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An Essential Role of a Terminal Peptide Segment of Three Hydrophobic Residues

Xiao-Ming Xia¹, J.P. Ding¹ and Christopher J. Lingle^1,2

¹ Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110
² Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, MO 63110

Address correspondence to C. Lingle, Department of Anesthesiology, Washington University School of Medicine, 600 S. Euclid Ave., Box 8054, St. Louis, MO 63110. Fax: (314) 362-8571; E-mail: clingle{at}morpheus.wustl.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An auxiliary ß2 subunit, when coexpressed with Slo subunits, produces inactivation of the resulting large-conductance, Ca²⁺ and voltage-dependent K⁺ (BK-type) channels. Inactivation is mediated by the cytosolic NH₂ terminus of the ß2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH₂ terminus in the inactivation process. The ß2 NH₂ terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2–4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH₂-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.

Key Words: inactivation mechanisms • inactivation domains • K⁺ channels • BK channels • Ca²⁺- and voltage-gated K⁺ channels

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rapid inactivation of Ca²⁺ and voltage-gated BK-type K⁺ channels arises from coexpression of the slo1 pore–forming subunits with particular auxiliary ß subunits (Wallner et al., 1999; Xia et al., 1999, 2000; Uebele et al., 2000; Lingle et al., 2001). Of the four members of the BK ß subunit family, inactivation arises from the short cytosolic NH₂ terminus of either the ß2 subunit (Wallner et al., 1999; Xia et al., 1999) or of particular splice variants of the ß3 subunit (Uebele et al., 2000; Xia et al., 2000; Lingle et al., 2001). Since slo1 and ß subunits assemble in a 1:1 stoichiometry (Knaus et al., 1994b; Wang et al., 2002), up to four inactivation-competent NH₂ termini can be present in any inactivating BK channel (Wang et al., 2002). Similar to inactivation of voltage-dependent K⁺ (Kv) channels mediated by NH₂-terminal domains of subunits (MacKinnon et al., 1993; Gomez-Lagunas and Armstrong, 1995), inactivation arises from the independent action of each NH₂ terminus (Xia et al., 1999; Wang et al., 2002). Thus, at least superficially similar elements would appear to contribute both to inactivation of Kv channels and BK channels.

Of the kinetic behaviors exhibited by voltage-gated ion channels, the phenomenon of rapid inactivation of Kv channels has perhaps been most amenable to a correlation of the structural elements of the channel with an actual mechanism of gating. For Kv channels, to produce inactivation, the cytosolic NH₂ terminus, either of the pore-forming subunits (Hoshi et al., 1990; Ruppersberg et al., 1991) or of cytosolic auxiliary ß subunits (Rettig et al., 1994), appears to move into a position that closely abuts the mouth of the ion permeation pathway. The close association of the Kv blocking domain and the ion permeation pathway is supported by the fact that cytosolic channel blockers compete with the blocking domain for occupancy of the channel (Choi et al., 1991; Demo and Yellen, 1991). Furthermore, once the inactivation domain occupies its blocking position, it impedes closure of the channels (Demo and Yellen, 1991; Ruppersberg et al., 1991). Yet, until recently the nature of the interaction between any inactivation domain and its target site has remained elusive. Now, an important advance has been the demonstration that the initial first four residues of the NH₂ terminus of an inactivating Kvß auxiliary subunit interact with specific residues in the pore-forming S6 segment of the Kv 1.4 subunit (Zhou et al., 2001). Thus, the initial residues of an inactivating NH₂ terminus appear to snake their way into the permeation pathway to occlude ion flux.

To what extent this molecular picture of Kv inactivation may apply to BK channels remains unclear. Several functional properties of BK inactivation clearly differ from Kv inactivation. For example, BK channel inactivation is not slowed by cytosolic blockers that bind to the mouth of the BK channel pore (Lingle et al., 1996; Solaro et al., 1997; Xia et al., 1999). Furthermore, unlike Kv inactivation (Demo and Yellen, 1991; Ruppersberg et al., 1991), BK channels do not reopen during recovery from inactivation, suggesting that when the inactivation domain resides in its blocking position, BK channels are not prevented from undergoing a normal open to closed conformational change (Solaro et al., 1997). These properties of BK channel inactivation seem more reminiscent of Na⁺ channel inactivation, in which occupancy by blockers of sites within the pore do not interfere with the inactivation mechanism (O'Leary and Horn, 1994; Kuo and Liao, 2000). However, BK channel inactivation shares with both ShakerB K⁺ channels (Gomez-Lagunas and Armstrong, 1994) and voltage-dependent Na⁺ channels (Kuo and Liao, 2000) a dependency on the concentration of extracellular permeant ions (Solaro et al., 1997). Thus, both similarities and differences exist between the rapid inactivation properties of BK channels and Kv channels and the extent to which the underlying molecular mechanism is similar is yet unresolved.

