Conversion of a Delayed Rectifier K Channel to a Voltage-Gated Inward Rectifier K Channel by Three Amino Acid Substitutions

Neuron, Vol. 16, 853 858, April, 1996, Copyright 1996 by Cell Press Conversion of a Delayed Rectifier K Channel to a Voltage-Gated Inward Rectifier K Channel by Three Amino Acid Substitutions A. G. Miller and R. W. Aldrich human hippocampus, both contain six putative transmembrane segments, including an S4 segment, and a Department of Molecular and Cell Physiology and Howard Hughes Medical Institute P region and are thus more closely related to the Shaker Stanford University family than to the IRK family. These S4-containing in- Stanford, California 94305 ward rectifiers raise an interesting question about the mechanism of voltage-dependent gating. How do channels with the same basic sequence topology and putative S4 voltage sensor (Liman et al., 1991; Papazian et Summary al., 1991, 1995; Yang and Horn, 1995) open in response to changes in voltage of opposite polarity? Single, double, and triple mutations progressively shift We have addressed this question by converting an Shaker activation to more hyperpolarized potentials, outwardly rectifying Shaker channel into a family of voltresulting in an increase in the fraction of inactivated age-gated inward rectifiers by combinations of three channels at negative resting voltages. The most nega- amino acid substitutions that progressively shift Shaker tively shifted mutation, the triple mutant, behaves like activation gating to more hyperpolarized voltages. In an inward rectifier. What is usually considered activa- these inward rectifier channels, what would normally be tion for an inward rectifier is, for the triple mutant, described as activation gating upon negative voltage recovery from inactivation, and what is usually consid- steps comes about from recovery of the channels from ered deactivation is inactivation. This conversion from fast N-type inactivation through the open state (see outward rectifier to inward rectifier does not rely on Demo and Yellen, 1991; Gomez-Lagunas and Arma difference in sign or direction of charge movement strong, 1994; Ruppersberg et al., 1991). of the voltage sensor, since activation of the Shaker The essential features of gating of the fast inactivating outward rectifier is due to a different gate than activa- Shaker channel can most simply be described by a kition of the triple mutant inward rectifier. Other voltage- netic scheme with three states (Hoshi et al., 1990): dependent inward rectifiers in the Shaker family may work by a similar mechanism. Introduction K channels have been classified as either outward rectifiers or inward rectifiers based on their opening in The closed to open transition (C O) corresponds to response to either depolarization (outward rectifier) or the Shaker activation gate, and the open to inactivated hyperpolarization (inward rectifier) (Hille, 1992). Identifi- transition (O I) corresponds to the Shaker inactivation cation of the genes encoding channels in both classes gate. Detailed steady-state and kinetic data have shown has shown that a distinction also holds, in part, at the that there are a number of transitions among closed molecular level. Voltage-dependent outwardly rectifying states before opening (Bezanilla et al., 1994; Koren et K channels, such as Shaker (Chandy and Gutman, al., 1990; Schoppa et al., 1992; Zagotta and Aldrich, 1995), have six putative transmembrane segments per 1990; Zagotta et al., 1994), which are represented in subunit, including a highly charged S4 segment, and an this scheme as a single C O transition. At the resting ion-conducting P region (Hartmann et al., 1991; MacKin- potential, and at more negative voltages, Shaker channon and Yellen, 1990; Yellen et al., 1991; Yool and nels will populate states to the left. After a step to more Schwarz, 1991). They exhibit voltage-dependent activa- positive voltages, the channels will move from the tion gating in response to positive voltage steps from closed state through the open state to the inactivated resting potentials. One class of inward rectifier channels, state. the IRK-related family, is composed of channels with This scheme could also accommodate inward rectifier smaller subunits containing only two putative mem- behavior if the channels were to occupy the inactivated brane-spanning regions in addition to the P region (Ho (I) state at rest. If the voltage range for activation were et al., 1993; Kubo et al., 1993). These channels activate much more negative, the equilibrium distribution among in response to negative voltage steps from resting po- states at the resting potential would be shifted toward tential due to voltage-dependent relief of block by