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Am J Physiol Cell Physiol 292: C1041-C1052, 2007. First published September 6, 2006; doi:10.1152/ajpcell.00274.2006
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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

SCAM analysis reveals a discrete region of the pore turret that modulates slow inactivation in Kv1.5

Cyrus Eduljee, Thomas W. Claydon, Vijay Viswanathan, David Fedida, and Steven J. Kehl

Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada

Submitted 18 May 2006 ; accepted in final form 1 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In Kv1.5, protonation of histidine 463 in the S5-P linker (turret) increases the rate of depolarization-induced inactivation and decreases the peak current amplitude. In this study, we examined how amino acid substitutions that altered the physico-chemical properties of the side chain at position 463 affected slow inactivation and then used the substituted cysteine accessibility method (SCAM) to probe the turret region (E456-P468) to determine whether residue 463 was unique in its ability to modulate the macroscopic current. Substitutions at position 463 of small, neutral (H463G and H463A) or large, charged (H463R, H463K, and H463E) side groups accelerated inactivation and induced a dependency of the current amplitude on the external potassium concentration. When cysteine substitutions were made in the distal turret (T462C-P468C), modification with either the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) or negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate reagent irreversibly inhibited current. This inhibition could be antagonized either by the R487V mutation (homologous to T449V in Shaker) or by raising the external potassium concentration, suggesting that current inhibition by MTS reagents resulted from an enhancement of inactivation. These results imply that protonation of residue 463 does not modulate inactivation solely by an electrostatic interaction with residues near the pore mouth, as proposed by others, and that residue 463 is part of a group of residues within the Kv1.5 turret that can modulate P/C-type inactivation.

electrophysiology; voltage-gated potassium channels; substituted cysteine accessibility method

KV1.5 IS A VOLTAGE-GATED K+ channel whose macroscopic currents activate rapidly upon depolarization before inactivating slowly over several seconds (18). This slow, P/C-type inactivation, is distinct from the fast, N-type inactivation seen with other potassium channels such as Shaker, which is due to occlusion of the inner pore mouth by a blocking peptide formed by the NH2 terminus (21, 48, 52). N-type inactivation can be conferred to Kv1.5, however, by coexpression with either a beta1 (19) or beta3 (31) regulatory subunit. Our current understanding of P/C-type inactivation is largely the result of studies of the S5-S6 region of the Shaker-IR (fast inactivation removed) potassium channel, which have shown that mutation of residue A463 in S6 (22), E418 at the distal end of S5 (30, 38), F425 in the S5-P linker (40), W434 in the pore helix (41), and T449 in the outer pore mouth (35) can alter the rate of inactivation. Increasing the external K+ concentration ([K+]o) has been shown to inhibit inactivation in Shaker-IR (35, 42), and together with studies involving the T449C mutant (46), which indicate that the outer pore may constrict during inactivation, has led to the hypothesis that binding of external K+ ions within the conducting pathway inhibits inactivation via a foot-in-the-door mechanism that prevents constriction of the outer pore mouth (35). Inactivation has also been demonstrated to lead to an immobilization of gating charge (17, 37). However, the finding that the Shaker-IR W434F mutant appears to be "permanently inactivated," and yet still displays charge immobilization, has led to the hypothesis that slow inactivation is comprised of two phases (34, 37, 44). In the initial pore (P)-type phase, the outer pore constricts, eliminating potassium conductance, whereas with continued depolarization, S4 enters a stable, immobilized conformation (C type) by interacting with the pore. We refer to these processes collectively as P/C-type inactivation.

Our previous work has shown that in Kv1.5, modification of residue 463 in the turret region (S5-P linker; Fig. 1, A and B) can also have profound effects on P/C-type inactivation. In particular, binding of extracellular H+ or Zn2+ ions to H463 or mutation to a glycine (H463G) increases the rate of depolarization-induced inactivation and reduces the macroscopic current amplitude (25, 50). The effects of H+ and Zn2+ binding could be antagonized by raising [K+]o: KD ~1 mM, similar to the [K+]o dependence of P/C-type inactivation in Shaker-IR (5), or by the R487V mutation (homologous to T449V in Shaker; Fig. 1B) (25). Therefore, we proposed that modification of turret residue H463 results indirectly in an enhancement of P/C-type inactivation.


Figure 1
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  		Fig. 1. The turret forms a rim at the perimeter of the outer pore mouth in K+ voltage-gated (Kv) channels. A: single subunit of the Kv1.5 channel highlighting the S5-P linker that forms the turret. B: two opposing subunits of the S5-S6 region of the Kv1.2 crystal structure (32) generated using Accelrys DS ViewerPro 5.0 are shown to indicate the location of the turret (black carbonyl backbone) and the approximate positions of residues homologous to H463 and R487. C: sequence alignment of the S5-P linker, P-loop, and P-S6 linker of Kv1.5 (NCB accession no. NM002234), Kv1.2 (NCB accession no. NM004974), Kv1.4 (NCB accession no. NM002233), Shaker (NCB accession no. P08510), and KcsA (NCB accession no. 1K4C). Conserved residues are indicated by a dash.