An additional challenge to our current understanding of rapid inactivation, both for Kv channels and BK channels, is that inactivation may involve kinetic complexity not previously accounted for by the simple, one-step open channel block model generally used to describe inactivation. Specifically, inactivation of BK channels mediated by the ß3b subunit involves two kinetic steps (Lingle et al., 2001) and a similar model has also been proposed for inactivation of Kv channels by NH₂-terminal inactivation domains (Zhou et al., 2001). For Kv channels, it was proposed that perhaps an initial movement of the inactivation structure (first step) then permits the hydrophobic blocking domain to enter the channel (second step) (Zhou et al., 2001). As part of this conceptualization, the first kinetic step was proposed to depend on the interaction of charged NH₂-terminal residues with charged residues lining the entryway to the channel, thereby appropriately positioning the hydrophobic segment for blockade. However, as yet there are no specific experimental results that support the idea that inactivation of Kv channels occurs with two distinct kinetic steps or to associate charge on the NH₂ terminus with a particular kinetic step. Similarly, the physical basis of each of the two kinetic steps involved in BK channel inactivation remains unknown (Lingle et al., 2001).

As part of our efforts to understand BK channel inactivation and to resolve the functional and structural differences between inactivation of Kv channels and BK channels, here we have undertaken a mutational analysis of inactivation of BK channels mediated by the ß2 auxiliary subunit. The NH₂ terminus of the ß2 subunit of the BK channel family contains 46 amino acids that are considered to be cytosolic to the first transmembrane (TM)^* segment (Wallner et al., 1999; Xia et al., 1999). For comparison, the noninactivating ß1 subunit (Knaus et al., 1994a) contains 15 cytosolic residues many of which are homologous to their counterparts (residues 31–45) in the ß2 subunit.

We address two different aspects of the role of the ß2 NH₂ terminus. First, we define the key segment of residues involved in producing inactivation. Second, we address the role of the linker between the key inactivation epitope and the first transmembrane segment (TM1) of the ß2 subunit. Our results clearly establish that residues 2–4 (FIW) of the NH₂ terminus are the critical inactivation epitope in the ß2 subunit. This critical inactivation segment appears to be both necessary and sufficient to produce inactivation. Our results also show that deletions involving residues from positions 4 through 36 are of minimal impact on the ability of the ß2 subunit to inactivate. Additional examination of the properties of the linker between the FIW epitope and TM1 shows that neither charge nor maintenance of any particular structural integrity conferred by residues from positions 5 through 41 is required to permit inactivation to occur. Thus, inactivation mediated by the ß2 subunit simply requires a set of three hydrophobic residues linked to TM1 by a spacer of rather nonspecific requirements.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Site-directed Mutagenesis
The pfu DNA polymerase was used in all PCRs to generate hß2 mutations and all constructs were verified by sequencing (Stratagene). In general, strategies followed standard procedures in use in this laboratory (Xia et al., 1998b, 1999). Here we explicitly describe procedures for generation of five categories of mutation employed in this paper: first, constructs with deletions in the NH₂ terminus; second, constructs with mutations of residues within or near the initial four residues of the NH₂ terminus; third, constructs with glutamine insertions; fourth, point mutations of charged residues; and fifth, constructs with artificial NH₂ termini.

Deletion Constructs
ß2 NH₂-terminal deletion constructs were generated by pfu PCR with two specific ß2 primers. For example, to generate 2–4 (FIW), a PCR was performed with pfu polymerase (primers 5'- ATCAGAATTCTCTAAGATGAGTGGCCGGACCTCTTCATC-3' and 5'-TACTAGTCGACAAAAATTATTTTATCCATTTTTG-3'). The product was then digested with EcoRI and SalI, and then ligated into EcoRI-SalI vector pBF. The deletion was verified by sequencing. Other deletions were obtained in a similar fashion.

Mutations of Residues within the Initial Four Residues
For mutations within the initial FIW region, a pair of complementary primers was used to generate each mutation. The primers contained the designated changes of codons (e.g., F to G, I to G, W to G). The PCR was performed on wild-type hß2 template (96°C for 2 min; 17–20 cycles of 96°C for 30 s, 50°C for 45 s, 68°C for 10 min; 96°C for 2 min). The reaction product was digested with DpnI for 2–3 h at 37°C, and then transformed to E. coli strain DH5 (Xia et al., 1998a). Mutants were identified by DNA miniprep and subsequently sequenced.