mag- the inactivated state. Hyperpolarizing voltage steps nesium and polyamines (Ficker et al., 1994; Lopatin et would then cause the channels to move from the inactial., 1994; Nichols et al., 1994). Other inward rectifiers, vated state through the open state, resulting in a relahowever, have been found that possess an intrinsic volt- tively large current, and then to the closed state. Conage-dependent activation that is not due to relief of versely, a positive voltage step would result in further an extrinsic blocking particle (Hille, 1992; Hoshi, 1995; inactivation and little or no current, since the channels Silver and DeCoursey, 1990). The KAT1 family of plant would already reside in the right-most inactivated state. inward rectifiers (Muller-Rober et al., 1995; Schachtman Such a channel will thus show inward rectification. The et al., 1992) and HERG (Sanguinetti et al., 1995; Trudeau above considerations suggest that an outward rectifier et al., 1995; Warmke and Ganetzky, 1994), cloned from could be converted into an inward rectifier by shifting

Neuron 854 Figure 1. Progressive Shift in the Midpoint of Activation (A) Shaker S4 sequence showing the position of the three amino acid replacements. (B) Representative normalized conductance voltage plots for ShB 4-46 (asterisks), R365N (open squares), R371I (closed circles), R365N:R371I (open circles), L366A:R371I (closed triangles), and R365N:L366A:R371I (closed double triangles) in the Shaker background. The constructs lack the N-terminal inactivation domain, a 40 amino acid segment from position 4 to 46 (Hoshi et al., 1990), so that the steady-state conductance voltage relationship can be measured without contamination by inactivation. Thus, ShB 4-46 denotes the wild-type ShB channel without the N-terminal inactivation domain. Similar shifts are seen in peak conductance voltage plots with the intact N-terminal domain (data not shown). Isochronal measurements were made approximately 0.5 1 ms after the step to the tail voltage. The tail current voltage was as follows: ShB 4-46 ( 80 mv), R365N ( 80 mv), R371I ( 120 mv), R365N:R371I ( 140 mv), L366A:R371I ( 140 mv), R365N:L366A:R371I ( 100 mv). Data were fit with a Boltzmann function raised to the fourth power. (C) Plot of the mean of the midpoint of activation for each of the six constructs. Error bars are the standard deviation. The midpoints (with n values) are as follows: ShB 4-46, 42.5 10.7 mv (8); R365N, 69.3 6.3 mv (5); R371I, 80.4 5.8 mv (4); R365N:R371I, 182.3 8.9 mv (3); L366A:R371I, 186.3 30.0 mv (3); R365N:L366A:R371I, 221.9 10.7 mv (7). the channels open and then inactivate, resulting in outward rectifying behavior. On the other hand, the triple mutant channels significantly populate the inactivated state at 80 mv, and thus a hyperpolarizing voltage step from 80 mv results in recovery from inactivation through the open state (Demo and Yellen, 1991; Gomez- Lagunas and Armstrong, 1994; Ruppersberg et al., 1991) and currents that resemble inward rectifiers (Muller- Rober et al., 1995; Schachtman et al., 1992). More positive voltage steps further populate the inactivated state, resulting in a smaller outward current relative to the inward current observed at hyperpolarized voltages. Thus, the triple mutant behaves like an inward rectifier. The single and double mutants have different relative amounts of inactivated channels at 80 mv resulting in current traces with characteristics intermediate to the outwardly rectifying wild-type channel and the inwardly rectifying triple mutant. As the peak conductance voltage curve shifts to more negative voltages, the amount of inward rectification increases. These six constructs thus represent a family of K channels with differ- ent degrees of inward rectification. The kinetic scheme used to describe the transition from an outward rectifier to an inward rectifier requires an inactive state. Removal of the inactivated state in the Shaker wild-type channel by deletion of the N-terminal region results in a noninactivating outward rectifier (Hoshi et al., 1990). Without the inactivation mechanism, shifting the midpoint of activation should not generate an inward rectifier. N-terminal deletions were generated for all six constructs, and the same protocols used in Figure 2A for the constructs with intact inactivation are shown in Figure 2B for the corresponding constructs without an N-terminal inactivation domain. In the absence of inactivation, there is no transition from an out- ward rectifier to an inward rectifier, but rather, as the midpoint of activation is shifted to more hyperpolarized the midpoint of activation of the outward rectifier far enough in the hyperpolarizing direction to populate the inactive state at resting