Fenestrations control resting-state block of a voltage- gated sodium channel Tamer M. Gamal El-Dina,1, Michael J. Lenaeusa,b,1, Ning Zhenga,c,2,3, and William A. Catteralla,2,3 aDepartment of Pharmacology, University of Washington, Seattle, WA 98195; bDivision of General Internal Medicine, Department of Medicine, University of Washington, Seattle, WA 98195; and cHoward Hughes Medical Institute, University of Washington, Seattle, WA 98195 Contributed by William A. Catterall, October 31, 2018 (sent for review September 4, 2018; reviewed by Ryan Hibbs, Michael C. Sanguinetti, and Joerg Striessnig) Potency of drug action is usually determined by binding to a specific Receptor Hypothesis posits that resting-state block is mediated receptor site on target proteins. In contrast to this conventional by drug entry from the lipid phase of the membrane into the drug paradigm, we show here that potency of local anesthetics (LAs) and receptor site in the pore, and rapid open-state block occurs as the antiarrhythmic drugs (AADs) that block sodium channels is con- drug enters the open pore from the cytoplasm (15). Both of these trolled by fenestrations that allow drug access to the receptor site forms of block are enhanced when the channel enters the inac- directly from the membrane phase. Voltage-gated sodium channels tivated state, which has high affinity for bound drug (15). It is initiate action potentials in nerve and cardiac muscle, where their well-established that LAs and AADs can reach their binding site hyperactivity causes pain and cardiac arrhythmia, respectively. LAs from the intracellular side if the activation gate is open, which and AADs selectively block sodium channels in rapidly firing nerve gives open-state block, and that inactivation increases the affinity and muscle cells to relieve these conditions. The structure of the for drug binding (15, 16). On the other hand, the hypothesis that ancestral bacterial sodium channel NaVAb, which is also blocked by direct access from the membrane phase causes resting-state block LAs and AADs, revealed fenestrations connecting the lipid phase of has lacked experimental support. the membrane to the central cavity of the pore. We cocrystallized Ancestral bacterial sodium channels are blocked by LAs and lidocaine and flecainide with NavAb, which revealed strong drug- AADs (17–20), and the structure of the bacterial sodium channel dependent electron density in the central cavity of the pore. Muta- NaVAb revealed fenestrations connecting the lipid phase of the tion of the contact residue T206 greatly reduced drug potency, confirm- membrane to the central cavity of the pore (21, 22), which are PHARMACOLOGY ing this site as the receptor for LAs and AADs. Strikingly, mutations conserved in eukaryotic NaV channels (23, 24) and are observed of the fenestration cap residue F203 changed fenestration size and in potassium channels (25). The discovery of fenestrations in had graded effects on resting-state block by flecainide, lidocaine, and NaVAb led to the hypothesis that they provide an access pathway benzocaine, the potencies of which were altered from 51- to 2.6-fold for resting-state block (21, 23, 24). We have tested this hypoth- in order of their molecular size. These results show that conserved esis by defining the receptor site for LAs and AADs through X- fenestrations in the pores of sodium channels are crucial pharma- ray crystallography, resetting the size of the fenestrations in NaVAb cologically and determine the level of resting-state block by widely BIOPHYSICS AND used drugs. Fine-tuning drug access through fenestrations provides Significance an unexpected avenue for structure-based design of ion-channel– COMPUTATIONAL BIOLOGY blocking drugs. Voltage-gated sodium channels initiate electrical signals in nerve and cardiac muscle, where their hyperactivity causes pain and voltage-gating | sodium channel | fenestration | antiarrhythmic drugs | local anesthetics cardiac arrhythmia. Local anesthetics and antiarrhythmic drugs selectively block sodium channels in rapidly firing nerve and muscle cells to relieve these conditions. We studied an ancestral bacterial oltage-gated sodium (NaV) channels are responsible for sodium channel to elucidate the structure of the drug-binding site Vgeneration of action potentials in electrically excitable cells (1). and the pathway for drug entry to the receptor site. We found that They encode and transmit information in the form of frequency- the drug-binding site is located in the center of the transmembrane modulated electrical signals. These channels are composed of four pore, through which sodium ions move and fenestrations form – identical subunits or four homologous domains (DI DIV), each an access pathway for drug entry directly from the cell membrane. containing six transmembrane segments (S1–S6) (2, 3). The first These results show how these widely used drugs block the sodium four segments (S1–S4) constitute the voltage-sensing module, which channel and have important implications for structure-based detects changes in membrane potential and transduces them to the design of next-generation drugs. pore (2, 3). The S5 and S6 segments and the P-loop between them form the pore, which is opened by an iris-like motion of the acti- Author contributions: T.M.G.E.