Insertion of a Glutamine Chain
Insertion of a chain of glutamine residues (poly-Q) was generated by linking two PCR fragments and subcloning into the oocyte expression vector pBF (Xia et al., 1998a). As an example, to generate the insertion of a 14 amino acid insert at position 46 (INS@46), two PCRs, A and B, were performed each with specific primers (A, 5'-ATCAGAATTCAGACCCTGGACCAACATTCTCTAAG-3' + 5'-CGGGATCCCTGCTGCTGCTGCTGCTGTCGGTCCTCTCCTGCCTTCAG-3'; B, 5'-GAAGATCTCAACAACAACAACAACAACGAGCTATTCTCCTGGGACTG-3' + 5'-TACTAGTCGACAAAAATTATTTTATCCATTTTTG-3'). The products were then purified with QIAGEN column, digested with EcoRI + BamHI (reaction A), and BglII + SalI (reaction B) overnight. After gel purification, three fragments, A + B + EcoRI-SalI vector pBF, were incubated for overnight ligation at 16°C, and then the ligation reaction was transformed to E. coli strain DH5. Clones with the correct insert size were identified by EcoRI + SalI digestion and then sequenced for verification. This generates the 14 amino acid insertion of QQQQQQGSQQQQQQ after the amino acid at position 45. Similar insertions were made following residues 8, 15, 27, and 35.

Point Mutations of Charged Residues
As an example of how point mutations of charged residues were generated, here generation of K33Q is described. Two complementary primers were synthesized (5'-CATGACCTCCTGGACCAAAGGAAAACAGTCACA-3' and 5'-TGTGACTGTTTTCCTTTGGTCCAGGAGGTCATG-3'), in which nucleotides at the site of the targeted codon were changed to nucleotides encoding the designated amino acid codon (AAA to CAA). The PCR was performed on pBF-hß2 template (96°C 2 min; 17–20 cycles of 96°C for 30 s, 50°C for 45 s, 68°C for 10 min; 96°C for 2 min), and the product was digested with DpnI for 2–3 h at 37°C, and then transformed to E. coli strain DH5 (Xia et al., 1998a). Single colonies were picked up for a subsequent DNA miniprep and sequencing was applied for mutant identification. Multiple point mutations could be obtained by repeating several rounds of the above.

Generation of Artificial NH₂ Termini
The artificial NH₂ termini, such as FIW-8Q, were generated by pfu PCR with a set of primers. The NH₂-terminal primer contained nucleotides encoding FIW-8Q after an initial ATG and then ß2 sequence from TM1 with a EcoRI site at 5' (5'-ATGAATTCTCTAAGATGCAGCAACAACAACAACAACAACGAGCTATTCTCCTGGGAC-3'); the COOH-terminal primer matched antisense sequence around the ß2 stop codon with a 5' SalI site (5'-ATCGTCGACAAAAATTATTTTATCCATTTTTGCAT-3'). The 634bp PCR fragment was purified, digested with EcoRI and SalI, cloned into pBF, and then verified by sequencing. The longer poly-Q chain constructs were generated by similar methods, although the shorter poly-Q chain constructs were used as the PCR template.

Expression in Xenopus Oocytes
SP6 RNA polymerase was used to synthesize cRNA for oocyte injection after DNA was linearized with MluI (Xia et al., 1999). 50 nl of cRNA (10–20 ng/µl) was injected into stage IV Xenopus oocytes harvested 1 d before. To ensure saturation of each BK channel with ß subunits, we injected and ß subunits at ratios of at least 1:2 by weight.

Electrophysiological Recording
Recordings from inside-out patches (Hamill et al., 1981) followed standard procedures in use in this laboratory (Xia et al., 1999; Lingle et al., 2001; Zhang et al., 2001). Currents were typically digitized at 10–20 kHz (Bessel low-pass filter; -3 dB). The pipette extracellular solution was (in mM) 140 potassium methanesulfonate, 20 KOH, 10 HEPES, and 2 MgCl₂, pH7.2. The usual test solution bathing the cytoplasmic face of the patch membrane contained (in mM) 140 potassium methanesulfonate, 20 KOH, 10 HEPES, pH 7.0, and 5 mM HEDTA with Ca²⁺ -methanesulfonate added to make 10 µM free Ca²⁺. Procedures for preparation of solutions with defined [Ca²⁺] have been described (Zeng et al., 2001; Zhang et al., 2001) and the solution applied over the cytosolic face of excised patches was controlled by a local perfusion system (Solaro et al., 1995, 1997). Voltage commands and the acquisition of currents were accomplished with pClamp 7.0 for Windows (Axon Instruments, Inc.).

The Evaluation of Inactivation Behavior of Different Mutant Constructs
For all constructs, the following functional characteristics were determined: (a) the G-V curve at 10 µM Ca²⁺ measured from peak current and also, for noninactivating variants, from tail current; (b) the time constant of inactivation (_on) at potentials from 40 through 160 mV at 10 µM Ca²⁺; and (c) the time constant of recovery (_off) from inactivation at -140 mV with 10 µM Ca²⁺. In addition, for some constructs, there was appreciable steady-state current at potentials where inactivation mediated by the ß2 subunit is essentially complete. In such cases, f_ss, the fractional amplitude of steady-state current relative to maximal activatable current (I_max), was determined. f_ss is potentially indicative of the equilibrium between blocking and unblocking transitions. I_max was determined in two ways: first, from fitting the current time course to a function including terms for both activation and inactivation and, second, from application of trypsin to directly define I_max. In cases where each method could be applied to the same constructs, both estimates of I_max were within 10%.