potentials. In this switch from an outward rectifier to an inward rectifier, the inactivation gate of the outward rectifier is the activation gate of the inward rectifier. Results and Discussion We tested this hypothesis in Shaker K channels by combining amino acid replacements that cause negative shifts in activation gating. Two point mutations in Kv1.1 (a noninactivating outward rectifier) and one point mutation in Shaker have been shown to shift the midpoint of activation to more hyperpolarized voltages (Liman et al., 1991; Lopez et al., 1991; Tytgat and Hess, 1992). These mutations (R365N, R371I, and L366A; see Figure 1A), all in the S4 region, were combined into two double mutants (L366A:R371I and R365N:R371I) and one triple mutant. Figure 1B shows a representative conductance voltage plot for each of the six constructs. Figure 1C shows their midpoints of activation. The effects of the mutations were cumulative, with progressive negative shifts of the midpoints of activation in the double and triple mutants. Figure 2A shows a family of current traces recorded in symmetrical K solutions during voltage steps from a holding potential of 80 mv for the six channel types, illustrating the progressive change from an outward rec- tifier (wild type, top) to an inward rectifier (triple mutant, bottom). As the midpoint of activation shifts to more hyperpolarized voltages, more channels populate the inactivated state at the holding potential of 80 mv. The wild-type Shaker channels rarely populate the inactivated state at 80 mv (all channels are closed), and thus there is no current change in response to a hyperpolarizing step. With sufficiently positive voltage steps,

Conversion of Shaker to an Inward Rectifier 855 Figure 2. Current Families of Constructs with and without an N-Terminal Domain (A) Mutants with an intact N-terminal domain show a gradual change from an outward rectifier to an inward rectifier. The six constructs are the same as in Figure 1, except that they all contain an intact N-terminal domain. From top to bottom: ShB, R365N, R371I, R365N:R371I, L366A:R371I, R365N: L366A:R371I. For all constructs except R365N:L366A:R371I, the oocyte was held at 80 mv, stepped through a range of voltages from 160 mv to 60 mv in increments of 20 mv for 300 ms, and then stepped to 40 mv for 100 ms (see inset voltage protocol at top). For R365N:L366A:R371I, the most positive voltage is 50 mv instead of 60 mv; all else is the same as for the other constructs; n values: ShB (6), R365N (7), R371I (6), R365N: R371I (4), L366A:R371I (4), R365N:L366A: R371I (9). (B) Mutants lacking the N-terminal domain show no switch from an outward rectifier to an inward rectifier. The six constructs are the same as in (A), except that a 40 amino acid segment from amino acid 6 to 46 has been deleted to remove inactivation (Hoshi et al., 1990). From top to bottom: ShB 4-46, R365N, R371I, R365N:R371I, L366A:R371I, R365N:L366A:R371I. For all constructs, the oocyte was held at 80 mv, stepped through a range of voltages from 160 mv to 60 mv in increments of 20 mv for 300 ms, and then stepped to 40 mv for 100 ms (see inset voltage protocol at top of [A]); n values: ShB 4-46(3), R365N (3), R371I (3), R365N: R371I (5), L366A:R371I (4), R365N:L366A: R371I (6). potentials, the channels become open over a larger voltage range. The triple mutant without the N-terminus has little rectification over a voltage range from 160 mv to 60 mv. External TEA block indicated that this triple mutant induced current was not a nonspecific leak (data not shown). In addition, the current due to this triple mutant shows little time dependence in response to either a positive or negative step. This confirms that activation (rising phase of the current upon hyperpolarization) and deactivation (current decay upon depolarization) of the triple mutant inward rectifier (with the N-terminal region) are due to the Shaker fast N-type inactivation gate. The difference in rectification between the constructs with or without the N-terminal region is further illustrated in Figure 3, which shows representative peak current voltage plots for all 12 constructs, normalized to the peak current at 60 mv. When the N-terminal inactivation domain is present (Figure 3A), there is a gradual change in the amount of inward rectification as the peak conductance voltage curve shifts to more negative voltages. This change is absent when the N-terminal region is deleted (Figure 3B) and there is no inactivated state. For all 12 channels, C-type inactivation (Hoshi et al., 1991) is slow relative to the pulse lengths considered here and can be ignored. However, a channel with a negative activation range and fast C-type inactivation could also behave as an inward