-D., M.J.L., N.Z., and W.A.C. designed research; T.M.G.E.-D. vation gate at the intracellular ends of the S6 segments (2, 3). and M.J.L. performed research; T.M.G.E.-D., M.J.L., and N.Z. analyzed data; and T.M.G.E.-D., M.J.L., and W.A.C. wrote the paper. Inherited or acquired dysfunction of NaV channels causes chronic pain, cardiac arrhythmia, periodic paralysis, and epilepsy (4–7). Reviewers: R.H., University of Texas Southwestern Medical Center; M.C.S., University of Utah; and J.S., University of Innsbruck. Local anesthetics (LAs) and antiarrhythmic drugs (AADs) are The authors declare no conflict of interest. used to prevent excessive electrical signaling by blocking NaV channels during therapy of pain and cardiac arrhythmia (8, 9). Published under the PNAS license. Both types of drugs are thought to bind to a receptor site in the Data deposition: The following data have been deposited in the Protein Data Bank (PDB), – www.wwpdb.org:NavAb/I217C, F203A (PDB ID code 6MVV); NavAb/I217C, F203W (PDB ID central cavity in the pore (1, 10 14). The complex therapeutic code 6MVW); NavAb/1217C, complexed with flecainide (PDB ID code 6MVX); and NavAb/ actions of these drugs derive from three interacting processes: I217C, 1-226, crystallized in the presence of lidocaine (6MVY). slow resting-state block, rapid open-state block, and high-affinity 1T.M.G.E.-D. and M.J.L. contributed equally to this work. inactivated-state block (15, 16). This state-dependent mode of 2N.Z. and W.A.C. contributed equally to this work. block is crucial for clinical use of these drugs, as it allows them to 3To whom correspondence may be addressed. Email: [email protected] or [email protected]. selectively prevent generation of high-frequency action potentials This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. characteristic of intense pain and cardiac arrhythmia, while having 1073/pnas.1814928115/-/DCSupplemental. less effect on normal electrical signaling (8, 16). The Modulated www.pnas.org/cgi/doi/10.1073/pnas.1814928115 PNAS Latest Articles | 1of6 Downloaded by guest on September 28, 2021 the term “potency” to describe drug block quantitatively, rather A than “affinity,” because our results show that both drug access and drug-binding affinity contribute substantially to the concentration- dependent drug block that we measure. Results Benzocaine Lidocaine Flecainide The Receptor Site for Lidocaine and Flecainide in NaVAb. We solved crystal structures of NavAb in complex with the LA/AAD lidocaine B (Fig. 1 A–C and SI Appendix,TableS1) and the AAD flecainide VSD SF VSDV (Fig. 2 A and B and SI Appendix,TableS1), two of the most widely prescribed NaV channel-blocking drugs. Complexes were prepared 90 by mixing the drugs with purified NaVAb protein before incorpo- CC CC FEN ration into bicelles, and cocrystals were formed in hanging drops as previously described for NaVAb alone (21). Structures determined AG A C - Lidocaine + Lidocaine SF 90 T206 T206 T206 T206 SF CC V213 V213 B D 100 MLidocaine 90 WT T175 IC = 135 M 50 M174 T206A T175 IC50 = 2.4 mM T206 T206 1 mM Lidocaine C 10 M Flecainide WT IC50 = 7 M T206A IC50 = 133 M Fig. 1. Binding site for lidocaine on NaVAb. (A) LA/AADs used in this study: 100 M Flecainide benzocaine, lidocaine, and flecainide. (B) The structure of NaVAb/I217C/Δ40 is shown in cartoon format. Residues homologous to those shown to be involved in LA/AAD binding are highlighted in magenta, and channel regions are labeled as VSD (voltage sensor), SF (selectivity filter), CC (central cavity), AG (activation gate), and FEN (fenestration). (C, Left) The structure of NaVAb/I217C/Δ40 when solved without any drugs in solution. The protein model is shown in cartoon format with the LA/AAD-binding site highlighted in magenta and sidechains of key residues (T206, V213) shown in stick format. Fo-Fc electron density contoured at Fig. 2. Binding site for flecainide on NaVAb. (A) Orthogonal views of fle- 3σ is shown as green mesh, as calculated from data deposited as Protein Data cainide in complex with NavAb/I217C. In each case the protein is displayed as cartoon helices, with the AAD binding site highlighted in magenta ribbon Bank ID code 5vb8. (C, Right) The structure of NaVAb/I217C/Δ40 when solved with lidocaine in solution. The protein and electron density are displayed as on and sidechain sticks. Functional areas of the channel labeled as in Fig. 1. = = = the Left. Lidocaine was manually placedintothismodel.(D) Effect of mutation of Flecainide is displayed as sticks, with yellow carbon, red oxygen, blue = T206 on resting-state block of Na Ab by lidocaine.