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First published online June 29, 2007
Journal of Experimental Biology 210, 2489-2500 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006361
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Properties and possible function of a hyperpolarisation-activated chloride current in Drosophila

Uwe Rose1,*, Christian Derst2, Mario Wanischeck1, Christiane Marinc1 and Christian Walther3

1 Institute of Neurobiology, University Ulm, Albert-Einstein-Allee 11, Ulm 89160, Germany
2 Institute for Integrative Neuroanatomy, Charite, Berlin, Germany
3 Institute of Physiology and Pathophysiology, Philipps University Marburg, Marburg, Germany


Figure 1
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  		Fig. 1. Currents recorded from a muscle fibre during prolonged hyperpolarising voltage commands. (A) Current electrode filled with 2 mol l–1 K-acetate. After the instantaneous jump, a small inward current develops within seconds. The superimposed traces, recorded 1 and 10 min after impaling the fibre, are nearly identical. Note that the lower trace represents the recorded voltage imposed by the pulse protocol. (B) Current–voltage relationships for the fibre in A. The instantaneous current (open symbols) is linear whereas the late current (filled symbols) exhibits slight inward rectification. (C) Recording from another fibre where current electrode filled with 3 mol l–1 KCl was used; voltage jump as in A. Although the current recorded is initially similar to that in A, the slow inward component is clearly increased 10 min after impalement. (D) I–V plot for the fibre recorded in C. The instantaneous current is practically linear, as in B, but the inward rectification of the late current is enhanced 10 min after impalement. Note inward-going tail currents after jumping from –110 to –60 mV. In both A and B the voltage electrode contained 3 mol l–1 KCl.

 

Figure 2
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  		Fig. 2. Hyperpolarisation-induced current in a Cl–-loaded muscle fibre. (A) Currents elicited by the hyperpolarising voltage commands shown in the inset. Note the pronounced tail currents after deactivating jumps to –60 mV. Small, short inward currents are miniature excitatory junctional currents. The recordings were performed after holding the fibre for 5 min at –50 mV with a holding current of {approx}–5 nA. The current electrode was filled with 3 mol l–1 KCl. (B) Current clamp recordings subsequently performed from the same fibre; currents were injected according to the protocol shown in the inset.

 

Figure 3
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  		Fig. 3. Reversal potentials of K+ current and hyperpolarisation-induced current. (A) Sample traces of tail currents recorded subsequently in one fibre. Left: deactivating K+ currents, mainly consisting of A currents. For K+ current activation the fibre was first depolarised from –70 to +10 mV for 80 ms, then the voltage was stepped to different levels as seen in B. Right: deactivation of hyperpolarisation-dependent currents. For activation the voltage was stepped from –50 mV to –120 mV for 4 s, followed by deactivating jumps to –50 mV or more positive levels as seen in B. (B) Mean current–voltage relationships of peak tail-currents obtained, as in A, from paired recordings of four fibres from two animals. Note that the ordinate gives relative sizes of the currents, i.e. normalised to IK at V=–30 mV and to hyperpolarisation-dependent current at –50 mV, respectively. [K+]o and [Cl–]o were standard, i.e. 10 mmol l–1 and 95 mmol l–1, respectively. The mean reversal potential was –55.2±4.1 mV for the K+ current and –20.5±4.0 mV for the hyperpolarisation-activated current (l=2, f=4). The current electrode contained 3 mol l–1 KCl; recordings of the hyperpolarisation-dependent currents were performed 5 to 10 min after impaling the fibre.

 

Figure 4
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  		Fig. 4 The reversal potential of the hyperpolarisation-induced current depends on the external Cl– concentration. Current–voltage relationships of tail currents as shown in Fig. 3A, right-hand side. Averages of paired measurements in three fibres, performed first with [Cl–]o=65 mmol l–1 and then with [Cl–]o=20 mmol l–1. The reduction of [Cl–]o led to a positive shift of the reversal potential, i.e. from –18.2±3.8 mV to +2.2±1.8 mV. K+ currents were blocked by means of 20 mmol l–1 TEA, 1 mmol l–1 4-aminopyridine (4AP) and 0.1 mmol l–1 quinidine.

