The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels

Figures

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  Figure 1. Sequence homology of Kv subfamily members with minimal gating diagram. (a) shows the sequence alignment of the P-loop (contributing to the SF) in KcsA, Shaker, hKv1.5, hKv2.1, and hKv3.1. The second threonine residue within this filter (indicated in bold) is replaced by alanine in this work. (b) depicts a minimal gating diagram with six states whereby the activated (A) and activated-inactivated (AI) states are intermediate states that the channel transitions to go from closed to open or inactivated and vice-versa. In the nonconductive closed state (C), the SF (S) has an open conformation, whereas the intracellular AG gate (G) is closed. Upon membrane depolarization, the AG converts into an activated and subsequently an open conformation, and the ion channel is conductive (O state). In channels displaying C-type inactivation, prolonged depolarization causes the channel to inactivate (I state) through SF rearrangements and ion conduction is inhibited. Finally, upon closing of the activation gate during repolarization, the channel occupies the closed-inactivated state (CI).

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  Figure 2. Activation kinetics of WT hKv2.1 and hKv3.1 and the alanine mutations hKv2.1-T377A and hKv3.1-T400A. Representative whole-cell current recordings of Ltk cells expressing WT channels or their corresponding T-to-A mutations, hKv2.1 (a) and hKv3.1 (b), are shown. Currents were elicited with the pulse protocols shown on top, and the horizontal bar at the start of the recordings indicates the zero-current level. After applying activating depolarizing pulses, the membrane potential was stepped back to a fixed value to elicit deactivating tail currents. Shown in (c) and (d) are the voltage dependence of activation for the hKv2.1 and hKv3.1 channels (open triangles) and their corresponding T-to-A mutations (solid circles). The conductance (G) versus voltage (V) relationship or GV curves were obtained by plotting the normalized G (obtained by normalizing the tail current amplitudes from pulse protocols shown in a and b) as a function of the voltage changes. Values are the means ± SEM with solid and dotted lines representing the average fit with a Boltzmann function (V1/2 and slope factor values are reported in Table 1). (e) and (f) display the activation and deactivation time constants…

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  Figure 3. hKv2.1-T377A and hKv3.1-T400A produced K+ selective channels. (a) and (b) show representative current recordings for hKv2.1 and its T377A mutation (a) and for hKv3.1 and its T400A mutation (b), respectively. Currents were elicited with the pulse protocol shown at the top, and the horizontal bar at the start of the recordings indicates the zero-current level. (c) and (d) show current (I) versus voltage curves (IV curves) that were obtained by plotting the normalized amplitude of the deactivating currents from recordings shown in (a) and (b) (normalized to the current amplitude at +60 mV) as a function of repolarizing potential. The IV curves of both hKv2.1-T377A and hKv3.1-T400A (solid circles) were comparable to those of the WT channels (open circles). WT and mutant channels displayed a similar reversal potential, i.e., the potential where the current is 0.

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  Figure 4. Noise analysis of hKv2.1-T377A and hKv3.1-T400A to determine channel conductance. (a) shows the representative current recordings of hKv2.1-T377A (left) and hKv3.1-T400A (right) elicited by repetitively applying a +40-mV pulse. The gray trace is the calculated average. (b) and (c) plot the calculated variance, obtained from analyzing the upstroke of the activating current recordings shown in (a), as a function of current amplitude. Fitting these relationships with a parabolic function (gray tracing) yielded a single-channel current i of 0.48 pA for hKv2.1-T377A (b) and 0.65 pA for hKv3.1-T400A (c), respectively. These single-channel currents correspond to a single-channel conductance γ, represented in parentheses.