SCHEME I

SCHEME II

The empirical measures of channel inactivation behavior, _on, _off, and f_ss are most useful if they can be related to specific molecular transitions in a blocking scheme.

The standard scheme used to characterize either inactivation or blockade by NH₂-terminal inactivation peptides is given in Scheme I. However, more recently it has been shown that inactivation mediated by the ß3b subunit involves two distinct kinetic steps (Lingle et al., 2001) and other work now shows that a similar model is also necessary to account for ß2 subunit–mediated inactivation (unpublished data). This model, given in Scheme II, involves formation of a preinactivated open state (O*) that precedes entry into inactivated states. A similar kinetic mechanism has been proposed to explain inactivation of Kv channels (Zhou et al., 2001), although direct evidence demonstrating the existence of two kinetic steps for Kv channels is still lacking. Because of the fact that Scheme II almost certainly applies to the mechanism of inactivation studied here (and perhaps to that of Kv channels; Zhou et al., 2001), there is simply no explicit way with the parameters we can measure to make definitive estimates of the underlying molecular transitions and the energetic changes caused by any given mutation. However, in lieu of such specific mechanistic information, here we employ three different empirical measures of the inactivation behavior that are of use in comparing the consequences of mutations.

First, for each construct we define ln[_on(mut)/_on(ß2)] and ln[_off(mut)/_off(ß2)], which allow comparison of the consequences of each mutation relative to the wild-type ß2 subunit in terms of units of kT. Irrespective of the molecular steps in the inactivation process, it is likely that _on at least qualitatively reflects primarily the factors that influence association of any inactivation domain with its blocking site while _off measured at -140 mV reflects, at least in part, dissociation of the inactivation domain. This approach has been also been used to evaluate the interaction of a Kv inactivation domain with the Kv1.4 subunit, in which it has also been proposed that a two-step mechanism of inactivation applies (Zhou et al., 2001). Irrespective of the mechanism of inactivation, ln[_on/_on(ß2)] and ln[_off/_off(ß2)] provide model-independent indicators of changes in the inactivation process that allows comparison among constructs.

Second, to allow comparison between constructs in which both _on and _off may change, we also determine ln[(_on(mut)/_off(mut))/(_on(ß2)/_off(ß2))], which yields a measure in units of kT of the amount of change in the stability of the inactivated state relative to the wild-type ß2 subunit. For inactivation of Kv1.4 by various mutations of the Kvß2 NH₂ terminus, which is also proposed to involve a similar two-step inactivation mechanism, ln[(_on(mut)/_off(mut))/(_on(ß2)/_off(ß2))] has been equated to ln[K_d(mut)/K_d(wt)] (Zhou et al., 2001). Although it is likely that the relative changes in this estimate of ln[K_d(mut)/K_d(wt)] caused by any mutation do reflect something about the true equilibrium constants of the inactivation process, they are clearly not true equilibrium constants, both because inactivation probably involves two steps and because inactivation onset and recovery are measured at different voltages. Yet, as one tool for comparing the consequences of any given mutation, this formulation is still useful. Here we use the term "inactivation stability" defined as K* = _on/_off for any given construct, such that ln[K*_mut/K*_ß2] = ln[(_on(mut)/_off(mut))/(_on(ß2)/_off(ß2))]. The parameter, ln[K*_mut/K*_ß2], which is in kT units, provides a sense of the magnitude of the overall energetic changes that arise from any given mutation, although it should not be taken as a true equilibrium constant. ln[K*_mut/K*_ß2] should probably be considered less useful when a construct exhibits appreciable steady-state currents (larger f_ss) at 100 mV.

Finally, as an additional tool for assessing inactivation stability among various constructs, we take advantage of both _on and f_ss. Scheme I allows explicit characterization of the underlying rates k₁ and k_-1, as given in the following pair of equations (Murrell-Lagnado and Aldrich, 1993b):

Thus, from Scheme I, the above equations provide a means of evaluating the effects of particular mutations directly on both the molecular association rate and the dissociation rate (Murrell-Lagnado and Aldrich, 1993b), where k₁ = 1,000/_on - k_-1 and k_-1 = f_ss*1000/_on. From this, we define an inactivation equilibrium constant, K = k_-1/k₁. Relative to wild-type ß2 behavior, this yields ln[K_mt/K_ß2]. Although f_ss is poorly defined for wild-type + ß2 currents, since the same value of K_ß2 is used for calculation of all estimates of ln[K_mt/K_ß2], it remains a useful tool for comparison among constructs. If a construct behaves in accordance with Scheme I, in which K defines a true binding affinity, ln[K_mt/K_ß2] defines the change in free energy of binding (G_mt-ß2) resulting from the mutation. For Scheme II, although K is not a true equilibrium constant, ln[K_mt/K_ß2] provides a simple qualitative estimate of the change in apparent efficacy of the inactivation process which is useful for comparison of different constructs, particularly when steady-state currents are appreciable.