 
In this study, we examined the effects of various substitutions at position 463 to understand the properties of this residue that are important in regulating Kv1.5 behavior. To determine whether residue 463 was unique in its ability to affect channel behavior, we then used the substituted cysteine accessibility method (SCAM) (20) to introduce cysteine mutations into each turret position (Fig. 1C) and tested for reactivity to the sulfhydryl-reactive [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) reagents. This was used to determine whether modification by either MTS reagent could affect current, and whether any MTS-induced inhibition of current might be linked to the P/C-type inactivation process. The results indicate that modification of position 463 does not disrupt inactivation via a purely electrostatic interaction with residues in the pore, as proposed by others (23), and suggest that position 463 is part of a discrete group of residues in the distal region of the turret (T462-P468) that are able to regulate P/C-type inactivation. These data are discussed in the light of reports on other channel types (e.g., TRPV5 and Kv2.1) that suggest a widespread role of the turret in the modulation of ion channel behavior.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Site-directed mutagenesis and cell preparation. Mutations were introduced into Kv1.5 in the pcDNA3 expression vector using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed using fluorescent terminator-dye sequencing (Applied Biosystems, Foster City, CA). Double mutations incorporating the R487V mutation were made by subcloning a cassette of DNA containing the mutation of interest into the R487V mutant vector using the BstEII and ClaI restriction enzymes. The wild-type (WT) Kv1.5 channel has one extracellular cysteine in the S1-S2 linker at position 268, and was present in all mutants, except for the C268V/T462C double mutant, which was made by subcloning the T462C mutant into the C268V vector using the BstEII and ClaI restriction enzymes. WT human Kv1.5 channels were studied in stably transfected human embryonic kidney (HEK)-293 and ltk– cell lines and maintained as previously described (25). Channel properties were found to be similar in both lines. HEK-293 cells were used for the H463 mutants. However, ltk– cells were used for the cysteine mutants because the expression of some cysteine substituted mutants was low (e.g., P468C), and as a result, the small currents could not be unambiguously studied in HEK-293 cells due to the presence of an endogenous K+ current in that cell line. In contrast, the lack of an endogenous K+ current in the ltk– expression system yielded clearly discernable mutant channel currents whose features could be analyzed without contamination by other currents. Cells were stably transfected with mutant Kv1.5 cDNA, as described previously (25), or transiently transfected with 1 µg of plasmid cDNA using 2–4 µl of Lipofectamine 2000 (Invitrogen) 1 day after being passaged, and used for experiments 1 to 3 days after transfection. Cells were also cotransfected with 1 µg of CD8 cDNA. Before experiments, cells were incubated with OPTI-MEM (Invitrogen) containing CD8 antibody-conjugated beads (Dynal Biotech, Oslo, Norway) for 30 min. Cells were then washed with OPTI-MEM and those with bound beads were selected for recording (24). Stable cell lines were used primarily to maximize the expression of those mutant channels whose transient expression was typically low.

Recording solutions. The standard bath solution was nominally K+ free and contained (in mM) 143.5 NaCl, 10 HEPES, 2 CaCl2, 1 MgCl2, 5 glucose, and its pH was adjusted to 7.4 with NaOH. The [K+]o was kept at 0 mM to eliminate the possibility of a Ko+-dependent relief of the MTS-mediated inhibition, except for the T462C mutation, where 3.5 mM Ko+ was required to sustain an appropriate level of resting availability. Where the effect of [K+]o on channel function was examined, KCl was substituted for NaCl. The standard patch pipette solution contained (in mM) 130 KCl, 4.75 CaCl2 (pCa2+ = 7.3), 1.38 MgCl2, 10 EGTA, and 10 HEPES, and was adjusted to pH 7.4 with KOH. Stock solutions of 2 M MTSET (Toronto Research Chemicals, Toronto, ON, Canada) and MTSES were made weekly in distilled H2O and kept at –20°C. Before experiments, a 2 mM working concentration was made from the stock by dilution with the standard bath solution and kept for no more than 2 h at 4°C before being used.

Signal recording and data analysis. Macroscopic currents were typically recorded in the whole cell configuration (25). Because of its high expression, the outside-out patch configuration was occasionally used for the H463C mutant. However, the use of this configuration was not found to affect channel properties. To construct a g-V curve, peak tail current was measured at –40 mV after 300-ms prepulses from –45 to +50 mV and fit to a single exponential function. After normalization, the data were fit to a Boltzmann function: y = A/{1 + exp[(V1/2 – V)/s]}, where y is the normalized current, reflecting conductance (g), A is the best fit of the maximal response, V1/2 is the half-activation voltage, V is the prepulse voltage, and s is the slope factor.

MTS-induced inhibition was quantified by perfusing 5 ml of the MTS reagent, after which the cell was washed with 5 ml of the standard bath solution to reverse any nonspecific effect of the MTS reagent. The peak current magnitude after MTS modification was subsequently normalized to the current amplitude from the same cell before treatment and expressed as a percentage of the pretreatment peak current amplitude.

Under normal cell culture conditions, currents from Kv1.5 T462C were small and inactivation was fast ({tau}inact = 121 ± 24 ms, n = 5, data not shown). Pretreatment with 0.5 mM of the reducing agent DTT (Fisher Scientific, Fairlawn, NJ) in OPTI-MEM for a minimum of 30 min was found to maximally increase current amplitude and slow the rate of inactivation (see GoGoFig. 4) and was used before all experiments with this mutant. After pretreatment, currents from the T462C mutant were recorded in the absence of DTT and the current phenotype was not observed to change during the control current recordings. The C268V/T462C double mutant was also DTT sensitive, suggesting that disulfide bond formation between T462C and C268 could not account for the effects of DTT observed in the T462C single mutant. DTT had no effect on the remaining cysteine-substituted mutants, and the basis for the DTT sensitivity of the T462C mutant was not addressed in this study.


