Hyposmotically activated chloride channels in cultured rabbit non-pigmented ciliary epithelial cells

Abstract

1. We used whole-cell patch-clamp recording techniques and noise analysis of whole-cell current to investigate the properties of hyposmotic shock (HOS)-activated Cl- channels in SV40-transformed rabbit non-pigmented ciliary epithelial (NPCE) cells. 2. Under conditions designed to isolate Cl- currents, exposure of cells to hyposmotic external solution reversibly increased the whole-cell conductance. 3. The whole-cell current activated with a slow time course (> 15 min), exhibited outward rectification and was Cl- selective. 4. The disulphonic stilbene derivatives 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 0.5 mM), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS, 0. 5 mM) and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS, 0.5 mM) produced a voltage-sensitive block of HOS-activated Cl- current at depolarized potentials, whereas niflumic acid produced a voltage-independent block of the current. 5. Under Ca2+-free conditions, HOS stimulation still reversibly activated the Cl- current, but the amplitude of current was reduced and the time course of current activation was slower compared with control (P < 0. 05). 6. The non-specific kinase inhibitor H-7 (100 microM), upregulated HOS-activated Cl- current amplitude in all cells tested (P < 0.05). 7. Noise analysis of whole-cell Cl- current indicated that cell swelling activated a high density of small conductance Cl- channels (< 1 pS). 8. We conclude that HOS primarily activates a high density of volume-sensitive small conductance Cl- channels in rabbit NPCE cells, and that Ca2+ and phosphorylation are involved in channel regulation.

Figure 1. Hyposmotic stimulation activates whole-cell current in NPCE cells

A, left panel shows patch-clamp recording from a representative NPCE cell taken 1 min after assuming the whole-cell configuration, in standard external solution and 90 mM [Cl⁻] internal solution (Control). The voltage protocol is shown at the top of the figure. Middle panel shows the same cell 30 min after superfusion with hyposmotic external solutions (HOS). The activation of a large current is apparent. The HOS-activated current recovered to the control levels (recovery, right panel) 15 min after return to standard external solution. B, time course for the activation of the HOS current shown in A. Current was measured at 2–15 min intervals at +58 mV from a holding potential of −62 mV in standard external solution or in external hyposmotic solution (HOS). C, current-voltage plot for the currents measured in the NPCE cell shown in A. Currents were measured 1 min after assuming whole-cell configuration in standard external solution (□), 30 min after superfusion with HOS (•) and 15 min after return to standard external solution (▴).

Figure 2. The HOS-activated current is Cl⁻ selective

A, current-voltage plots for 3 representative NPCE cells are shown for [Cl⁻]_i of 30 (▪), 60 (○) or 90 mM (▴). The reversal potential of the HOS current was shifted when [Cl⁻]_i was altered by substitution with Trizma base, from +2 mV (90 mM Cl⁻) to −15 mV (60 mM Cl⁻) and to −29 mV (30 mM Cl⁻). B, the mean (±s.e.m.) reversal potential of the HOS current was measured in 3–15 cells in which [Cl⁻]_i was altered by substitution with Trizma base and plotted against the theoretical E_Cl. The least-squares fit of the data (dashed line) had a slope of 0·998 corresponding to a 58 mV per 10-fold change of [Cl⁻]_i. The continuous line has a slope of 1, predicted for a Cl⁻-selective current.

Figure 3. HOS current is sensitive to Cl⁻ channel blockers

A, whole-cell currents recorded from a representative NPCE cell. Currents were measured from a holding potential of −62 mV in response to voltage steps from −102 to +118 mV in standard external solution (Control) and hyposmotic external solution, in the absence (HOS) and in the presence of 0·5 mM DIDS (HOS + DIDS). B–E, mean (±s.e.m.) current amplitude of the HOS Cl⁻ current. Current was measured at +58 and −62 mV before (Control; ▪) and after superfusion with, or application of, the Cl⁻ channel blockers (□) DIDS (B), SITS (C), DNDS (D), or niflumic acid (NFA; E). Data have been normalized for cell capacitance (*P < 0·05; **P < 0·01).

Figure 4. Ca²⁺ is involved in the regulation of the HOS Cl⁻ current

A, mean (±s.e.m.) time course for the HOS Cl⁻ current measured from a V_H of −62 mV in low Ca²⁺ external solution and 10 mM BAPTA internal solution in 8 NPCE cells. B, mean (±s.e.m.) current amplitude of the HOS Cl⁻ current measured 50 min after superfusion with hyposmotic external solution at +58 and −62 mV. HOS Cl⁻ current amplitude was measured in standard external and internal solutions (▪) and Ca²⁺-free external and internal solution (□). All data have been normalized for cell capacitance (*P < 0·05).

Figure 5. Role of protein phosphorylation in regulation of the HOS-activated Cl⁻ current

Bar graph showing mean (±s.e.m.) current amplitude of the HOS-activated Cl⁻ current measured at +58 and −62 mV, 30 min after superfusion with hyposmotic external solution, in cells in the absence of H-7 (▪) and in cells pre-treated for 20 min and superfused with H-7 (□). All data have been normalized for cell capacitance (*P < 0·05).

Figure 6. Estimation of HOS-activated single channel properties by noise analysis

A shows noise variance versus membrane current during the development of HOS-activated Cl⁻ current in one cell, fitted by eqn (1) with γ= 0·53 pS and N = 18 000. The increased measurement variability at intermediate current values reflects the relatively short duration and more rapid current change experienced at these values. B, measured current versus time for the same data with superimposed open probablility calculated from eqn (2). Note that HOS almost completely activated the channels.