 

Figure 5
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  		Fig. 5. Steady-state activation of the chloride-conductance, GCl,H. (A) Mean G–V relationship at two different internal Cl– concentrations. Paired measurements of currents and of ECl were performed in six fibres after moderate and strong Cl– loading of the fibre (the respective recordings were performed 1 and 5 min after impalement; for details of the approach and for the voltage-clamp protocols employed, cf. Materials and methods). Currents were converted to conductance, G. The conductance is greatly enhanced at raised [Cl–]i. This effect is largely because of the increase in maximal conductance. (B) Normalised G–V relationships for the data shown in A. The rise in [Cl]i led to some positive shift of the curve on the voltage axis. Curves in A and B are Boltzmann-fits. For further analysis, cf. Table 2. Error bars indicate s.e.m.

 

Figure 6
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  		Fig. 6. Effects of pH changes on time course and amplitude of ICl,H. (A) A change from pH 7.2 to 8.4 leads to a faster time course of the hyperpolarisation-induced Cl– current. The amplitude, obtained by extrapolating the current until it reached steady state, was somewhat reduced. Voltage jump was as shown in B. (B) At pH 6.0 the amplitude became greatly reduced. The time course of the current appears to be slowed, yet in fact it remained unaffected, as found on fitting double exponentials to the current traces. Both pH effects were rapid; they were recorded {approx}1 min after changing the bath solution. The recordings labelled `wash' were made 3 to 5 min after changing back to pH 7.2. For a more detailed analysis of these effects see Table 1.

 

Figure 7
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  		Fig. 7. Potentiation of ICl,H in hypotonic saline. (A) Currents recorded from one fibre on jumping the voltage from –50 to –120 mV. The intervals between the different recording conditions lasted 8–10 min. (B) Amplitudes of tail currents following hyperpolarising prepulses as in A. Data from the same fibre as in A. The lines connecting the data points represent Boltzmann-fits with the following parameters, presented in the sequence control-hypotonic wash: Imax: 5.2, 8.5, 3.4 (nA); V0.5: –110, –106, –106 (mV); k (slope factor): 13.1, 11.8, 10.0 (mV). (C) Mean osmotically induced increase in tail currents recorded after –120 mV prepulses as shown in A (f=4, l=2, N=4).

 

Figure 8
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  		Fig. 8. Effect of 9-AC. (Ai) Representative recordings from one fibre performed 3 min (control), 8 min (9-AC) and 17 min after impaling the muscle fibre. 9-AC, at –120 mV, blocks ICl,H in a time-dependent manner and reduces the tail current on jumping back to –50 mV. Prolonged washing led to almost full recovery of ICl,H. (Aii) Means and their standard errors of normalized ICl,H from four experiments. (B) The effect of 1 mmol l–1 9-AC on the resting membrane conductance measured by voltage ramps. 9-AC significantly lowered the membrane conductance of muscle fibres by approximately 20%. (C) Synaptic currents were consistently decreased by 1 mmol l–1 9-AC to approximately 70% of the control value (N=4). This effect was reversed by washing with saline.

 

Figure 9
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  		Fig. 9. Effect of 9-AC on stimulus evoked muscular contraction. (A,B) Muscular contractions were consistently augmented when 1 mmol l–1 9-AC was applied to the bath. Washing with saline almost reversed this effect. The 9-AC effect was basically similar for single contractions (twitch) or tetanus (5 Hz, 10 Hz) contractions, although it seemed to be less pronounced for twitch contractions. However, no significant difference was detected between twitch and tetanus. 9-AC did not change the time course (50% relaxation time) of twitch contractions.

 

Figure 10
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  		Fig. 10. RT-PCR analysis of the expression of ClC channels in Drosophila melanogaster muscle preparations. (A) RT-PCR analysis of body wall RNA preparations. Two independent PCR reactions were performed for each Drosophila ClC channel (primers F1-B1 in left lane and F2-B2 in right lane). (B) DmClC-a RT-PCR analysis (primers F2-B2) of five different pure muscle RNA preparations, Mu1-5 and CNS. Grey arrows indicate RT-PCR products; white arrows indicate PCR products amplified from genomic DNA; *, an unspecific amplification (as verified by DNA sequencing). DNA marker: M1, {lambda}DNA/Eco47I; M2, pBR322 DNA/BsuRI; M3, 100 bp ladder; WP, water control. `DmClC-a' corresponds to `DmClC-2', which is chiefly used in the text and which has been introduced by Flores et al. (Flores et al., 2006Go). (C) Localisation of the ClC-2 channel in larval longitudinal muscles by immunohistochemistry showed distinct bands of antibody staining. ClC-2-positive staining corresponds to the Z-line of the sarcomere.

 

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© The Company of Biologists Ltd 2007