Figure 2
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  		Fig. 2. Some mutations of turret residue 463 alter inactivation and confer sensitivity of the current magnitude to external K+ concentration ([K+]o). Wild-type (WT) and 463 mutant currents were evoked by a depolarization to +50 mV from a holding potential of –80 mV. Tail currents were measured at –40 mV. [K+]o ranged from 0 to 140 mM. In this and subsequent figures, the dashed line denotes the 0 current level. Currents from WT, H463Q, H463Y, and H463C mutants showed little inactivation over a 300-ms period in the absence of [K+]o (0 mM). Inactivation of H463R, H463K, and H463G mutant currents was rapid and well fit by a single exponential fast inactivation time constant ({tau}inact) = 67 ± 3 ms, n = 3, in 140 mM Ko+; 323 ± 33 ms, n = 6, in 140 mM Ko+ and 73 ± 8 ms, n = 4, in 3.5 mM Ko+ [data taken from Fig. 7 in Ref. 25], respectively. H463G, H463K, and H463R did not conduct in 0 mM Ko+, but the current level could be titrated by increasing [K+]o. H463A and H463E currents inactivated with a {tau}inact of 390 ± 33 ms, n = 6, in 3.5 mM Ko+ and 227 ± 18 ms, n = 3, in 3.5 mM Ko+, respectively, and although [K+]o-sensitive, did not collapse completely in 0 mM Ko+.

 

Figure 3
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  		Fig. 3. Cysteine substitution in the turret has modest effects on activation and deactivation. A–C: activation time constants ({tau}act) were measured by fitting the activating phase (50–100% of maximal) of currents at +50 mV to a single exponential function (49) represented by the superimposed dashed line. Tail currents were measured at –40 mV. Compared with WT Kv1.5 (A) ({tau}act 3.0 ± 0.7 ms, n = 5), the {tau}act of D458C (B) and P468C (C) were significantly slower at 14 ± 2 ms (n = 15) and 10 ± 2 ms (n = 3), respectively (activating WT current was scaled and shown as a dotted line in B and C). D: average activation and deactivation ({tau}deact) time constants are shown for the WT and cysteine-substituted channels. The {tau}deact was measured at –80 mV by fitting a single exponential. The S465C mutation significantly slowed deactivation, {tau}deact = 17 ± 4 ms (n = 5), compared with 8.7 ± 0.7 ms (n = 7) for WT Kv1.5, whereas the P468C mutation increased the rate of deactivation to 3.4 ± 0.3 ms (n = 5). In this and subsequent figures, a significant difference with respect to WT Kv1.5 (*P < 0.05) behavior is shown, a dotted line indicates the WT Kv1.5 value, and n.s.e. indicates there was no surface expression. E: D458C, S466C, and P468C mutations were also associated with a significant depolarizing shift of the g/V curve and an increase in the slope factor. The V1/2 and slope factor of Kv1.5 were –19.3 ± 0.8 mV and 4.7 ± 0.4 mV (n = 7), compared with –5 ± 2 mV and 7.9 ± 0.4 mV (n = 5) for D458C, –10 ± 3 mV and 9.2 ± 0.6 mV (n = 5) for S466C, and –7 ± 3 mV and 11 ± 1 mV (n = 3) for P468C.

 

Figure 4
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  		Fig. 4. Slow inactivation was largely unaffected by cysteine substitution. A: representative WT and mutant currents in response to a depolarization to +50 mV for 5 or 6 s. Current decay was best fit to a double exponential to quantify the extent and rate of slow inactivation, except for D458C and P468C, where no fast phase of inactivation was detectable and a single exponential was used. Currents were recorded in the whole cell configuration, except for the H463C mutant, for which outside-out patches were used due to its high current expression. B: residual component from the double exponential fit is shown as a percentage of the peak current, and in S465C, was significantly smaller (18 ± 6%; n = 10), compared with WT Kv1.5 (47 ± 2%; n = 11). C: average {tau}slow and {tau}fast of inactivation are shown for each mutant, except those that did not express, as denoted by n.s.e. The fast phase of inactivation in N459C was significantly slowed to 852 ± 162 ms (n = 4, P < 0.05) compared with 281 ± 46 ms (n = 11) for the WT. {tau}slow was 2.4 ± 0.3 s (n = 11) for the WT channel and was not different from the cysteine mutants.

 
Data are expressed as means ± SE, except for the values obtained by nonlinear least-squares fitting routines, which are expressed as means ± SD (Igor, Wavemetrics, OR). A one-way ANOVA and Dunnett's test were used to test for significance between the control and test groups. A P value ≤0.05 was considered significant.