rectifier. The demonstration that small changes in sequence can convert the Shaker channel into an inward rectifier provides a partial explanation for sequence similarities between Shaker, HERG, and the KAT1 family of plant inward rectifiers. The strong voltage-dependent inward rectification of the triple mutant is similar to that seen in KAT1. HERG channels can activate in response to depolarizations from rest, but behave as inward rectifiers after a prepulse to positive potentials due to a fast C-type inactivation (Sanguinetti et al., 1995; Trudeau et al., 1995; Smith et al., 1996). With a similar pulse protocol, the behavior of the R371I channels is strikingly simi- lar to that of HERG. However, in contrast with HERG, R371I channels rectify by an N-type inactivation mecha- nism. Figure 4 shows a prepulse protocol for ShB, R371I, and the two double mutants. Under these conditions, all four channels, including the wild-type ShB channel, show large inward currents upon hyperpolarization from 40 mv. As the Shaker activation range becomes more negative, there is a progressive increase in the rate of

Neuron 856 Figure 3. Peak Current Voltage Relationships Representative normalized peak current voltage for all constructs, with (A) and without (B) an N-terminal domain. The current was normalized to the value at 60 mv. For clarity, the wild-type data (ShB and ShB 4-46) and that from the triple mutant (with and without N-terminus) are shown with lines connecting the points. Leak current is not subtracted. The following are n values and corresponding symbols for the constructs with the N-terminal domain: ShB (6) (asterisks), R365N (7) (open squares), R371I (6) (closed circles), R365N:R371I (4) open circles), L366A:R371I (4) (closed triangles), R365N:L366A:R371I (9) (closed double triangles). The following are n values and corresponding symbols for the constructs without the N-terminal domain: ShB 4-46 (3) (asterisks), R365N (3) (open squares), R371I (3) (closed circles), R365N:R371I (5) (open circles), L366A:R371I (4) (closed triangles), R365N:L366A:R371I (6) (closed double triangles). considered activation for an inward rectifier is, in the case of the triple Shaker mutant, recovery from inactiva- tion, and what is traditionally considered deactivation for an inward rectifier is inactivation. Since activation of the two channels is due to two different gating mechanisms, it is not necessary to alter the charge on the voltage sensor or the direction it moves across the electric field to convert one into the other (Suchyna et al., 1993; Verselis et al., 1994). These results compliment both early work on anomalous rectification in squid axon by relief of internal TEA block (Armstrong and Binstock, 1965) and recent work suggesting an inactivation-medi- ated inward rectification for the HERG channel (San- guinetti et al., 1995; Shibaski, 1987; Trudeau et al., 1995; Smith et al., 1996). recovery from inactivation (I O) relative to the rate of deactivation (O C), thus generating currents similar to HERG when challenged with the same pulse protocols (Sanguinetti et al., 1995; Trudeau et al., 1995). HERG currents can also display outward rectification if the prepulse is negative ( 70 mv) (Sanguinetti et al., 1995). This dependency of rectification on prepulse potential is also observed for the R371I mutant, which shows outward rectification when the prepulse is negative ( 80 mv, see Figure 2A). Differences in kinetic and steady- state properties between HERG and R371I could be accounted for by a difference in the relative rates of activation (, scheme I) and inactivation (, scheme I), even though the molecular mechanisms of inactivation are different between the two channels. These results demonstrate that small alterations in amino acid sequence can change a Shaker-delayed rectifier channel into a voltage-gated inward rectifier. Mechanistically, this can be explained by a shift in activation gating to more negative potentials so that the channels populate inactivated states at rest. What is traditionally Experimental Procedures Electrophysiology All data were collected without leak subtraction, since the hyperpo- larized shifted mutants are open over a large voltage range. Approximately 0.8 1 ms at the beginning of each step was blanked. The Figure 4. Prepulse Protocols to Inactivate the Channels Maximally Prepulse protocols to 40 mv for ShB, R371I, R365N:R371I, and L366A:R371I, all with an intact N-terminal inactivation domain. The ooctye was held at 80 mv stepped to 40 mv for 100 ms and then through a range of voltages from 160 mv to 60 mv in increments of 20 mv for 190 ms; n values: ShB (6), R371I (7), R365N:R371I (6), L366A:R371I (8).

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