It should be noted that ln[K*_mt/K*_ß2] and ln[K_mt/K_ß2], although both reflect something about the stability of the inactivation mechanism, are calculated from different conditions and, although relative changes between constructs would be expected to be similar, exact values are expected to differ.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Properties of the ß2 NH₂ Terminus
The sequence of the ß2 NH₂-terminal residues that precedes the predicted first TM1 is given in Fig. 1 along with the NH₂-terminal residues for other BK ß subunits. The ß2 NH₂ terminus consists of a total of 46 residues, including the initiation methionine that extend cytosolically from the beginning of the postulated TM1 sequence. The NH₂ terminus contains six positive and four negative amino acids in the first thirty amino acids, resulting in a net charge on the initial 31 amino acids of +2, ignoring the terminal methionine. ß2 sequence following the initial 31 residues shares similarity with the ß1 NH₂ terminus, which does not exhibit inactivation. Thus, residues in positions 31–46 of the ß2 subunit are unlikely to participate directly in inactivation.

View larger version (27K): [in this window] [in a new window]   FIGURE 1. Sequence of NH2 termini of the BK auxiliary ß subunit family. (A) NH2 termini of the four known auxiliary ß subunits are shown. For the ß3 subunit for which four alternatively spliced NH2 termini have been identified (Uebele et al., 2000), the rapidly inactivating ß3b variant is shown. TM1 designates the proposed beginning of the first TM segment. Positive and negative residues in the ß2 subunit are in blue and red, respectively. Boxed sets of ß2 residues (11–17 and 20–30) are thought to adopt relatively helical structures in an isolated peptide, while other portions of the NH2 terminus are relatively disordered (Bentrop et al., 2001). (B) The initial 20 residues of several inactivating NH2 termini are compared, showing the common theme of a hydrophobic segment at the NH2 terminus and the downstream hydrophilic region.

The ß2 subunit shares some common features with many other NH₂-terminal inactivation domains of both and ß subunit of Kv channels. In general, a segment of largely hydrophobic residues (Fig. 1 B) is followed by a more hydrophilic segment often containing both positive and negative charges. Among different NH₂ termini, there is no clear pattern of charge, although most inactivating NH₂ termini contain net positive charge.

Deletion of Amino Acids in Positions 2–4, but not in Positions 5–31, Abolish Inactivation
Our first goal was to define residues or regions of the NH₂ terminus of the ß2 subunit that might be critical to the inactivation process. Therefore, a series of constructs was generated in which residues were deleted from various positions in the NH₂ terminus. Two protocols were used to characterize each construct: first, an activation protocol involving a depolarizing command step to various potentials from -100 through 180 mV and, second, a paired pulse recovery protocol in which two depolarizing voltage steps to 100 mV were separated by a variable recovery interval at -140 mV. As shown in Fig. 2 A1 for wild-type ß2 currents, the activation protocol allows measurement of a time constant of inactivation (_on) at different potentials and also the fraction of noninactivating current at steady-state (f_ss) at a given potential. The paired pulse protocol (Fig. 2 A2) yields a time constant of recovery from inactivation (_off).

View larger version (32K): [in this window] [in a new window]   FIGURE 2. Deletions spanning positions 5–36 do not abolish inactivation. In A1, currents resulting from subunits coexpressed with wild-type ß2 subunits were activated by the indicated voltage protocol. In A2, currents were activated by a paired pulse protocol (activation steps to 100 mV) separated by steps of different duration to -140 mV. Currents during the initial activation step were truncated to allow better visualization of the recovery time course. In B, removal of Phe, Ile, and Trp in positions 2–4 (FIW) results in removal of inactivation. In C1 and C2, currents arising from a ß2 subunit with amino acids in positions 5–16 deleted (5–16) are shown. The first 10 amino acids in this construct are MFIWEKRNIY. Note the steady-state current in this construct that may arise from the influence of charged residues in positions 5–7. In D1 and D2, currents arising from a construct with residues 16–25 deleted (16–25) are shown. In E1 and E2, currents are shown for a construct with residues 27–36 deleted (27–36). In F, the currents show that deletion of residues 5 through 35 (5–35) results in removal of inactivation. In 5–35, the total length of the cytosolic portion of the NH2 terminus is 14. In G, inactivation time constants (on) for ß2 (, 4 patches), 5–16 (, 3 patches), 16–25 (, 4 patches), and 27–36 (, 4 patches) are plotted as a function of activation potential showing a similar weak voltage-dependence of on for each construct. Each point is the mean and SD of 4–7 patches. In H, the recovery time course at -140 mV defined from the paired pulse protocol is shown for a set of patches for each construct. For ß2 (, 4 patches), the fitted off is 23.4 ± 2.3 ms; for 5–16 (, 3 patches), off is 5.13 ± 0.19 ms; for 16–25 (, 4 patches), off is 9.30 ± 0.55 ms; for 27–36 (, 3 patches), off is 6.19 ± 0.33 ms. Vertical calibration bar corresponds to: A1, 3 nA; A2, 2 nA; B, 6 nA; C1, 4 nA; C2, 3 nA; D1, 6 nA; D2, 4 nA; E1, 1.5 nA; E2, 1.2 nA; F, 8 nA.