Expression of cysteine mutants and proteinase K experiments. While the majority of cysteine mutants displayed robust currents, <5% of P468C-transfected cells expressed currents. These currents were routinely <1 nA at +50 mV, except in the presence of the R487V mutation, which increased current amplitude and the proportion of expressing cells. No current was detected in cells transfected with either E456C, F464C, or I467C cDNA, so a proteinase K assay (14) and immunoblot (7) were conducted to examine the surface expression of these mutants. The results indicated that the E456C, F464C, and I467C mutants were not fully glycosylated and did not express on the cell surface (not shown).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Addition of a positive or negative charge or a decrease of volume of the side chain at position 463 increases the rate of slow inactivation and induces [K+]o dependence. Our previous work (25, 50) has shown that the H463G mutation increases the rate of Kv1.5 inactivation and almost completely eliminates current when potassium is removed from the extracellular solution. This motivated us to examine the effect of other substitutions at position 463 to determine whether a change of side chain volume or charge was an important determinant of the functional changes observed. Figure 2 illustrates typical currents in channels where H463 was mutated to glutamine (Q), tyrosine (Y), cysteine (C), arginine (R), lysine (K), glycine (G), alanine (A), or glutamate (E). Compared with WT Kv1.5, the H463Q, H463Y, and H463C mutations did not alter the rate of inactivation (see Fig. 4 for quantification of inactivation in H463C) and the current amplitude was not decreased by removing extracellular K+. However, as with the H463G mutation, the H463R and H463K mutations increased the rate of inactivation and caused the current to collapse in 0 mM Ko+. The H463A and H463E mutants inactivated with a time course similar to that of H463K, but the sensitivity of current to removal of [K+]o was comparatively smaller. These results indicate that substitutions of large (cysteine = 109 Å3; tyrosine = 194 Å3; glutamine = 144 Å3; histidine = 153 Å3), neutral (at pH 7.4) sidegroups at position 463 have little effect on channel function, whereas small (<100 Å3), neutral side groups as well as large (>100 Å3), charged side groups increase the rate of inactivation and induce a [K+]o-dependence of current amplitude.

Since several mutations at position 463 altered the rate of inactivation, we asked whether other residues in the turret region could also affect inactivation. An alignment of the S5-S6 linkers from hKv1.2, hKv1.4, hKv1.5, Shaker, and KcsA channels reveals a large degree of homology at each end of the turret, while the intervening region shows considerable variation (Fig. 1C). To investigate which turret residues might be important for regulating inactivation, a SCAM analysis was performed by substituting cysteines at each turret position, from E456 to P468. The effect of cysteine substitution alone was characterized before assessment of the functional consequences of the covalent modification by either the MTSES or MTSET reagent.

Cysteine substitutions have modest effects on macroscopic properties of Kv1.5. Activation kinetics were largely unaffected by turret mutations. However, for D458C and P468C, the {tau}act was significantly slowed compared with the WT channel (Fig. 3, A–D). This slowing of activation was correlated with a depolarizing shift of the half-activation voltage (V1/2) (Fig. 3E). The V1/2 of the S466C mutant also displayed a significant depolarizing shift, but V1/2s of the other mutants were not significantly affected. The slope factor obtained from the fit to a Boltzmann equation was significantly larger in the D458C, S466C, and P468C mutants, compared with the WT channel, but unchanged in the remainder of mutants. As with activation, the deactivation kinetics were unaffected in the majority of the cysteine mutants. However, {tau}deact was significantly slowed in S465C and sped in P468C, compared with the WT channel (Fig. 3D). While the {tau}deact of the Q460C and G461C mutants suggested a trend toward slowing, this change was not found to be significant. Together, our results indicate that in Kv1.5, turret residues D458, S466, and P468 can affect activation kinetics and voltage sensitivity. This is consistent with studies that have suggested that the Shaker turret may interact with S4 to affect gating (16, 29, 34, 47) and structural data from the Kv1.2 channel indicating that S4 is in close proximity to the pore domain (33). Alternatively, mutation of these residues may alter the coupling between activation and channel opening (47).

Representative traces of inactivation in WT Kv1.5 and the cysteine mutants are shown in Fig. 4A. Analysis of the fast and slow components of inactivation of most mutants indicated that the majority were best fit by the sum of two exponentials and were not significantly different from WT Kv1.5 (Fig. 4C). Only the Kv1.5 N459C mutation demonstrated a significant slowing of the fast component of inactivation compared with that of WT Kv1.5 (Fig. 4C). No fast component of inactivation was observed in the D458C and P468C mutations, perhaps due to their slower activation (see above). The residual current at the end of a 5-s pulse to +50 mV was also not significantly altered in most mutants, except for Kv1.5 S465C, where it was significantly reduced relative to the WT channel (Fig. 4B). Analysis of the recovery from inactivation indicated that compared with the WT channel (Fig. 5A), H463C was significantly slowed and the A457C mutant did show a modest, but nonsignificant, change in recovery kinetics (Fig. 5B). P468C was not tested due to a typically small current amplitude, and the remainder of cysteine mutants were not significantly affected (Fig. 5C).


Figure 5
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  		Fig. 5. The H463C mutation slows recovery from the inactivated state. A and B: recovery from inactivation was determined by inactivating channels with a 5 s prepulse to +60 mV and then repolarizing to –80 mV for 40 s. Throughout the recovery period, current amplitude was assessed without producing additional inactivation by applying 15-ms pulses to +60 mV, starting 25 ms after repolarization. Only the first 12 recovery pulses are shown for clarity (top). Peak currents elicited during the recovery period were then normalized to the peak of the prepulse, plotted against the time elapsed since the end of the prepulse and fit to an exponential to obtain the time constant of recovery ({tau}recov) (bottom). {tau}recov was 3.9 ± 0.4 s (n = 6) for WT Kv1.5 (A) and slowed to 7.7 ± 0.8 s (n = 5) for H463C (B; *P < 0.05 in C). C, The {tau}recov is shown for WT Kv1.5 and the cysteine-substituted mutants, except those that did not express (n.s.e.). Due to low expression, the {tau}recov was not determined (n.d.) for P468C.