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  Figure 5. Effect of prolonged depolarization on the current amplitude. Panels show representative current recordings for WT hKv2.1 (a), WT hKv3.1 (b), and their corresponding hKv2.1-T377A and hKv3.1-T400A mutants. Current recordings were elicited with the pulse protocols shown on top. (a) A prolonged depolarization induces U-type inactivation in hKv2.1, which manifests as a time-dependent reduction in current amplitude (black trace). This decrease in current amplitude can be quantified by the current elicited during a subsequent test pulse at +60 mV, after a conditioning test pulse to +40 mV (black trace) or to −80 mV (gray trace). Inset on the right shows an enlarged view of the currents elicited during the test pulse to +60 mV. The magnitude of the current reduction during the test pulse at +60 mV is proportional to the size of the black arrow. For the mutant hKv2.1-T377A, the current amplitude slowly increased during membrane depolarization at +40 mV (red trace), noticeable by the increase in current amplitude (red arrow) during the +60 mV test pulse between a conditioning prepulse to −80 (blue) or +40 mV (red). (b) The current of WT hKv3.1, alike to hKv2.1, decreased in a…

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  Figure 6. For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.bpj.2020.03.032. Inactivation kinetics of hKv2.1, hKv3.1 and their T-to-A mutants. (a) and (b) show representative current recordings for hKv2.1 (a) and hKv3.1 (b) channels with their T-to-A mutations represented on the right, respectively. Currents were elicited with the pulse protocols shown on top, and the zero-current level is indicated by the horizontal bar at the start of the recordings. Prolonged depolarizations induce inactivation in WT channels, resulting in a gradual decrease in current amplitude. The degree of channel inactivation was determined by applying an activating test pulse to +60 mV, which elicited a current amplitude that reports directly on the number of channels that did not inactivate. Note, the current amplitude of hKv2.1-T377A (a) gradually increased upon prolonged depolarization or remained constant for hKv3.1-T400A (b). (c) and (d) display the voltage dependence of channel inactivation for the WT hKv2.1 and hKv3.1 channels (open circles). Curves were obtained by plotting the normalized current amplitude at the +60 mV test pulse at (I/Imax) as a function of the 5-s…

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  Figure 7. Increased external K+ results in larger macroscopic currents in hKv2.1-T377A and hKv3.1-T400A. (a) A representative hKv2.1-T377A recording showing the effect of increasing the external K+ concentration on current amplitude using a pressurized fast perfusion system. During a prolonged depolarization to +40 mV, starting from −80-mV holding potential, the external K+ concentration was changed gradually from 4 to 148 mM, as indicated on top. Elevating the external K+ concentration increased T377A current amplitude; despite that, the outward directed electrochemical driving force for K+ is reduced. Switching the extracellular K+ concentration back to 4 mM resulted in a transient increase in amplitude, which is in agreement with a stronger outward electrochemical driving force, followed by a current decrease. The gray line represents the current recorded in standard 4 mM extracellular solution. Horizontal bar at the start of the recording indicates the zero-current level. (b) A representative current recording for hKv3.1-T400A upon increasing gradually the extracellular K+ concentration is shown.

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  Figure 8. Elevated external K+ increases current amplitude in hKv2.1-T377A and hKv3.1-T400A channels. (a) shows example recordings from WT Kv2.1 and Kv2.1-T377A, using the pulse protocol on top and in three different external [K+]. (b) and (d) show the current density at a +60-mV test pulse as a function of the conditioning 5-s prepulse potential for the WT hKv2.1 (n = 3) and hKv3.1 (n = 3) channels, (c) and (e) for hKv2.1-T377A (n = 3), and hKv3.1-T400A (n = 3), respectively. Represented values are the means ± SEM. Although both WT channels clearly demonstrate inactivation, U-type inactivation is only observed for hKv2.1. Stronger depolarizations are necessary to reveal U-type inactivation in hKv3.1. Increasing the extracellular [K+] reduces the current density at +60 mV. The hKv2.1-T377A mutant lacks inactivation and even displays an initial increase in current density at 4 mM (white circles) and 18 mM (gray inverted triangles) external [K+]. Moreover, the current amplitude of hKv2.1-T377A increased with elevated external [K+] and was largest with 145 mM external [K+] (black triangles). Similar to hKv2.1-T377A, there is no decrease in current amplitude for hKv3.1-T400A with elevated…