View larger version (40K): [in this window] [in a new window]   FIGURE 3. Deletions within positions 2–4 of the NH2 terminus reduce and abolish inactivation. In A1, activation of wild-type + ß2 currents are illustrated while, in A2, the time course of recovery from inactivation at -140 mV is shown. In B1 and B2, currents resulting from a construct with deletion of Phe in position 2 (F) are shown, along with the time course of recovery from inactivation for that construct. Note the appearance of some steady-state current at all activation potentials. In C1 and C2, currents resulting from a construct with deletion of Phe and Ile in positions 2 and 3 (FI) are shown. More substantial steady-state current is observed along with more rapid recovery from inactivation. In D, on is plotted as a function of activation potential for each of the three inactivating constructs (ß2: , 4 patches; F: , 4 patches; FI, , 8 patches). In E, the time course of recovery from inactivation determined at -140 mV is illustrated for the three constructs. For ß2, off is given in Fig. 2; for F (3 patches), off is 4.5 ± 0.3 ms; for FI (4 patches), off is 2.99 ± 0.33 ms.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Consequences of replacement or displacement of FIW residues with GGG. In A, currents arising from a construct in which residues FIW were replaced with GGG are shown. No inactivation is observed, and trypsin application resulted in no increase in outward current. In B, currents are shown for a construct in which GGG was appended to the initial FIW sequence. The apparent stability of inactivation is reduced, but inactivation still occurs. In C, currents are shown for a construct in which GGG was inserted between FIW and the remainder of the NH2 terminus. In this case, steady-state inactivation is than for wild-type, but still substantial.

Currents from constructs in which each of the three NH₂-terminal residues were substituted with glycine are shown in Fig. 5, B–D. In each case, introduction of a single glycine, although weakening the apparent affinity of the inactivation process, did not abolish the inactivation process. With two glycines (Fig. 5, E–G), the efficacy of the inactivation process was further reduced. However, either a single F or single W were sufficient to maintain some inactivation, while construct GIG did not exhibit inactivation. This suggests that residues F and W and/or positions 2 and 4 are more critical to the stability of the inactivated state than residue I in position 3.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Consequences of replacement of one or two residues in the FIW epitope. In A, inactivating currents resulting from wild-type ß2 subunits are shown. In B–D, glycine was individually substituted for each residue in the FIW epitope. In each case, this resulted in a small weakening of inactivation, with the strongest effect arising from the F2G substitution. In E–G, two glycines were substituted for a pair of residues in the FIW epitope. In F, replacement of both F and W with G abolished inactivation, while the presence of a single F (E) or W (G) appears sufficient to maintain some fast inactivation. In H–K, the consequences of increasing the separation between F and W are illustrated. In H, the presence of two glycines between F and W results in currents similar to those with an FGW epitope, suggesting that W can still contribute to the stability of the inactivated state when there are two glycines interposed. In I and J, three and four glycines are interposed between F and W, in both cases resulting in currents in which steady-state inactivation is comparable to that resulting from FGG (E). This suggests that, in FGGGW (I) and FGGGGW (J), W may not substantially participate in defining the stability of the inactivated state. In K, the introduction of two negative charges between F and W abolishes inactivation.

Examples of the consequences of introduction of positive (Arg) or negative (Glu) charge into each of the three positions are provided in Fig. 6. In general, the introduction of a glutamate was more effective at disrupting inactivation than the introduction of an arginine, although in all cases inactivation still occurs. Furthermore, charges in position 2 (F) were more disruptive of inactivation than at positions 3 or 4.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Introduction of single charges in the inactivation segment reduces but does not abolish rapid inactivation. In A–C, each residue in the inactivation segment was replaced individually with glutamate. Replacement of F with E (A) produced the most marked disruption of inactivation, with substantial steady-state current observed at all potentials. In D–F, the consequences of replacing each residue with arginine are illustrated. Arginine is less effective in each case at disrupting inactivation than glutamate, although at each position arginine produces some reduction in the stability of the inactivated state. Similar to the action of glutamate, replacement of F with R (D) had the strongest effects in disrupting inactivation. Vertical calibration: A, 4 nA; B, 1.5 nA; C, 5 nA; D, 6 nA; E, 5 nA; F, 4 nA.

