Mechanism of inhibition of cyclic nucleotide-gated ion channels by diacylglycerol

Abstract

Cyclic nucleotide-gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409-417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (alpha subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (P(o)), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the P(o) but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer-channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.

Figure 1

Inhibition by DAG is more pronounced for rod than for olfactory CNG channels. Data were measured from multichannel, inside-out patches of homomultimeric (α only) rod and olfactory channels. The families of cGMP-activated currents were recorded in response to voltage jumps ranging from −100 to +100 mV in steps of 50 mV, from a holding potential of 0 mV. Currents measured in the absence of cGMP were subtracted from all traces. 2 mM cGMP and 100 μM cGMP are saturating levels of cGMP for rod and olfactory channels, respectively. (A) Inhibition at saturating cGMP: rod channels, 42% inhibition; and olfactory channels, 10% inhibition. (B) Inhibition at low cGMP concentrations: rod channels, 10 μM cGMP, I/I _max = 0.2 before DAG application, and 93% inhibition by DAG; olfactory channels, 2 μM cGMP, I/I _max = 0.6 before DAG application, and 72% inhibition by DAG.

Figure 2

Dose–response curves for rod and olfactory channels demonstrate differential inhibition by DAG. Bath contained low divalent NaCl solution with or without 0.5 μM DAG and various concentrations of cGMP. Steady-state, cGMP-activated currents were measured at +100 mV from a single patch containing either rod or olfactory channels and were normalized to the I _max without DAG. Smooth curves were drawn by fitting the averaged data with the Hill equation, I/I _max = [cGMP]ⁿ/(K_1/2 ⁿ + [cGMP]ⁿ), where I is the cGMP-activated current, I _max is the cGMP-activated current obtained at saturating cGMP, K_1/2 is the concentration of cGMP giving half maximal activation, and n is the Hill coefficient. In fitting the data, this relation was scaled to match the measured maximal I/I _max (e.g., the maximal I/I _max for the rod channel in the presence of 0.5 μM DAG was only 0.71 here, rather than 1). The Hill relation was used only for an empirical description of the data to quantify changes in the apparent affinity and maximal activation (or inhibition; see Fig. 3); it is not meant to suggest a mechanism of channel activation or inhibition by DAG. (A) Rod channels. Top curve (without DAG): I/I _max = 1, n = 2.25, and K_1/2 = 24 μM. Bottom curve (with DAG): I/I _max = 0.71, n = 2.25, and K_1/2 = 76 μM. (B) Olfactory channels. Top curve (without DAG); I/I _max = 1, n = 2.8, and K_1/2 = 1.8 μM. Bottom curve (with DAG): I/I _max = 0.97, n = 2.8, and K_1/2 = 4.0 μM.

Figure 3

Rod channel currents show complete suppression by DAG at saturating cGMP concentration, whereas olfactory channel currents show only partial suppression; inhibition by DAG is greater at low than at high cGMP concentration. Averaged data obtained at saturating cGMP were fit with the Hill equation, IN/IN_max = [DAG]ⁿ/(IC₅₀ ⁿ + [DAG]ⁿ), where IN is percent inhibition, IN_max is maximal inhibition, IC₅₀ is the concentration of DAG required to achieve half maximal inhibition, and n is the Hill coefficient. As for Fig. 2, the relation was scaled to reflect the measured maximal IN/IN_max. Data points at low cGMP are from a single rod or olfactory patch at a concentration of cGMP that gave ∼40% of maximal current. This finding was consistent in several other patches of each type. Data points at saturating cGMP (2 mM for rod and 100 μM for olfactory) are averaged values, plotted with SD (error bars) of data from 11 patches for rod and 13 patches for olfactory channels. Note the different scales of the two figures. (A) Rod channels. (Closed circles) At ∼30 μM cGMP, IN_max = 100%, IC₅₀ = 0.41 μM, and n = 1.3. (Open circles) At 2 mM cGMP, IN_max = 100%, IC₅₀ = 0.83 μM, and n = 1.7. Triangles represent data from native rod channels (two patches), included for comparison with the cloned channels. (B) Olfactory channels. (Closed squares) At 1.5 μM cGMP, IN_max = 100%, IC₅₀ = 0.47 μM, and n = 1.4. (Open squares) At 100 μM cGMP, IN_max = 31%, IC₅₀ = 3.24 μM, and n = 1.4.

