Electrophysiological Properties of Endogenous Single Ca2+ Activated Cl- Channels Induced by Local Ca2+ Entry in HEK293

Abstract

Microdomains formed by proteins of endoplasmic reticulum and plasma membrane play a key role in store-operated Ca²⁺ entry (SOCE). Ca²⁺ release through inositol 1,4,5-trisphosphate receptor (IP₃R) and subsequent Ca²⁺ store depletion activate STIM (stromal interaction molecules) proteins, sensors of intraluminal Ca²⁺, which, in turn, open the Orai channels in plasma membrane. Downstream to this process could be activated TRPC (transient receptor potential-canonical) calcium permeable channels. Using single channel patch-clamp technique we found that a local Ca²⁺ entry through TRPC1 channels activated endogenous Ca²⁺-activated chloride channels (CaCCs) with properties similar to Anoctamin6 (TMEM16F). Our data suggest that their outward rectification is based on the dependence from membrane potential of both the channel conductance and the channel activity: (1) The conductance of active CaCCs highly depends on the transmembrane potential (from 3 pS at negative potentials till 60 pS at positive potentials); (2) their activity (NPo) is enhanced with increasing Ca²⁺ concentration and/or transmembrane potential, conversely lowering of intracellular Ca²⁺ concentration reduced the open state dwell time; (3) CaCC amplitude is only slightly increased by intracellular Ca²⁺ concentration. Experiments with Ca²⁺ buffering by EGTA or BAPTA suggest close local arrangement of functional CaCCs and TRPC1 channels. It is supposed that Ca²⁺-activated chloride channels are involved in Ca²⁺ entry microdomains.

Keywords: CaCC; HEK293; IP3R; SOCE; TMEM16; TRPC1; anoctamin; single channel.

Figure 1

Electrophysiological properties of endogenous Ca²⁺-activated chloride channels (CaCC) in HEK293 cells. (A) Representative recording of currents, registered at different membrane potentials, within one experiment before and after bath application of 2.5 μM IP₃ (inositol 1,4,5-trisphosphate). The expanded panels depict inward IP₃-induced Ca²⁺ currents followed by Ca²⁺-activated outward currents. The baseline was appropriately adjusted for voltage switches. (B) Examples of CaCC current recordings obtained after IP₃-induced Ca²⁺ entry at different membrane potentials. (C) Current–voltage relationship of IP₃-induced TRPC1 (transient receptor potential-canonical) Ca²⁺ currents (n = 3–11). The linear fit to the data points from −100 to −40 mV yielded single channel conductance (γ) of 21 pS. (D) Left: Current–voltage relationship of Ca²⁺-activated chloride channels recorded in panel B (n = 3–4). Right: The dependence of the conductance of Ca²⁺-activated chloride channels on the membrane potential. (E) The dependence of the activity of Ca²⁺-activated chloride channels, expressed as NPomax30, on the membrane potential (n = 4). (F) An open-time histogram of CaCC channel was constructed from 2563 single-channel opening events. Single exponential fit (solid line) corresponds to a time constant of 5.7 ± 0.3 ms. The current trace demonstrates a typical fragment of the current recording used for the histogram at +30 mV and filtered at 1 kHz.

Figure 2

Induction of CaCCs activity by IP₃-mediated Ca²⁺ entry in HEK293 cells. (A) Current recordings after bath application of 2.5 µM IP₃ to inside-out patches with 105 Ca²⁺ in pipette solution (top), 105 Ba²⁺ in pipette solution (middle), and 140 Na⁺ in pipette solution (bottom). Patches were held at +20 mV membrane potential. (B) IP₃-induced CaCC currents in inside-out patches after switching to positive (top) or to negative (bottom) potential. (C) Current recordings after bath application of 100 µM UTP to cell-attached patches with 105 Ca²⁺ in pipette solution (top), 105 Ba²⁺ in pipette solution (middle), and 105 Ca²⁺ in the presence of 100 µM NFA in pipette solution (bottom). (D) The summary plot of the frequency of CaCCs observation (top) and the CaCC open channel probability (bottom) for series from the panels A–C. The frequency of CaCCs observation reflects a proportion of positive experiments to the total number of experiments. Differences are considered significant when p values are <0.05 (*).

Figure 3

Endogenous CaCCs activated by direct Ca²⁺ application in HEK293 cells. (A) Representative recordings of channels activated by the increase in intracellular Ca²⁺ concentration. Shown are traces at compressed and expanded scale recorded in inside-out configuration at holding potential of +40 mV. (B) Representative traces of channels activated by the increase in intracellular Ca²⁺ concentration recorded at the same conditions as in panel A but in the presence of 100 µM NFA and 100 µM DIDS in the recording pipette. (C) Current–voltage relationship for CaCCs activated by direct 100 µM Ca²⁺ application (n = 3). (D) A summary plot of the CaCCs open channel probability in inside-out recordings at holding potential of +40 mV. The data are presented for different cytoplasmic Ca²⁺ concentration, and with or without chloride channel blockers NFA (100 µM) and DIDS (100 µM). (E) The frequency of CaCC observation in patches is plotted as a proportion of positive experiments to the number of experiments in the series. The data are presented for experiments with bath application of 100 µM Ca²⁺ with or without 100 µM NFA and 100 µM DIDS in the pipette. Data are shown as means ± S.E. Differences are considered significant when p values are <0.05 (*), and <0.01 (**).

Figure 4

Properties of Ca²⁺-activated chloride channels upon using fast Ca²⁺ chelator 10 mM BAPTA. (A) Examples of single channel recordings through the TRPC1 channels induced by the bath application of 2.5 μM IP₃ in inside-out patches. Membrane potentials were held at 0 mV (left) and −70 mV (right). (B) Current–voltage relationship of store-operated TRPC1 channels is induced by 2.5 µM IP₃ application. Approximation of conductance gives 21 pS (n = 3–7). (C) Current recordings of CaCCs-activated following IP₃-induced Ca²⁺ entry registered at different membrane potentials +50 mV, 0 mV, −50 mV (top to bottom). (D) Left: The current–voltage relationship of CaCCs in the BAPTA containing bath solution (n = 4–8). Right: The dependence of the CaCC conductance on the membrane potential (n = 4–8). (E) The dependence of the CaCC activity, expressed as NPomax30, on the membrane potential (n = 4). (F) Histogram of the open state lifetime distribution of the CaCCs. The membrane potential is +30 mV. An exponential fit gives an average lifetime of 0.3 ± 0.1 ms. The total number of events is n = 7946. Typical fragment of the current used for the histogram, monitored at +30 mV and filtered at 1 kHz.