A homology model of the pore domain of a voltage-gated calcium channel is consistent with available SCAM data

Abstract

In the absence of x-ray structures of calcium channels, their homology models are used to rationalize experimental data and design new experiments. The modeling relies on sequence alignments between calcium and potassium channels. Zhen et al. (2005. J. Gen. Physiol. doi:10.1085/jgp.200509292) used the substituted cysteine accessibility method (SCAM) to identify pore-lining residues in the Ca(v)2.1 channel and concluded that their data are inconsistent with the symmetric architecture of the pore domain and published sequence alignments between calcium and potassium channels. Here, we have built K(v)1.2-based models of the Ca(v)2.1 channel with 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET)-modified engineered cysteines and used Monte Carlo energy minimizations to predict their energetically optimal orientations. We found that depending on the position of an engineered cysteine in S6 and S5 helices, the ammonium group in the long flexible MTSET-modified side chain can orient into the inner pore, an interface between domains (repeats), or an interface between S5 and S6 helices. Different local environments of equivalent positions in the four repeats can lead to different SCAM results. The reported current inhibition by MTSET generally decreases with the predicted distances between the ammonium nitrogen and the pore axis. A possible explanation for outliers of this correlation is suggested. Our calculations rationalize the SCAM data, validate one of several published sequence alignments between calcium and potassium channels, and suggest similar spatial dispositions of S5 and S6 helices in voltage-gated potassium and calcium channels.

Figure 1.

The extracellular (A) and cytoplasmic (B) views of the K_v1.2 x-ray structure, with the C^α-C^β bonds of positions i15–i29 shown as sticks. The S5 and S6 helices are shown as strands and ribbons, respectively. The P loops are not shown for clarity.

Figure 2.

The extracellular and side views of ^mC¹ⁱ¹⁵ (A and B) and ^mC¹ⁱ¹⁹ (C and D) in the open Ca_v2.1 channel. The side chains of the ^mC residues in different conformations within 4 kcal/mol from the apparent global minima are superimposed and shown as gray sticks with blue nitrogen and yellow sulfur atoms. Native residues are shown in the lowest energy conformation as pale orange sticks with red oxygens, blue nitrogens, and yellow sulfur atoms. The P loops and S6s in repeats I, II, III, and IV are cyan, orange, green, and violet, respectively. For clarity, P loops in A and C, IIS6 in B, and S5s in A–D are not shown. The ammonium group of ^mC¹ⁱ¹⁵ is inside the pore (A) between levels i15 and i18 (B). The ammonium group of ^mC¹ⁱ¹⁹ is close to the pore axis (C) approaching either the focus of P helices or level i22 (D). The red cross at A and C indicates the pore axis.

Figure 3.

The extracellular views of various orientations of ^mCⁱ¹⁸ residues in Ca_v2.1. For clarity, the P loops are not shown. The red cross indicates the pore axis. (A) Cation–π interactions with Y²ⁱ¹⁶ stabilize the repeat interface orientation ^mC¹ⁱ¹⁸; the pore orientations have higher energy. (B) In the most preferable conformations, ^mC²ⁱ¹⁸ is oriented in the pore. (C) Cation–π interactions with F⁴ⁱ¹² and F³ⁱ²² stabilize orientation of ^mC³ⁱ¹⁸ in the repeat interface; the pore orientations are less preferable. (D) Both pore and repeat interface orientations of ^mC⁴ⁱ¹⁸ are energetically favorable.

Figure 4.

The extracellular views of ^mC¹ⁱ²² (A), ^mC²ⁱ²² (B), ^mC³ⁱ²² (C), and ^mC³ⁱ²² (D). The red cross indicates the pore axis. P loops are not shown for clarity. Orientations of the ^mCⁱ²² side chains in the pore are energetically more preferable than repeat interface orientations.

Figure 5.

The cytoplasmic (A and C) and side (B and D) views of the environment for C¹ⁱ²² in the Shaker (A and B) and Ca_v2.1 (C and D) channels. Side chains in positions i19, i22, i23, and i26 are space-filled with gray carbon and black sulfur atoms.

Figure 6.

(A) The extracellular view of ^mC¹ⁱ¹⁶ that orients either along IVS6 or toward IS5 and IVS5. (B) The cytoplasmic view of three possible orientations of ^mC²ⁱ²⁰. (C) The cytoplasmic view of ^mC²ⁱ²⁹ interacting with N^3o6, N^3o9, and N³ⁱ²⁰. The red cross indicates the pore axis.

Figure 7.

The extracellular view of possible orientations of ^mC residues in S5s. ^mC^2o10 (A) and ^mC^4o10 (C) can extend their ammonium groups toward the pore. (B) ^mC^3o10 is stabilized inside the repeat interface by cation–π interaction with F³ⁱ¹⁸ and Y^3o14. The red cross indicates the pore axis.

Figure 8.

The residual current upon MTSET application correlates with the distance of the MTS atom N⁺_^mC (A and B), but not atom C^β_^mC (C and D) from the pore axis. (A and C) The experimental values of the current inhibition with standard deviations (Zhen et al., 2005) are plotted against the predicted distances of atoms N⁺_^mC (A) or C^β (C) from the pore axis. Data are shown for channels with engineered cysteines in positions i15–i21, i23–i25, and o10. Black dots represent the apparent global minima of channels in which all minimum energy conformations of ^mC side chains are unambiguously oriented in respect to the pore (e.g., inside the pore for channels ^mCⁱ¹⁵ or outside the pore for channels ^mCⁱ¹⁷). Blue dots represent the apparent global minima of the channels in which the ^mC side chain adopts low energy conformations with distinct orientation in respect to the pore (e.g., channels ^mCⁱ¹⁸). A green dot represents a local minimum (within 2 kcal/mol from the apparent global minimum) of a channel in which the ^mC side chain adopts conformations with distinct orientation in respect to the pore (e.g., channels ^mCⁱ¹⁶). Horizontal lines show the N⁺_^mC atom mobility in conformations within 2 kcal/mol from the apparent global minimum (Table S1). Note a smooth decrease of the current inhibition with increase of the distance between the MTS nitrogen and the pore axis. The current inhibition of ∼20% at distances >16 Å corresponds to MTSET block of the “control channel,” in which eight native cysteines in the α₁ subunit have been replaced with alanines and no engineered cysteines have been introduced (Zhen et al., 2005). (B and D) The extracellular view of Ca_v2.1, with atoms ^mC_N⁺ (B) and ^mC_C^β (D) shown as spheres. P loops are omitted for clarity. Yellow and blue spheres represent the respective atoms in the channels, which are inhibited by MTSET by >30 and ≤30%, respectively. (B) In most of the channels, which are strongly inhibited by MTSET, the yellow-colored ammonium nitrogen (^mC_N⁺) is located either close to the pore axis or at the inner surface of the pore, whereas in the channels, which are weakly inhibited by MTSET, the blue-colored ammonium nitrogen is not inside the pore. (D) Location of β carbons does not correlate with the level of current inhibition by MTSET. Both yellow and blue spheres are randomly distributed at different sides of the inner helices.