The fact that inactivation still occurs after rather extensive mutagenesis of the FIW segment suggests that a specific structure defined by this triplet of residues is probably not critical to inactivation. Therefore, we also examined three constructs in which the positions of the F, I, and W were rearranged: FWI, IWF, and WIF. In each case, these constructs inactivated similarly to wild-type ß2 currents (Table II). _off was also comparable to the wild-type FIW construct, although recovery from inactivation of construct FWI exhibited two exponential components.

To compare the consequences of alterations in the FIW region, the magnitude of the changes in _off resulting from each mutation is compared along with the magnitude of the changes in _on in Fig. 7. In terms of kT units, most mutations generally disrupt _off more than _on, consistent with the idea that the major effect of the mutations is to promote faster dissociation of the inactivation domain from its binding site. Changes in _on are much smaller, although not absent. However, for those mutations in which f_ss is appreciable, some of the change in _on may also reflect a small contribution of dissociation to the _on relaxation. The apparent change in the stability of the inactivated state for each mutant was also plotted in terms of ln(K_mt/K_ß2) (Fig. 7 C), which reflects an apparent affinity calculated from the fraction of steady-state current (f_ss) and _on.

View larger version (36K): [in this window] [in a new window]   FIGURE 7. Relationship of inactivation parameters to alterations in the FIW triplet. In A, changes in off relative to inactivation mediated by wild-type ß2 subunits is expressed kT units. Mutations are grouped into those at position 2 (F2), those at position 3 (I3), those at position 4 (W4), constructs with deletions or multiple glycines in the NH2 terminus, and then a set of repeated residues in the initial triplet (FFF, III, WWW). Error bars reflect standard errors for measurement of the mutant construct expressed relative to the mean ß2 estimate. In B, changes in on are shown for each construct. Except for the slowing in inactivation resulting from the WWW mutation, most mutations have minimal effects on inactivation onset. In C, effects of mutations are compared in terms of ln(Kmt/Kß2), which is calculated based on the steady-state current at 100 mV (fss) and on (see MATERIALS AND METHODS).

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Dependence of inactivation onset and recovery on properties of residues in the initial triplet. In A1, on (on the left) and off (on the right) is plotted as a function of the mean surface area of the residue in position 2 that would be buried on transfer from solvent to a folded protein (Rose et al., 1985). In A2, off is plotted as a function of area what would be buried. The lines correspond to linear regressions [(area) = B*exp(C*area)] fit through only the uncharged residues. In B1 and B2, on and off are plotted with respect to area buried on transfer of a residue from solvent to a folded protein in regard to the residue in position 3 in the inactivation epitope. In C1 and C2, on and off are plotted with respect to area buried on transfer of a residue from solvent to a folded protein in regard to the residue in position 4 in the inactivation epitope.

View larger version (20K): [in this window] [in a new window]   FIGURE 9. Dependence of inactivation parameters on bulk hydrophobicity within the initial triplet. In A, off for constructs with mutations within the FIW triplet is plotted as a function of the mean surface area (for the three residues in positions 2–4) that would be buried on transfer from solvent to a folded protein (Rose et al., 1985). The solid line is a linear regression [off(A) = 0.018 * exp(0.012*A)] for all constructs involving uncharged residues. Error bars are SD for a least three determinations. , F2G, F2A, F2L; , I3T, I3A, I3G; , W4G, W4A, W4L; , FGG, GGW; , FFF, III, WWW; red , F2R, I3R, W4R; blue , F2E, I3E, W4E; red , FWI, WIF, IWF. In B, on is plotted as a function of area of residue buried on transfer to a folded protein. Symbols are as in A. The solid line was a fit to constructs with no charges in the initial triplet [on(A) = 0.002 exp(0.016A) + 8.0].

View larger version (37K): [in this window] [in a new window]   FIGURE 10. NH2 termini with artificial polymeric linkers support inactivation of BK channels. In A and H, wild-type ß2 currents are shown at two different time bases for comparison to mutant constructs. In B, an NH2 terminus with a polymeric chain length of 30 glutamine residues separating FIW from R46 results in currents that exhibit inactivation. In C, an NH2 terminus consisting solely of 30 glutamine residues (30Q) does not inactivate. In D, an NH2 terminus with a polymeric chain length with 10 glutamine residues separating FIW from R46 results in currents that do not inactivate. In E, when the chain length reaches 12 residues, fast time-dependent block is observed at potentials positive to 140 mV, while at more moderate potentials the fast kinetics of block result in an apparent increase in current activation rate. In F and G, traces show inactivating currents resulting from linkers of 14 and 20 glutamine residues, as indicated. In H, wild-type ß2 currents are shown on a different time base. In I, the NH2 terminus contained a linker with 10 proline residues. In J, the linker contained 12 proline residues. In K, the linker contained 14 proline residues. In L, the linker contained 14 residues, an alternating sequence of 7 alanine-arginine pairs.

