Ion channel breakthrough (2019)

Voltage-dependent ion channels (Na, K, Ca) are nature's transistors for electrical and calcium signaling. These channels control our muscles, brain activity, heart rhythm, theta rhythm, memory and sleep. They are associated with many diseases. A great leap forward in understanding these channels is made by controling the gates with tunneling electrons. The gating mechanism and structure for controlling ion channel conduction and timing is encoded in the amino acid sequence. The first published amino acid sequence was for the sodium channel of an electric eel. It was determined by a group at Kyoto University led by S. Numa (Noda et al., 1984). An arginine or lysine amino acid was found at every third residue in four S4 segments. They proposed that positively charged arginine and lysine on the S4, moving across the membrane electric field, could be associated with gating. For more than 30 years all sodium and potassium ion channel gating models published in peer reviewed journals have been based on this idea and there is still no satisfactory gating mechanism. The positively charged arginine and lysine approach is unworkable.

Only the negatively charged electron can solve the ion channel gating problem.
The potassium channel needs four independent gates to give the Hodgkin and Huxley open channel probability n4 and the inactivation gates and timing need to be modeled. A breakthrough in decoding the amino acid sequence is shown in Fig. 1. The much-studied Shaker B potassium channel is ideal for decoding the sequence because each subunit has an activation gate, a fast N-type inactivation gate, and a C-type inactivation gate. That is a total of 12 gates and it only needs one tunneling electron in each subunit to control all the gates and give the published experimental time constants. Ion channels researchers have published many experimental observations on potassium channels and this data was used to configure the structure.


Fig. 1. Architecture required for 4 tunneling electrons to control activation and inactivation in Shaker K+ channels.
(A) Amino acid sequence shows α-helices S1 to S6 aligned for controlling activation and inactivation gates in one of four identical subunits. An electron tunnels across arginine (R) and lysine (K) NH3 sites on S4 increasing the energy barrier at adjacent gating cavity. The α-helix cross-section angles φ (in black) are for the residue backbone center with 180° furthest from the channel center. Arg/lys NH3 tunneling sites (q1, q2, etc.) have cross-section angles φ (in red) with 0 degrees nearest the channel center. Angles in italic are for α-helices end termini. Gating cavities U1, U2, U3 need additional residues to close the cavity and maximize the gating energy barrier. Residues T442 and V474 close one side of cavities U1 and U2. Open ends for U1 and U2 are closed by a cuff formed by residues 431-435 and 478-482.
(B) Outside view showing alignments for subunits KA to KD. The subunits are incremented by 1/4 α-helix pitch (1.35 Å) along channel z-axis to form four sequential cavities for U1, U2, U3. Only U1 cavities are shown. Efflux K+ Ions and water sequentially pass through activation-gate cavities (U1A to U1D) until an electron tunnels to a q1 control site blocking ion transit (Fig. 1.A). Negatively charged glutamate (E) side chains on S1, S2, S3 are adjacent the lipid membrane and likely provide an energy landscape to optimize tunneling. Intersubunit distances between cysteine termini (gray boxes) are within ±10% of reported Shaker intersubunit distances for eight sites. The architecture is based on many published experimental observations, which are referenced in the book: Electron-Gated Ion Channels: With Amplification by NH3 Inversion Resonance (2005).

      Fig. 2. Schematic for electron gating and timing of Shaker potassium channels.
      Summary of time constants for Shaker B and Shaker A channels

      TL        Activation lag time
      TN        N-type inactivation time constant
      TC        C-type inactivation time constant
      TD        Deactivation time constant
      TO        Open-gate time for Shaker B (all switches closed)
      TO+T7   Open-gate time for Shaker A (all switches closed)
      TOD      Open-gate time during deactivation (subunit only)
      T7        7-residue tunneling time constant for q7 to qF7
      T7D      7-residue tunneling time constant for qF7 to q7
      T3        3-residue tunneling time constant for qF7 to qF10
      T3D      3-residue tunneling time constant for qF10 to qF7

