Functional characterization and optimization of a bacterial cyclic nucleotide-gated channel

Abstract

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening (i.e. gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability (P_o). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the P_o in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the P_o in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.

Keywords: allosteric regulation; allostery; bacteria; channel activation; cyclic nucleotide; cyclic nucleotide gating; electrophysiology; potassium channel; prokaryotic ion channel; voltage-gated ion channel.

Figure 1.

SthK structure and allosteric gating scheme. A, cartoon representation of SthK cryo-EM structure (Protein Data Bank (PDB) code 6CJU) with the functional domains labeled. cAMP is displayed as spheres. PD, pore domain; VSD, voltage-sensing domain. B, MWC gating scheme with C and O representing closed and open states, respectively. cAMP is indicated as a. Equilibrium constants are shown near arrows.

Figure 2.

SthK expression and patch-clamp recording in E. coli spheroplasts. A, method for preparing E. coli spheroplasts. B, bright-field and fluorescence images of spheroplasts expressing GFP-tagged SthK. C, top, non-leak-subtracted currents in the absence of cNMP from inside-out patches of SthK-expressing spheroplasts in response to the voltage protocol at the top. Middle and bottom, leak-subtracted currents in the presence of 1 mm cAMP or 5 mm cGMP.

Figure 3.

Cyclic nucleotide-dependent gating of wtSthK. A, dose-response curve of wtSthK channels for cAMP. The solid black line represents fit of the Hill equation with K_1/2 = 1.5 μm and h = 1.5. Points and error bars represent mean ± S.E., respectively, from three patches. B, single-channel currents from a patch containing 150–200 wtSthK channels held at −60 mV in the absence of cNMP (upper trace) and in the presence of 5 mm cGMP (lower trace). The inset shows zoom of an ∼1-s region. Data were filtered at 0.5 kHz for display. C, concentration jump from 0 to 1 mm cAMP while holding the patch at −40 mV. The inset shows an ∼500-ms region. The thin red line represents fit of a single exponential with a time constant of 11.0 ms.

Figure 4.

Voltage dependence of wtSthK gating. A, leak-subtracted current family for wtSthK in the presence of 1 mm cAMP. B, normalized G–V curve calculated from the instantaneous tail current at +100 mV (black points). The solid blue line represents fit of a Boltzmann equation with V_1/2 = 22 and zδ = 0.55. Red points represent P_o from single-channel recordings (right axis). Points and error bars represent mean ± S.E., respectively, from three to 10 patches. C, representative single-channel recording of wtSthK in 1 mm cAMP at −60 mV. An all-points histogram is shown on the right. The blue line represents fit with a sum of Gaussians with P_o = 0.63 for this patch. D, representative single-channel recording of wtSthK in 1 mm cAMP at +60 mV. An all-points histogram is shown on the right. The blue line represents fit with a sum of Gaussians with P_o = 0.91 for this patch. E, leak-subtracted single-channel voltage ramp from −120 to +120 mV in 500 ms.

Figure 5.

Properties of Cys-free SthK. A, leak-subtracted current family for cfSthK in the presence of 1 mm cAMP (red, top) and 5 mm cGMP (green, bottom). The voltage protocol is the same as in Fig. 2C. B, dose-response curve of cfSthK channels for cAMP. The solid black line represents fit of the Hill equation with K_1/2 = 1.1 μm and h = 1.5. The dashed black line represents cAMP dose response for wtSthK. Points and error bars represent mean ± S.E., respectively, from three patches. C, representative single-channel recordings for cfSthK at −60 mV in 1 mm cAMP. An all-points histogram is shown on the right. The blue line represents fit with a sum of Gaussians to give P_o = 0.58 for this patch.

Figure 6.

Toxicity of cfSthK expression in E. coli. A, growth curves for C43 cells with expression of cfSthK or cfSthK-R377A. Points and error bars represent mean ± S.D., respectively, from three cultures. B, crystal structure of SthK CNBD bound to cAMP (PDB code 4D7T). cAMP and residues in direct contact with cAMP are shown as sticks. C, leak-subtracted currents in 1 (black trace) and 15 mm (red trace) cAMP from a patch expressing cfSthK-R377Q. The inset shows single-channel openings at −60 mV in the presence of 1 mm cAMP. D, left, cartoon showing the strategy for making C43 cyaA⁻ cells. Right, gel showing colony PCR of the region between H1 and H2 of C43 and C43 cyaA⁻ cells. E, growth curves comparing nontransformed C43 cyaA⁻ cells in the absence and presence of cAMP with nontransformed C43 cells. F, growth curves for C43 cyaA⁻ cells with expression of cfSthK or cfSthK-R377A. Points and error bars represent mean ± S.D., respectively, from three cultures.

Figure 7.

Characterization of cfSthK-3QV construct. A, cartoon representation of SthK (PDB code 6CJQ in the middle and left panels and PDB code 4D7T on the right) showing locations of three Gln residues and one Val residue in the 3QV construct. B, representative single-channel recording of cfSthK-3QV in 1 mm cAMP. An all-points histogram is shown on the right. The blue line represents fit to a sum of Gaussians with P_o = 0.92 for this patch. C, left, leak-subtracted currents in cAMP (red traces) and cGMP (green traces) for cfSthK-L422Q and cfSthK-3QV channels. Right, currents at −60 mV in cGMP relative to cAMP for cfSthK-L422Q, cfSthK-3Q, and cfSthK-3QV. Bars and error bars represent mean ± S.E., respectively, for three patches. D, dose-response curves for cfSthK-3QV in cAMP (red circles) and cGMP (green squares). The solid black line represents Hill fit with K_1/2 = 32 nm and h = 2.2 and with K_1/2 = 5.5 μm and h = 1.9 for cAMP and cGMP, respectively. The dashed black line represents cAMP dose response for wtSthK. Points and error bars represent mean ± S.E., respectively, for three patches. E, growth curves of C43 and C43 cyaA⁻ cells with cfSthK and cfSthK-3QV. Expression of the indicated SthK construct was induced between an OD₆₀₀ of 0.4 and 0.6. Points and error bars represent mean ± S.D., respectively, for three cultures.