 
MTSET and MTSES irreversibly inhibit currents of cysteine-substituted mutants in the distal turret. Having established that the majority of cysteine substitutions had only a modest effect on macroscopic current behavior, we investigated the consequences of covalent modification of the cysteine mutants by either the positively charged MTSET or negatively charged MTSES reagent. On the basis of the crystal structures of KcsA (13) and Kv1.2 (32), we have assumed that all introduced cysteines are accessible from the extracellular medium. In both WT and mutant channels, treatment with either MTS reagent caused a nonspecific inhibition that reversed after washout with reagent-free medium. A second component of inhibition observed in some mutants could not be washed off, even with prolonged perfusion of control solution (not shown). This inhibition was attributed to a nonreversible, covalent modification of one or more of the four introduced sulfhydryl groups per channel, and was quantified for each mutant as shown in Fig. 6. The magnitude of this inhibition was the same, regardless of whether or not depolarizing steps were applied during the MTS application (not shown), indicating that both the open and closed states were accessible to the MTS reagents and that an accumulation of inactivation did not contribute to the inhibition. However, because of the incomplete inactivation of the cysteine mutants (Fig. 4A), we were not able to accurately determine whether the MTS-modification rate was different for the inactivated state, relative to the open or closed states. Cumulative inactivation was further accounted for by adjusting the cycle length of the depolarizations used to assess the peak current amplitude based on the recovery from inactivation time data from Fig. 5. Furthermore, the degree of current inhibition following covalent modification was not affected by long periods of hyperpolarization (~5 min; not shown).


Figure 6
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  		Fig. 6. 2 mM [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) and sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) inhibited currents in mutants where the cysteine substitution was in the distal turret. The effect of 2 mM MTSET or MTSES on peak current amplitude was determined by pulsing channels to +50 mV for 300 ms every 10 s (20 s for H463C, to allow complete recovery from inactivation). Tail currents were measured at –40 mV. A: sample traces from a single cell are shown for the control (a), MTSET application (b) and washout (c) for the T462C mutant. The exponential fit of the current inactivation after MTSET modification (c) is also shown scaled up (d) to allow comparison with the control trace. The {tau}inact was 124 ± 12 ms, n = 6, after MTSET modification. B: peak currents from the same cell as in A were plotted against time, and the steady-state level after washout was used to quantify the extent of current inhibition. Letters correspond to the traces in A. Inhibition was complete within 1–2 min of the application of MTSET, but a small component of the inhibition was reversed during the washout with control solution. C and D, as for A and B, but for a single cell treated with MTSES. After MTSES modification, {tau}inact was 66 ± 5 ms, n = 7. E: average inhibition due to MTSET and MTSES modification in the WT and cysteine mutant channels is shown. For WT Kv1.5, the mean percent inhibition by MTSET and MTSES was 15 ± 2 (n = 13) and 0 ± 3 (n = 6); 1 ± 4 (n = 4) and 20 ± 4 (n = 5) for A457C; 15 ± 4 (n = 3) and 30 ± 4 (n = 5) for D458C; 9 ± 5 (n = 4) and 1 ± 2 (n = 6) for N459C; 47 ± 3 (n = 6) and 18 ± 3 (n = 8) for Q460C; 19 ± 2 (n = 8) and –6 ± 4 (n = 5) for G461C; 81 ± 3 (n = 6) and 87 ± 3 (n = 7) for T462C; 82 ± 3 (n = 5) and 80 ± 4 (n = 4) for H463C; 24 ± 2 (n = 3) and 31 ± 4 (n = 4) for S465C; 94 ± 4 (n = 4) and 68 ± 4 (n = 5) for S466C; and 96 ± 4 (n = 3) and 99 ± 5 (n = 3) for P468C. [K+]o was 3.5 mM for T462C and 0 mM for all other mutants. n.s.e. denotes those mutants that did not express and thus were not tested.

 
Peak WT Kv1.5 currents were irreversibly inhibited by 15 ± 2% (n = 13) after application and washout of 2 mM MTSET (Fig. 6E). This degree of inhibition was not significantly different from that seen in the D458C, N459C, and G461C mutants. Surprisingly, A457C was not inhibited to any degree by MTSET. In contrast, most cysteine substituted mutants at positions 462 through 468 were inhibited by 80–96% by MTSET (Fig. 6E). Representative traces of the effect of MTSET on T462C are shown in Fig. 6, A and B. Letters beside each trace indicate the current in control solution (a), at the end of MTS application (b), and after wash out with 5 ml of control solution (c). A notable exception to the high MTSET sensitivity, however, was S465C which was not significantly inhibited.

To determine whether the inhibition by MTSET was due to its positive charge, and as such mimicked the effect of H+ or Zn2+ binding to H463 in WT Kv1.5, the effect of MTSES, which carries a negative charge, was examined. Inhibition by MTSES followed roughly the same pattern as that of MTSET (Fig. 6E). WT, N459C, and G461C currents were not inhibited by MTSES, while A457C, D458C, and Q460C mutants were significantly inhibited, but to a maximum of only ~30% (Fig. 6E). As with MTSET treatment, Kv1.5 T462C current was dramatically reduced by MTSES (Fig. 6, C and D). In the seven cells tested, there was an 87 ± 3% (P < 0.05) reduction in peak current (Fig. 6E). H463C, S466C, and P468C currents were also significantly inhibited by 80–99% (Fig. 6E). Again, as with MTSET, the sensitivity of S465C to MTSES was much less than for cysteine substitutions of neighboring residues. T462C was unique among those mutants inhibited by MTS reagents in that currents recorded after treatment with either MTSET or MTSES exhibited a faster rate of inactivation as shown in Fig. 6, A and C. Consistent with an effect due to covalent modification, application of 0.5 mM of the reducing agent DTT reversed the inhibition due to either MTSET or MTSES. Representative traces from the H463C mutant (Fig. 7) illustrate that the inhibition of the current magnitude after MTSET inhibition (b) compared with the pretreated current (a) was alleviated following DTT application and washout (c). A similar recovery of the current magnitude was observed with DTT treatment in the other cysteine mutants following either MTSET or MTSES modification.


