Independent movement of the voltage sensors in KV2.1/KV6.4 heterotetramers

Abstract

Heterotetramer voltage-gated K⁺ (K_V) channels K_V2.1/K_V6.4 display a gating charge-voltage (Q_V) distribution composed by two separate components. We use state dependent chemical accessibility to cysteines substituted in either K_V2.1 or K_V6.4 to assess the voltage sensor movements of each subunit. By comparing the voltage dependences of chemical modification and gating charge displacement, here we show that each gating charge component corresponds to a specific subunit forming the heterotetramer. The voltage sensors from K_V6.4 subunits move at more negative potentials than the voltage sensors belonging to K_V2.1 subunits. These results indicate that the voltage sensors from the tetrameric channels move independently. In addition, our data shows that 75% of the total charge is attributed to K_V2.1, while 25% to K_V6.4. Thus, the most parsimonious model for K_V2.1/K_V6.4 channels' stoichiometry is 3:1.

Figure 1. Sequence alignment of the Shaker, K_V2.1 and K_V6.4 S4 region.

The underlined arginine residues in Shaker represent those that contribute to the gating charge. The bold valine residues were substituted for cysteines in K_V2.1 (K_V2.1(V296C)) and K_V6.4 (K_V6.4(V335C)). In red are shown residues that are conserved among the three channel sequences, while blue residues represent those conserved in only two sequences. The remaining ones are shown in black.

Figure 2. MTSET modification of K_V2.1 subunits.

(a) Representative current recordings to determine whether K_V2.1(V296C) homotetramers and K_V2.1(V296C)/K_V6.4 heterotetramers are modified by MTSET in the closed state (left) or open state (right). The applied pulse protocols and modification are given on top. (b) Time course of modification. Symbols represent normalized current reductions by MTSET modifications of K_V2.1(V296C) (diamond) and K_V2.1(V296C)/K_V6.4 (triangle down) channels at +60 mV (open symbols) and −120 mV (closed symbols). Black symbols denote normalized values before modification (c) Voltage dependence of the modification rate of K_V2.1(V296C) (square) and K_V2.1(V296C)/K_V6.4 (circle). The solid lines represent sigmoidal fits. The best-fit parameter values for V_1/2 for the K_V2.1(V296C) and K_V2.1(V296C)/K_V6.4 data were −5.4 mV and −23.9 mV, respectively. Numbers above symbols represent the number of cells analyzed at each voltage. Data are represented as the mean ± SEM (shown when it is larger than the size of the symbol).

Figure 3. Gating charge displacement in K_V2.1(V296C) homotetramers and K_V2.1(V296C)/K_V6.4 heterotetramers.

(a) Representative gating current recordings of K_V2.1(V296C) homotetramers (left) and K_V2.1(V296C)/K_V6.4 heterotetramers (right) at −120 mV, −100 mV, −80 mV, −60 mV, −40 mV, −20 mV and 0 mV. The applied pulse protocol is given on top. (b) Q_V distribution of K_V2.1(V296C) (square) and K_V2.1(V296C)/K_V6.4 (circle) channels. The Q_V distributions were obtained by plotting the area under the recorded ON-gating currents as a function of voltage. For comparison, the normalized MTSET modification curves of K_V2.1(V296C) and K_V2.1(V296C)/K_V6.4 (dashed and solid green line, respectively) are shown. Data are represented as the mean ± SEM (shown when it is larger than the size of the symbol).

Figure 4. MTSET modification of K_V6.4 subunits.

(a) Representative current recordings to determine whether K_V2.1/K_V6.4(V335C) channels are modified by MTSET in the closed state (left) or open state (right). The applied pulse and modification protocols are given on top. (b) Time course of modification. Symbols represent current normalized to the value at time 0. Open symbols depict MTSET modifications at + 60 mV while closed symbols represent modification at −120 mV. Black symbols symbolize normalized values before modification. (c) Voltage dependence of the modification rate of K_V2.1/K_V6.4(V335C). Solid line represents a sigmoidal fit. The best-fit parameter values for V_1/2 and k were −71.3 mV and 16.5, respectively. Numbers above symbols represent the number of cells analyzed at each voltage. Data are represented as the mean ± SEM (shown when it is larger than the size of the symbol).

Figure 5. Gating charge displacement in K_V2.1/K_V6.4(V335C) channels.

(a) Representative gating current recordings of K_V2.1/K_V6.4(V335C) channels at −120 mV, −100 mV, −80 mV, −60 mV, −40 mV, −20 mV and 0 mV. The applied pulse protocol is given on top. (b) Q_V distribution of K_V2.1/K_V6.4(V335C) channels. The Q_V distribution was determined as described in Fig. 3b. For comparison, the normalized MTSET modification curve (dashed green line) is shown. Data are represented as the mean ± SEM (shown when it is larger than the size of the symbol).