or more different cells for each condition. Upper row: currents obtained at +80 and −80 mV. Lower row: corresponding current-voltage relationships constructed with data obtained from +80 to −80 mV in 20 mV steps.

Figure 3. Concentration-response curves to Tris-ATP and Bz-ATP

Currents were sampled at +80 mV at different agonist concentrations using 400 ms square pulses applied from 0 mV. Concentration-response curves obtained from single cells were normalized using the maximum current calculated by fitting the Hill equation. Normalized currents were then averaged, plotted as a function of agonist concentration and fitted with Hill eqn (2) (see Methods). EC₅₀ and Hill coefficient values are given in the inset.

Figure 4. Anion dependence of the ATP-activated current

A, whole-cell current recorded at +80 mV. The current was activated by perfusing 5 mm Tris-ATP during the time indicated by the double-headed arrow. External Cl⁻ was reduced from 140 to 1 mm by replacement with glutamate during the indicated time. ATP-activated current magnitude was reduced from +0.51 to +0.16 nA by decreasing the extracellular Cl⁻ concentration. B, current-voltage relationships obtained from a single cell bathed in solutions containing 141 mmCl⁻ (▪), 140 mm SCN⁻ (•), 140 mm I⁻ (▴), or 140 mm NO₃⁻ (▾). Current magnitudes were measured at the end of the 400 ms pulse using data like that depicted in Fig. 2B. Current-voltage curves were fitted with a third-order polynomial function (continuous lines) to calculate the reversal potentials (E_r, inset).

Figure 5. CFTR, ClC-2, ClC-3 and Ca²⁺-activated Cl⁻ channels are not ATP-activated channels

Currents were activated by applying 5 mm ATP during the times indicated by the double-headed arrows. The membrane potential was changed from 0 to +80 mV approximately 5 s prior to the addition of ATP. Currents were recorded from acinar cells isolated from Cftr^-/- (A), Clcn2^-/- (B), Clcn3^-/- (C) and wild-type (WT, D) mice. Currents from the WT cell (D) were obtained from the same cell dialysed with 20 mm EGTA without Ca²⁺ and bathed in a solution containing 0.5 mm Ca²⁺ (0.5 mm [Ca²⁺]_o) or 0 Ca²⁺ (0 Ca²⁺+ 20 mm EGTA).

Figure 6. Volume-activated Cl⁻ channels are not involved in the ATP-activated current

A, whole-cell currents at +80 mV were sampled every 5 s from a cell bathed in a hypertonic medium (370 mmol kg⁻¹). 1,9-dideoxyforskolin (0.1 mm DD-FKL) was applied 45 s before perfusing with 5 mm Tris-ATP. B, whole-cell currents were measured every 5 s using a 40 ms test pulse to +80 mV from a holding potential of 0 mV. Volume-activated channels were activated by decreasing the bath tonicity from 370 to 240 mmol kg⁻¹ during the indicated time. DD-FKL (0.1 mm) was applied to inhibit the volume-sensitive current and then 5 mm Tris-ATP was added at the indicated times. Insets: whole-cell currents obtained at the time indicated by the corresponding numbers in the main figure.

Figure 7. Effects of Cibacron Blue 3GA on the ATP-activated Na⁺ and Cl⁻ currents

A, current-voltage relationship of the ATP-activated Na⁺ current in the absence (▪) and presence (•) of 0.5 mm Cibacron Blue. B, current-voltage relationship of the ATP-activated Cl⁻ current in the absence (▪) and presence (•) of 0.5 mm Cibacron Blue. Insets: whole-cell currents obtained at +80 mV in the absence of ATP (control, smallest currents), in the presence of ATP (largest currents), and in the presence of ATP + Cibacron Blue (indicated by the arrows). Na⁺ currents were recorded from cells dialysed and bathed with solutions containing Na-glutamate. Cl⁻ currents were recorded from cells dialysed and bathed with solutions containing TEA-Cl.