Figure 7
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  		Fig. 7. Application of the reducing agent DTT relieved the MTS-mediated inhibition. The effect of 0.5 mM DTT was determined following treatment of the H463C mutant with 2 mM MTSET by pulsing to +50 mV every 30 s in 0 mM [K+]o. Tail currents were measured at –40 mV. A: representative traces are shown for the control current (a), the current after MTSET treatment (b), and the current following application of DTT and wash-out with the control solution (c). B: peak current amplitude from the same cell is plotted against time. Letters correspond to the sample traces shown in A.

 
These data indicate that substituted cysteine residues from positions T462-P468 of the turret, which we will refer to as the distal turret, are susceptible to covalent modification by MTS reagents, and that this modification results in a loss of current. The goal of the following series of experiments was to determine the mechanistic basis for the MTS-mediated inhibition of current.

R487V mutation or elevation of [K+]o antagonizes the MTS-mediated inhibition of current. Inhibition of WT Kv1.5 by H+ or Zn2+ is antagonized either by raising [K+]o or by the R487V mutation (25, 51). Similarly, the [K+]o dependence of Kv1.5 H463G currents is mitigated by the R487V mutation as implied in Fig. 5 of Ref. 42a. Given that both of these manipulations, i.e., changing [K+]o or mutating R487, have been linked to effects on P/C-type inactivation (see the introduction), we examined the effect of MTSET and MTSES either in the presence of 140 mM Ko+ or with the addition of the R487V mutation, in the four mutants (T462C, H463C, S466C, and P468C) that showed the greatest sensitivity to MTS treatment (>65% inhibition, Fig. 6E).

Representative traces for each of these four mutants before (–) and after MTSET exposure and wash off (+) in the presence of 140 mM Ko+ are shown in Fig. 8A. As indicated in Fig. 8B, the inhibition of each of the four mutants by MTSET in the presence of 140 mM Ko+ was significantly lower than that measured in 0 or 3.5 mM Ko+. A similar effect of 140 mM Ko+ on the MTSES-mediated inhibition was observed in all four mutants (Fig. 8B).


Figure 8
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  		Fig. 8. Inhibition by MTSET and MTSES can be antagonized by raising [K+]o or by the addition of the R487V mutation in the P-S6 linker. A: MTSET inhibition was examined, as described in Fig. 6, in the T462C, H463C, S466C, and P468C mutants but in the presence of 140 mM Ko+ or in double mutants, including the R487V mutation (0 mM Ko+, except for T462C, where 3.5 mM was used; c.f. Fig. 6). Sample traces on the left indicate current in 140 mM Ko+ before (–) and after incubation with and subsequent washout of MTSET (+). Representative traces in A, right, indicate the current before (–) and after MTSET incubation and washout (+) in R487V double mutants. B: average percent inhibition of the peak current amplitude by MTSET and MTSES reagents in 140 mM Ko+ or with the R487V mutation (in 0 mM Ko+, except for T462C, where 3.5 mM was used) are shown vs. the inhibition from Fig. 6. The percent inhibition by MTSET and MTSES was 32 ± 9 (n = 5) and 18 ± 3 (n = 5) in 140 mM Ko+ and –4 ± 2 (n = 4) and –24 ± 4 (n = 5) with R487V for T462C; 4 ± 1 (n = 5) and –3 ± 2 (n = 4) in 140 mM Ko+ and 4 ± 2 (n = 4) and 0 ± 2 (n = 4) with R487V for H463C; 17 ± 9 (n = 4) and 6 ± 5 (n = 5) in 140 mM Ko+ and 4 ± 5 (n = 4) and 4 ± 3 (n = 6) with R487V for S466C; 52 ± 9 (n = 4) and 35 ± 8 (n = 4) in 140 mM Ko+ and 25 ± 1 (n = 5) and 9 ± 3 (n = 5) with R487V for P468C.

 
Figure 8A also shows representative traces of double mutants containing either T462C, H463C, S466C, or P468C together with the R487V mutation, before (–) or after (+) MTSET treatment. Compared with the degree of inhibition observed in the single cysteine mutants at the same [K+]o, the sensitivity of the double mutants to MTSET was significantly reduced (Fig. 8B). A similar result was observed with MTSES in the double mutants and, in the case of T462C-R487V, even produced a modest 24 ± 4% (n = 5) potentiation (Fig. 8B). Together, these results imply that modification by MTS reagents of cysteine residues substituted in this region of the turret inhibits current by facilitating a process akin to P/C-type inactivation.