Figure 4

DAG affects P_o rather than single-channel conductance of the rod channel. Currents recorded from an inside-out patch containing two α-homomultimeric mutant rod channels. These channels contained two point mutations (H468Q and A483V) that increased their cAMP efficacy to ∼40%. However, like wild-type rod channels, they were very sensitively (IC₅₀ = 0.33 μM DAG) and fully inhibited by DAG even in the presence of saturating cGMP. Holding potential +80 mV, sampling rate 25 kHz after filtering at 5 kHz. Partial records are shown from five traces of 1 s duration. Patches were bathed in low divalent NaCl without cGMP, with saturating cGMP, and with two different DAG concentrations at saturating cGMP as designated. Amplitude histograms from full records (5 s) are shown to the right of each set of current traces. The histograms in the two middle panels were fit by the sum of two Gaussians, with means of 2.1 and 4.2 pA, suggesting a single-channel amplitude of 2.1 pA, with and without 0.25 μM DAG. The smooth lines represent the Gaussian fits, and the shaded areas indicate the histograms that were constructed from the data.

Figure 5

DAG affects P_o rather than single-channel conductance of the mouse olfactory channel. Currents were recorded and data are presented in the same manner as in Fig. 4; the patch contains a single olfactory channel. Partial records are shown from three traces of 1 s in length. Amplitude histograms from full records (3 s) are shown. The histograms were fit with Gaussians, indicating a mean single-channel current of 3.2 pA at +80 mV, with and without DAG.

Figure 6

Inhibition by DAG is not voltage-dependent. Addition of DAG mimics a reduction in cGMP concentration, producing increased current–voltage rectification consistent with lower P_o. Steady state current–voltage relations constructed from data obtained from single patches; all data in a given plot are from the same patch. Open circles represent cGMP-activated currents at saturating cGMP in the absence of DAG. Closed circles represent currents at saturating cGMP in the presence of 2 μM DAG. For comparison, the triangles show the currents at lower cGMP concentration, without DAG: 10 μM cGMP for rod and 5 μM for olfactory. (A) Rod channels. (B) Olfactory channels.

Figure 7

Examples of chimera channel behavior in response to DAG. This figure shows the amount of channel inhibition as a function of increasing DAG concentration for three chimeric constructs (structures depicted in Fig. 8). Points are mean values, plotted with SD (error bars). CHM 11: IN_max = 100%, IC₅₀ = 0.63 μM, and n = 1.6 (five patches). CHM 13: IN_max = 100%, IC₅₀ = 1.80 μM, and n = 2.8; (six patches). CHM 3: IN_max = 68%, IC₅₀ = 3.30 μM, and n = 1.6; 4 patches.

Figure 8

K_1/2 (cGMP) and IC₅₀ (DAG) show an approximately inverse relationship among chimeras. In the chimera illustrations, the regions shown in black are from the olfactory channel, and the regions shown in gray are from the rod channel. A general channel diagram showing more detail is included at the top of the figure. The data for chimeras shown in this figure, but not included in Fig. 7, were measured from the following numbers of patches: CHM18 (six); CHM22 (six); CHM16 (five); CHM12 (three); CHM4 (five); and CHM15 (four). Chimeras are arranged from top to bottom in the order of increasing cGMP K_1/2 values. Points represent values of cGMP K_1/2 (triangles) and DAG IC₅₀ (circles) obtained by fitting Hill curves to averaged data of each type (e.g., as in the cGMP dose–response curves without DAG shown in Fig. 2 and as in the DAG dose–response data at saturating cGMP shown in Fig. 3 and Fig. 7).

Figure 9

Maximal inhibition by DAG is greatest for chimeras whose transmembrane segments and loops are mostly of the rod channel type. Values for maximal inhibition were obtained from fits to the Hill equation while determining the IC₅₀ values for Fig. 8.

Figure 10

Cartoons depicting two possible mechanisms for the action of DAG. The actual mechanism may lie somewhere between these two extremes. Multiple DAG molecules stabilize the closed state(s) of both rod and olfactory channels, although to different extents. Channels in this diagram are assumed to be fully liganded with cGMP. (See Discussion for further information.) (A) Direct interaction of DAG with the channels. DAG is hypothesized to bind in hydrophobic crevices in closed channels, thereby allosterically hindering channel opening. The allosteric opening transition of the olfactory channel is more energetically favored than that of the rod channel (Gordon and Zagotta 1995b). Thus, the olfactory channel can sometimes open in spite of DAG occupancy, whereas the rod channel generally cannot. (B) Bilayer deformation model. DAG is hypothesized to insert into the bilayer, altering its mechanical properties and its interaction with the channels. In one possible scenario, the closed states of the channel protein have a smaller hydrophobic exterior surface than the open states. The surrounding bilayer is depicted as having more curvature in the presence of DAG, suggesting a thinner hydrophobic core that is better matched to the size of the closed channel's hydrophobic surface. In this way, the insertion of DAG into the lipid bilayer may make channel closure more energetically favorable.