Fig. 2. A brief description of timing: Arginine and lysine NH3 groups function as electron tunneling sites on the S4 voltage sensor of the four potassium channel subunits (KA, KB, KC, KD). When a gating electron tunnels to a control site (q1, q7, qF7, or qF10) the electric field across the adjacent cavity (U1, U2 or U3) creates an energy barrier that traps an ion in the cavity. The electron-activated energy barrier, for each gating cavity, is represented by a switch. Subunit KA is used to illustrate the timing. When a gating electron tunnels to a control site, the associated switch opens, thus blocking current flow. At −80 mV resting potential, the gating electron is at q1, causing switch S1A to be open. When the membrane is depolarised to +30 mV at time t0 there is a brief lag time TL and then the electron tunnels across 6 Arg/Lys sites to the Shaker B control site q7. The electron's field causes switch S3A to open, resulting in N-type inactivation. The N-type inactivation time constant is reported to be about 2-4 ms and the gate open time is TO = TN − TL. For Shaker A, there is no N-type inactivation, thus q7 is not a control site and switch S3A remains closed. After an interval T7, the electron tunnels across the 7-residue distance to the inactivation control site qF7 and opens switch S2A. It then tunnels on to qF10, keeping S2A open. The inactivation time constant TC for C-type inactivation, reported to be 10-30 ms, is determined by the tunneling distance between q7 and qF7. When membrane voltage returns to −80 mV at time t1 the membrane field causes the gating electron to return to q7 after an interval T3D + T7D. Then the electron tunnels back to q1. During time interval TOD the gate is open (all switches closed) for the subunit, but since the tunneling time has a random component, an electron in one subunit can return to q1 while in another subunit it is still at inactivation site qF7 blocking current flow. The deactivation time constant TD can be much greater than inactivation time constant TC, because of a local field from positively charged arginine (R394, R309) and other charges near the far sites qF7 and qF10.

Table 1.  Alignments for functional gating in Shaker K+ channels
Experimentally determined amino acid sequences for gating        Requirements for optimized gating
1. Shaker family channels have 7 Arg/Lys on S4 with 3-residue spacing. This gives an 18-residue spacing between q1 and q7. This 18-residue spacing gives q1 = 0° and q7 = 0°, maximizing gating barriers for both activation and N-type inactivation.
2. There is an 18-residue spacing between Shaker selectivity filter center residue Y445 and key residue A463 for N-type inactivation. This aligns cavity U1 and U3 with tunneling sites q1 and q7 maximizing gating barrier for both activation and N-type inactivation (Fig. 1.A).
3. There is a 5-residue distance between Shaker L7 and charged E12, D13. This critical distance aligns L7 in crevice with U3 cavity. Charged residues E12 and D13 remain at cytoplasm/membrane interface.
4. Shaker N-terminus residue L7 is hydrophobic with moderately long side chain. This allows side chain penetration near U3 cavity, increasing gating energy barrier for N-type inactivation.
5. Shaker family channels have a key residue I470 spaced 25 residues from Y445. (Published experiments place I470 at the center of a cavity for inactivation). This spacing places I470 at the center of gating cavity U2 and in alignment with R387 and K390 for C-type inactivation.
6.Shaker R387 is 7 residues from K380. This spacing sets tunneling distance for a fast C-type inactivation time constant (10−30 ms).
7. In Kv1 channels, with Q387 instead of R387, Arg R390 is 10 residues from K380.(Kv1.1, Kv1.2, Kv1.4, sqKv1A) This spacing sets tunneling distance for a slow C-type inactivation time constant (0.2−5 s).

Fig. 3.  New BFP spectra confirms that Arg/Lys NH3 group is Inverting
Fig. 3.  Blue Fluorescent Protein microwave thermal fluorescent spectrum. The rotational J,K states show alignment with scaled frequencies fG1 in Table 2. More than 20 inversion lines are within 1% of the scaled down NH3 gas phase inversion lines.
This is a big improvement over the spectra published in my book, Electron-Gated Ion Channels. The NH3 inversion is required for amplified electron tunneling. It should be present in all life forms and I am developing an instrument to measure it in other biological specimens.