Elevated K+ does not alter cysteine accessibility. A possible explanation for the relief of the MTS-mediated inhibition in the presence of elevated [K+]o is that there was a change in the accessibility of the MTS reagents to the substituted cysteine residues. To address this possibility, T462C, H463C, S466C, and P468C mutants were first exposed either to MTSET or MTSES in the presence of 140 mM Ko+ using the same protocol outlined in Fig. 8, and following MTS washout, were then perfused with low Ko+ (0 or 3.5 mM). Representative current traces from Kv1.5 T462C in response to MTSES treatment are shown in Fig. 9. Letters beside each trace in Fig. 9, left indicate the current before (a) and after (b) MTSES application in 140 mM Ko+, after switching Ko+ to 3.5 mM (c) and then returning to 140 mM Ko+ (d). A plot of the peak current amplitude over time is shown in Fig. 9, right. Currents recorded in 140 mM Ko+ were inhibited during MTSES exposure but this inhibition reversed on washout. Subsequent lowering of Ko+ to 3.5 mM caused the current amplitude to decrease, despite an increase of driving force, and was consistent with the conclusion that the covalent modification had indeed occurred during the MTSES treatment in 140 mM Ko+. A similar pattern of [K+]o dependence was seen for both MTSET and MTSES in all four mutants (not shown). As with the MTS-mediated inhibition in low Ko+, the prevention of the inhibition by 140 mM Ko+ was independent of whether or not channels were pulsed during the MTS treatment. These results suggest that high Ko+ antagonizes the inhibition promoted by modification with the MTS reagents, but does not affect cysteine accessibility.


Figure 9
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  		Fig. 9. Increasing [K+]o does not relieve MTS inhibition by preventing covalent modification of sulfhydryl residues in cysteine-substituted mutants. The T462C mutant was exposed to MTSES in the presence of 140 mM Ko+, after which Ko+ was reduced to 3.5 mM to determine whether the MTS-mediated inhibition could be restored. Sample traces of T462C in response to depolarizations to +50 mV every 10 s (tail currents evoked by repolarization to –40 mV) are shown on the left and the peak current amplitude is plotted against time on the right. Letters in the diary plot correspond to the sample traces shown. Control currents recorded in 140 mM Ko+ (a) are compared with those after MTSES incubation (in 140 mM Ko+) and washout with 140 mM Ko+ (b) to establish that minimal inhibition occurred as a result of MTS modification in the presence of 140 mM Ko+. The Ko+ concentration was then lowered to 3.5 mM (c) to determine whether currents were modified during the preceding MTS wash in, after which Ko+ was changed back to 140 mM (d; trace shown as dashed line for clarity) to demonstrate the reversibility of the effect.

 

   DISCUSSION
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We have previously shown that turret residue H463 plays a key role in the H+ inhibition of Kv1.5. In this study, we further explored the role of the Kv1.5 turret by examining which properties of position 463 are important in modulating channel behavior and by demonstrating that residue H463 is part of a discrete set of residues in the distal half of the turret that can affect slow, P/C-type, inactivation.

Mutation of H463 speeds the rate of inactivation and induces [K+]o sensitivity. Figure 2 demonstrates that several substitutions at position 463 increased the rate of depolarization-induced inactivation and were associated with a decrease in macroscopic current in the absence of [K+]o. This latter phenomenon has also been observed at pH 7.4 in WT Kv1.4 (39) and fast-inactivating T449 Shaker-IR mutants (35, 42) and has been suggested to represent a loss of channel availability due to closed-state inactivation (35). However, as with T449 mutations in Shaker-IR (35, 42), there was no clear correlation between the properties of substituted side chains at position 463 in Kv1.5 and the effects on the rate of inactivation or the [K+]o dependence of current amplitude. For example, both charged (H463K and H463E) and neutral substitutions (H463G) had similar effects on channel function. These results are inconsistent with the hypothesis that residue H463 interacts with position R487 by a purely electrostatic mechanism, as suggested by others (23). Instead, modification of residue 463, either by mutation or by protonation, appears to disrupt other interactions within the outer pore region that regulate inactivation gating. In support of this view, recent experiments have suggested that inactivation in KcsA is partially dependent on the ability of the side chain at position D80 of the GYGD signature sequence (equivalent to Kv1.5-D485) in the outer pore mouth to become reorientated into an outward, or "flipped", conformation (9, 10). Analysis of the KcsA crystal structure indicates that the side chain of residue D80 in the flipped conformation is directed toward the side chain of the equivalent turret residue of Kv1.5-H463 (Q58), and the closest atoms from both side chains are within 3 Å of each other. This suggests that in Kv1.5, the effects of the turret on inactivation may be mediated in part by an interaction between H463 in the turret and D485 in the outer pore mouth.

Similar to the [K+]o-dependent relief of slow inactivation in Shaker-IR (4, 35), raising external K+ also slowed the rate of depolarization-induced inactivation and increased the macroscopic conductance in several of the Kv1.5-H463 mutants. Interestingly, the estimated equilibrium dissociation constant (KK) for these two effects of Ko+ varied between mutants, i.e., KK = ~1 mM and ~103 mM for the [K+]o dependence of macroscopic conductance and the rate of inactivation in H463G, vs. ~14 M and ~190 mM in H463R (15, 50). These differences suggest that these are inactivation-related, but not equivalent, processes. However, whether there are two K+ binding sites that individually regulate these processes, or if a single "foot-in-the-door" site can account for both effects, is still unknown.

MTS reagents inhibit current by enhancing slow P/C-type inactivation. Using the SCAM technique, we found that MTS modification of cysteine-substituted residues in the distal region of the turret inhibited channel current to a greater degree than those in the remainder of the turret (Fig. 6). As with the substitutions at position 463, the sign of the charge on the MTS reagent did not affect the current inhibition. Thus, in addition to position 463, modification of a discrete set of turret residues appears capable of inhibiting channel activity.

