Stoichiometry and Arrangement of Subunits in Rod Cyclic Nucleotide–Gated Channels  Iman M Shammat, Sharona E Gordon  Neuron  Volume 23, Issue 4, Pages 809-819 (August 1999) DOI: 10.1016/S0896-6273(01)80038-6

Figure 1 Coexpression of the α and β Subunits Increases the Efficacy of cAMP (A) Currents activated by voltage jumps from a holding potential of 0 mV to −100 mV through +100 mV, in steps of 20 mV. The current families on the left are from the same α-only patch and the current traces on the right are from the same α/β patch, in which an RNA injection ratio of 1:4 α:β was used. Current families on the top are in response to 2 mM cGMP. Current families on the bottom are in response to 16 mM cAMP. (B) Box plot of the current activated by 16 mM cAMP divided by the current activated by 2 mM cGMP, all at +100 mV. The horizontal line represents the median of the data, the box represents the middle half of the data, between the 25th and 75th percentiles, and the bars show the range of the data. Each box represents data from at least three patches. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 4 Ni2+ Potentiation of α-Only and α/β Channels The cartoons at the top represent the type of channel examined in the panels below. The gray circles represent the α subunit, the white circles represent the β subunit, and the black circles represent subunits that could be either α or β. The letter within each circle denotes the identity of the amino acid at the 420/546 position. The small black dot represents a Ni2+ ion. (A and B) Dose–response curves for activation by cGMP. The solid symbols were recorded in the absence of Ni2+, and the open symbols were recorded in the presence of 10 μM Ni2+. Currents were normalized to their value at 2 mM cGMP, and concentrations were normalized to the value of K1/2 in the absence of Ni2+. Solid curves are fits to the Hill equation using the following parameters (K1/2 values are before normalization): (A) K1/2 = 66 μM and n = 1.8 in the absence of Ni2+, and K1/2 = 5.1 μM and n = 1.8 in the presence of 10 μM Ni2+; (B) K1/2 = 69 μM and n = 1.8 in the absence of Ni2+, and K1/2 = 9.9 μM and n = 1.8 in the presence of 10 μM Ni2+. The dashed curves in (B) are reproduced from the solid curves in (A). (C and D) Currents activated by either 2 mM cGMP or 16 mM cAMP, as labeled, in response to a voltage pulse from a holding potential of 0 mV to +100 mV. The thick traces were recorded in the absence of Ni2+, and the thin traces were recorded in the presence of 10 μM Ni2+. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 2 Coexpression of the α and β Subunits Increases the Voltage Dependence of Activation (A and B) Dose–response curves for activation by cGMP for α-only channels (A) and α/β channels (B). Open symbols were recorded at −100 mV and solid symbols were recorded at +100 mV. For each voltage, currents were normalized to the current response to 2 mM cGMP. Smooth curves are fits with the Hill equation: using the following parameters: (A) K1/2 = 70 μM and n = 1.9; (B) K1/2 = 68 μM and n = 2.3 at +100 mV, and K1/2 = 128 μM and n = 2.3 at −100 mV. (C) Box plot of the ratio of K1/2 at −100 mV to K1/2 at +100 mV for α-only and α/β channels. Each box represents data from eight patches. The difference between the two channel types is statistically significant (Student's t test, p < 0.02). Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 3 Alignment between One Region of Sequence of the α and β Subunits The horizontal bar indicates the S6 region, the black box indicates the Ni2+-binding site, and the gray boxes show amino acid identities. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 8 Energy of Potentiation of cAMP Activation for Six Different Channel Types Box plot of the difference between ΔG0 in the presence of Ni2+ and ΔG0 in the absence of Ni2+, calculated using Scheme 1. The number of patches used were as follows: α, 6; α/β, 4; H420Q-α/N546H-β, 3; H420Q-α, 10; H420Q-α/β, 5; and α/N546H-β, 4. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 9 Cartoons of Alternative Models of Ni2+ Coordination Each gray circle represents an α subunit, each white circle represents a β subunit, and each black dot represents a Ni2+ ion. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 5 A Histidine at the 420 Position Is Required for Ni2+ Potentiation in Both α-Only and α/β Channels Cartoons have the same meaning as in Figure 4. (A and B) Dose–response curves for activation by cGMP. The solid symbols were recorded in the absence of Ni2+ and the open symbols were recorded in the presence of 10 μM Ni2+. Currents and concentrations were normalized as in Figure 4. The dashed curves are reproduced from the solid curves in Figure 4A. Before normalization, the data were fit with the Hill equation using the following parameters: (A) K1/2 = 51 μM and n = 1.7; (B) K1/2 = 45 μM and n = 2. (C and D) Currents activated by either 2 mM cGMP or 16 mM cAMP, as labeled, in response to a voltage pulse from a holding potential of 0 mV to +100 mV. The thick traces were recorded in the absence of Ni2+, and the thin traces were recorded in the presence of 10 μM Ni2+. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 6 A Histidine in the β Subunit Rescues Ni2+ Potentiation Cartoons have the same meaning as in Figure 4. (A and B) Dose–response curves for activation by cGMP. The solid symbols were recorded in the absence of Ni2+ and the open symbols were recorded in the presence of 10 μM Ni2+. Currents and concentrations were normalized as in Figure 4. The dashed curves are reproduced from the solid curves in Figure 4A. Before normalization, the data were fit with the Hill equation using the following parameters: (A) K1/2 = 53 μM and n = 2.1 in the absence of Ni2+, and K1/2 = 20 μM and n = 1.2 in the presence of 10 μM Ni2+; (B) K1/2 = 40 μM and n = 2.1 in the absence of Ni2+, and K1/2 = 5.0 μM and n = 1.4 in the presence of 10 μM Ni2+. (C and D) Currents activated by either 2 mM cGMP or 16 mM cAMP, as labeled, in response to a voltage pulse from a holding potential of 0 mV to +100 mV. The thick traces were recorded in the absence of Ni2+, and the thin traces were recorded in the presence of 10 μM Ni2+. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Figure 7 Effect of Subunit Composition on Channel Affinity for Ni2+ (A) Time course of onset of Ni2+ potentiation. Currents in response to 16 mM cGMP at +100 mV. At time t = 0, the solution was switched from one containing EDTA and no Ni2+ to one containing 10 μM Ni2+. Data from one patch each of α-only, α/β, α/N546H-β, and H420Q-α/N546H-β are shown. The currents were normalized to their value at steady state in the presence of Ni2+. The smooth curve is a fit to all the data with a single exponential, with a time constant of 15 s. (B) Time course of recovery from Ni2+ potentiation. At time t = 0, the solution was switched from one containing 10 μM Ni2+ to one containing EDTA and no Ni2+. Data from one patch each of α-only, α/β, α/N546H-β, and H420Q-α/N546H-β are shown. The currents were normalized to their value at steady state in the absence of Ni2+. The smooth curve is a fit to all the data with the sum of two exponentials, with time constants of 70 and 310 s. Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)

Neuron 1999 23, 809-819DOI: (10.1016/S0896-6273(01)80038-6)