Slob, a Slowpoke channel-binding protein, modulates synaptic transmission

Abstract

Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel-binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission.

Figure 1.

Slob-null and RNAi flies show reduced Slob protein expression in adult fly heads. (A) In mut^IP1 flies (lane 2), there is greatly reduced Slob expression compared with the WT^P41 line (lane 1). The remaining staining near 57 kD in lane 2 is a result of the presence of a cross-reacting band not related to Slob. Slob expression driven by a ubiquitous (lane 3) or nervous system–specific elav-GS (lane 4) Gal4 driver in the mut^IP1 background is able to restore Slob expression in the fly head. (B) In Slob-RNAi flies, expression of Slob-RNAi driven by the ubiquitous Gal4 driver reduces the expression of Slob protein (lane 2). In contrast, Slob-RNAi expression driven by either the muscle-specific Gal4 driver (lane 3) or the nerve-specific Gal4 driver 1407-Gal4 (lane 4) does not affect Slob levels in the adult head. WT¹ and WT² are the uncrossed parental lines used as WT controls (refer to Materials and methods); WT³ is a yellow-white fly line used as an additional control.

Figure 2.

Slob expression at the Drosophila NMJ. HRP antibody stains nerve surface and presynaptic terminals (red; left column). White arrows in A point to individual synaptic boutons. Polyclonal antibody to Slob (green; middle column) stains Slob in nerve and nerve terminals and to a more limited extent in muscle. Overlay (yellow-orange; right column) illustrates colocalized Slob and HRP staining. (A) Staining of the WT line in the absence of primary antibody to Slob. (B) Staining of the WT line. (C) Staining of the Slob-null line. Bar, 5 µm.

Figure 3.

Slob can be restored to specific locations in a Slob-null background. Staining as for Fig. 2. (A) The Slob-null line as control. (B) Ubiquitous rescue of Slob. (C) Rescue of Slob expression in nerve. (D) Rescue of Slob expression in muscle. Bar, 5 µm.

Figure 4.

Slob can be disrupted in a tissue-specific manner using Slob-RNAi and specific Gal-4 drivers. Staining as for Figs. 2 and 3. (A) The uncrossed WT¹ line (refer to Materials and methods) as control. (B) Ubiquitous disruption of Slob. (C) Disruption of Slob expression in nerve. (D) Disruption of Slob expression in muscle. Bar, 5 µm.

Figure 5.

Evoked synaptic transmission is increased in Slob-null flies. (A) Representative recording of the muscle cell membrane potential during voltage clamping and sample traces. The EJC was measured with the muscle cell voltage clamped at −60 mV (top). The stimulation artifact arises from stimulation of the segmental nerve. Sample EJC traces from WT control, two Slob-null lines, and one ubiquitous rescue fly line are shown. (B) Pooled data. Peak amplitude of EJC is increased significantly in mut^IP1 and mut^K162 flies (black and dark gray bars) compared with the WT^P41 control (white bar). In addition, ubiquitous expression of Slob57 in a Slob-null background rescues synaptic transmission to the WT level (light gray bar).

Figure 6.

mEJC frequency and amplitude are increased in Slob-null flies. The muscle cell was voltage clamped at −60 mV as for Fig. 5, but no stimulus was delivered to the segmental nerve. (A) Sample mEJC traces from control and Slob-null flies in the absence or presence of 1 µM TTX. (B) mEJC frequency and amplitude distributions in WT^P41 and mut^IP1 flies. Bin sizes are 0.15 Hz and 0.05 nA, respectively. The distributions for mut^IP1 are shifted dramatically to higher frequency and amplitude. (C) Pooled data. Frequency and amplitude of mEJCs are increased significantly in mut^IP1 line (black bar) compared with WT^P41 flies (white bar) in the absence or presence of TTX. In mut^IP1rescue^all flies (gray bar), both mEJC frequency and amplitude are rescued to the level of the WT^P41 flies.

Figure 7.

Evoked and spontaneous synaptic transmission are increased in Slob-RNAi flies. (A) Sample EJC traces from two control lines and one Slob-RNAi line. (B) Pooled data. Peak amplitude of EJC is increased significantly when Slob-RNAi is expressed ubiquitously (black bar) compared with controls (white bars). (C) Sample mEJC traces from two control lines and one Slob-RNAi line. (D) Pooled data. Frequency and amplitude of mEJCs are increased significantly in the Slob-RNAi^all line (black bars) compared with controls (white bars). Refer to Materials and methods for the definition of the WT¹ and WT² lines.

Figure 8.

Genetic or pharmacological disruption of dSlo eliminates the differences in synaptic transmission between mut^IP1 and WT^P41 flies. (A) Averaged EJC traces from Slob-null and control lines. The difference in the EJC (A) is not observed in the Slo4 genetic background (B) or in the presence of 1 mM TEA (C). (D) Sample mEJC traces from Slo4 flies crossed to either Slob WT or null flies, or from control and Slob-null lines in the absence or presence of 1 mM TEA. Pooled data are shown in Table II.

Figure 9.

Targeted nerve expression of Slob-RNAi enhances evoked and spontaneous synaptic transmission. (A) Sample EJC traces from one control line and two Slob-RNAi lines. The Slob-RNAi^nerve line expresses Slob-RNAi in nerves, whereas the Slob-RNAi^muscle line expresses Slob-RNAi in muscle. (B) Pooled data. Peak amplitude of EJC is increased significantly when Slob-RNAi is expressed in nerves (black bar) compared with the uncrossed WT¹ control (white bar). EJC in the Slob-RNAi^muscle line (gray bar) is not significantly different from the EJC in the WT¹ control. (C) Sample mEJC traces from one control line and two Slob-RNAi lines. (D) Pooled data. Frequency and amplitude of mEJCs are increased significantly in the Slob-RNAi^nerve line (black bar), but not in the Slob-RNAi^muscle line (gray bar), compared with the uncrossed WT¹ control (white bar). Refer to Materials and methods for the definition of the WT¹ line.

Figure 10.

Rescue of Slob in nerves rescues the alterations in synaptic transmission. (A) Sample EJC traces from WT, Slob-null, and two rescue lines (rescue in muscle or nerve, respectively). (B) Pooled data. The enhanced EJC peak amplitude in Slob-null flies (black bar) is rescued by adding Slob back to the presynaptic nerve (dark gray bar), but not to the postsynaptic muscle (light gray bar). (C) Sample mEJC traces from WT, Slob-null, and two rescue lines (rescue in muscle or nerve, respectively). (D) Pooled data. Enhanced frequency and amplitude of non-evoked synaptic transmission (black bars) are rescued by adding Slob back to the presynaptic nerve (dark gray bars), but not to the postsynaptic muscle (light gray bars).

Figure 11.

Calcium dependence of synaptic transmission, and postsynaptic cell properties, in Slob-null and WT flies. (A–C) Slob modulates various aspects of synaptic transmission: (A) EJC amplitude, (B) mEJC amplitude, and (C) mEJC frequency over a range of calcium concentrations. Input resistance (D) and cell capacitance (E) of the postsynaptic muscle cells are similar in WT^P41 (white bar) and mut^IP1 (black bar) lines.