To date, studies employing the SCAM technique to probe the outer pore mouth of ion channels have not directly addressed the basis for the MTS-mediated inhibition, although some have provisionally attributed it to an occlusion of the outer pore mouth (27). However, the crystal structure of Kv1.2 (32) predicts that the {alpha}-carbons of turret residues equivalent to positions T462 to P468 in Kv1.5 are ~16–21 Å from the central axis of the pore. Similar turret dimensions were also estimated for Kv1.3 using a functional (toxin-binding) assay (1). This suggests that when bound to the substituted cysteines, the 6–10 Å long MTS reagents (2, 3) are too far away to occlude the outer pore mouth. Without unitary current analysis of MTS-modified channels, however, we cannot exclude the possibility that MTS reagents cause a fast channel block as expected for a blocker tethered near the pore (6). Nevertheless, based on the following observations, we propose that at least in Kv1.5, the MTS inhibition is related to an enhancement of P/C-type inactivation, similar to the effects of select mutations and protonation of position 463.

The addition of the R487V mutation reduced the extent of MTS-mediated inhibition in the cysteine mutants tested (Fig. 8). Similarly, in Shaker-IR, the homologous T449V mutation alleviated the enhanced inactivation observed in the W434F (26, 43), E418C (38), and A463V (36) mutations. Therefore, if the R487V mutation antagonizes inactivation similarly to T449V, as previously suggested (18), then the relief of the MTS-mediated inhibition by R487V suggests the involvement of the P/C-type inactivation pathway in this process. Figure 8 illustrates that raising [K+]o also relieves the MTS-mediated inhibition. Because P/C-type inactivation in Shaker-IR is classically defined by its sensitivity to [K+]o (4, 35, 42), the Ko+ sensitivity observed here also suggests that the MTS-mediated inhibition may represent an enhancement of inactivation.

Together, these results suggest that the inhibition due to MTS modification of residues in the distal turret represents an enhancement of closed-state P/C-type inactivation. This may occur either because each of these residues interacts with the inactivation gate, and MTS-modification affects this interaction, or because modification of each residue disrupts a common interaction between the turret and outer pore, such as the suggested interaction between H463 and D485. The formation of one or more interactions between the turret and pore is compatible with previous suggestions that the turret and pore may behave as a rigid structure in Shaker-IR (34). As a result, conformational changes within the pore during slow inactivation are detected by a fluorophore bound at turret position Shaker-S424C (homologous to Kv1.5-T462C). Therefore, it is plausible that in Kv1.5 a reciprocal effect may occur, such that modification of turret residues results in a conformation change within the pore leading to an enhancement of inactivation. Furthermore, the rigid interaction between the pore and turret appears to reach an upper limit at position 462. Interestingly, modification of S424C in the Shaker-IR turret by tetramethylrhodamine maleimide has little effect on currents (34), whereas modification of the homologous Kv1.5 T462C mutation by either tetramethylrhodamine maleimide or propyl-MTS does (unpublished observations). This lack of sensitivity to turret modification in Shaker-IR is also reflected by the comparatively smaller effect of pH on channel function when a histidine is substituted at the equivalent position of H463 in Kv1.5 (40). These differences likely result from variations in the primary structures of these channels (Fig. 1C) and suggest that a complex network of interactions is responsible for the phenotype of slow inactivation in Kv channels.

Functional role of the turret in other channels. The presence of a titratable side group in the turret appears to mediate pH sensitivity in other channels, including H508 in fKv1.4 (8), the F425H mutation in Shaker (40), H117 in Kir2.3 (11), and E522 in the TRPV5 Ca2+ channel (45). A compelling feature of this pH sensitivity is that in every case the titratable side group is at the position homologous to H463 in Kv1.5, suggesting that this position plays an important regulatory role in several diverse channel types.

A similar functional role of the turret is especially evident with regard to the TRPV5 channel, whose pore is hypothesized to exist in a conformation similar to that of KcsA (12). Single channel analysis of TRPV5 (45) showed that, as for Kv1.5 (28), there is a decrease in the open probability, but not the single channel conductance in low pH. This suggests that as with Kv1.5, an enhancement of closed-state inactivation may underlie the H+ inhibition of this channel, and that the turret is functionally coupled to the pore. In addition, SCAM analysis of the TRPV5 (12) turret revealed that cysteine substitution results in robust current inhibition following MTS modification at positions similar to those that inhibit Kv1.5. These findings raise the possibility of a conserved structure-function relationship of the turret across a wide range of ion channels.

In conclusion, these data indicate that a discrete group of residues in the distal turret are involved in regulating channel activity in Kv1.5, and perhaps other channel types, and suggest that this occurs by a modulation of slow, P/C type, inactivation via interactions with the pore mouth.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (to S. J. Kehl and D. Fedida) and from the Heart and Stroke Foundation of British Columbia and Yukon (to D. Fedida). T. W. Claydon was supported by the Focus on Stroke strategic initiative from The Canadian Stroke Network, the Heart and Stroke Foundation, the CIHR Institute of Circulatory and Respiratory Health, and the CIHR/Rx&D Program along with AstraZeneca Canada. C. Eduljee was in receipt of a Natural Sciences and Engineering Research Council scholarship.


   ACKNOWLEDGMENTS
 
We thank Kakee Chiu for cell culture services.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Kehl, Dept. of Cellular and Physiological Sciences, Univ. of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada (e-mail: skehl{at}interchange.ubc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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