Heteromultimeric CLC chloride channels with novel properties

Abstract

The skeletal muscle chloride channel CLC-1 and the ubiquitous volume-activated chloride channel CLC-2 belong to a large gene family whose members often show overlapping expression patterns. CLC-1 and CLC-2 are coexpressed in skeletal and smooth muscle and in the heart. By coexpressing CLC-1 and CLC-2 in Xenopus oocytes, we now show the formation of novel CLC-1/CLC-2 heterooligomers that yield time-independent linear chloride currents with a chloride-->bromide-->iodide selectivity sequence. Formation of heterooligomeric CLC channels increases the number and possible functions of chloride channels.

Figure 1

Voltage-clamp traces of oocytes injected with CLC-1 cRNA (B), CLC-2 cRNA (C and D), and coinjected with both RNAs (E and F). (A) Voltage-clamp pulse protocol. Test pulses were applied in −20 mV steps starting at +40 mV (holding potential: −30 mV). The tail pulse was varied depending on the cRNA injected. Solid line, CLC-2; dashed line, CLC-1; dotted line, coinjection. (B) Typical current traces of an oocyte expressing CLC-1, showing the characteristic inward rectification at positive voltages and the deactivation at negative voltages. (C and D) Typical currents of an oocyte expressing CLC-2. Long pulses (C) elicit a slowly activating current at negative voltages, which deactivates relatively fast when stepping to +40 mV. Short pulses (as in B, E, and F) does not significantly activate CLC-2 (D). (E and F) Typical currents of an oocyte coinjected with CLC-1 and CLC-2 cRNA at a 1:1 concentration ratio. Treatment with 0.2 mM 9-AC (F) abolishes a remaining deactivating component at negative voltages, as well as the gating seen in the tail current at a potential of −140 mV. This is more obvious when oocytes injected with an about 10-fold excess of CLC-1 over CLC-2 cRNA (G) are treated with 9-AC (H).

Figure 2

I–V relationships and ion selectivity of currents of oocytes expressing CLC-1 (A and D), CLC-2 (B and E), or both (C and F). (Upper A–C) Steady-state I–Vs in ND96 (104 mM Cl). (Lower D–F) Instantaneous I–Vs (open pore properties) with different anions in the bath. Measurements were performed in ND96 having 104 mM Cl (○), or in ND96 where 96 mM Cl were replaced by equal amounts of Br (□), glutamate (▵), or I (▿). This leaves 8 mM Cl in the medium. Data of one representative oocyte each are shown as a plot against the test potential. (A) Steady-state I–V of CLC-1. The current was measured at the end of a 100-ms test pulse. (B) Quasi-steady-state I–V of CLC-2 (measured at the end of a 6-s test pulse). (C) Steady-state I–V of CLC-1/CLC-2 heterooligomers (measured after treatment with 9-AC to block CLC-1 homooligomers). (D) Instantaneous I–V of CLC-1. Currents were extrapolated to the beginning of the test pulse by fitting the sum of two exponential functions. (E) Open-channel I–V of CLC-2. The oocyte was held at −140 mV until the current saturated before 200-ms test pulses were applied. (F) Instantaneous (equivalent to steady-state) I–V of the heterooligomer currents measured after 9-AC treatment.

Figure 3

Generation of CLC-1/CLC-2 heteromeric conductance as a function of cRNA concentration ratios. (A) Slope conductance at 50 mV measured in ND96 is plotted vs. the fraction of CLC-2 cRNA in the CLC-1/CLC-2 coinjection. Slope conductance of uninjected oocytes was subtracted. Conductance at +50 mV is predominantly due to heterooligomers. (B) Same as A (different oocytes from the same batch), but measured after incubation in 0.2 mM 9-AC that blocks CLC-1 homooligomers. Error bars = SEM, n = 4–5.

Figure 4

Dominant negative effect of the CLC-1 mutation P480L on wild-type CLC-2. (A) Current traces of an oocyte expressing CLC-2 (Left) or coexpressing CLC-2 and CLC-1_P480L (Right). (B) Absolute current at the end of the test pulse to −140 mV (error bar = SEM, n = 9). Background current of noninjected oocytes was subtracted. RNA was coinjected at a 1:1 ratio, with a constant total amount of RNA.