Polymeric NH₂ Termini Place Constraints on the Distance Between TM1 and the Interaction Site of the Inactivation Epitope
The ability of artificial NH₂ termini to produce inactivation suggests that we can place additional limits on the properties of inactivation-competent NH₂ termini. A series of NH₂ termini with different polyglutamine (poly-Q) linkers were constructed. At poly-Q lengths of 8, 10 (Fig. 10 D), and 11, no inactivation was observed. In all cases, NH₂ termini with poly-Q lengths from 12 to 30 supported inactivation (Fig. 10, E–G; Table III). At a chain length of 12 residues, direct time-dependent inactivation was observed only at potentials more positive than 140 mV, whereas the low affinity of the inactivation equilibrium and the rapidity of inactivation resulted in currents with a faster apparent activation time course at other potentials (Fig. 10 E). In comparison to the native ß2 NH₂ terminus, all constructs with the poly-Q linkers exhibited a faster onset of inactivation and a faster rate of recovery from inactivation, although with longer linker lengths the rates begin to approach those of the wild-type ß2 NH₂ terminus. With a linker of 12 residues, the total number of residues from the initiation methionine preceding the R at the beginning of TM1 is 16. It is interesting that noninactivating ß1 and ß4 NH₂ termini have 14 and 15 cytosolic residues, respectively, suggesting that their terminal residues would rarely approach the position at which the ß2 NH₂ terminus acts (Fig. 1 A).

It seems remarkable that a linker as short as 12 residues should support inactivation given that the TM1 of the ß2 subunit presumably resides further from the channel axis than the subunit S0–S6 segments. Can any inferences be made about the length and structure of the peptide segment required for inactivation? Polymeric chains of amino acids are probably best treated as a random coil. In such a case, the rms end-to-end distance for a chain of N residues is given approximately by (Creighton, 1993), such that a chain of 12 residues should, on average, extend 39.5 Å and a chain of 20 residues, 51 Å. For comparison, an -helical coil of 12 residues should extend 18 Å (1.5 Å/residue) and a ß-sheet 38–40 Å (3.2 Å/residue).

A particularly informative linker would be based on poly-proline (poly-P). Proline adopts neither an nor ß helical shape, but forms its own more rigid helical structures, with a polyproline II conformation (3.33 residues per turn; 3.12 Å per residue) favored in aqueous media (Creighton, 1993). Similar to the poly-Q linkers, a chain of 10 proline residues did not support inactivation, while chains of 12, 13, and 14 residues all supported inactivation (Fig. 10, I–K). Thus, uncharged chains formed by either the rigid proline or the more flexible glutamine exhibit a similar cut-off in terms of the minimum number of residues required to ensure that the inactivation segment reaches a blocking position. For a 12 residue poly-P chain, a polyproline II conformation predicts a length of 37.4 Å. This is remarkably similar to the average end-to-end distance for a random coil, which is likely to apply to the poly-Q chains.

If the poly-Q linkers adopt a helical structure rather than a random coil, how the FIW inactivation epitope is presented to its interaction site might depend on the fractional rotation of the epitope dependent on the number of turns conferred by different chain lengths. We therefore plotted _on and _off as a function of the number of residues in a linker (Fig. 11, A and B). Over a series of poly-Q linkers from 12 through 30 Q, _on and _off varied in a continuous fashion, suggesting that the ability of the FIW epitope to produce inactivation was not particularly constrained by any aspect of the linker.

View larger version (31K): [in this window] [in a new window]   FIGURE 11. Dependence of inactivation properties on lengths of artificial NH2 termini and altered ß2 NH2 termini. In A, on for poly-glutamine (Poly-Q) and poly-proline (poly-P) linkers is plotted as a function of linker length. In B, off for poly-Q and poly-P linkers is plotted as a function of linker length. In C, ln(K*mt/K*ß2) is plotted as a function of linker length. In D, off is plotted as a function of the length of the ß2 NH2-terminal linker for various deletion constructs, as indicated. In E, off is plotted as a function of ß2 linker length for various deletion constructs. In F, ln(K*mt/K*ß2) is plotted as a function of linker length for deletion constructs. Dotted lines correspond to values for the wild-type ß2 construct.

The differences in the kinetic properties of currents with the poly-P and poly-Q linkers may be explainable in terms of chain flexibility. The poly-Q linker will adopt lengths both shorter and longer than 38 Å and exhibit substantial flexibility, whereas with the more rigid poly-P linker there may be constraints in terms of how the FIW epitope can reach its site of action.

Two other polymeric linkers were also examined. A linker with a series of seven alanine/arginine repeats permitted inactivation (Fig. 10 L). A linker of 14 alanine residues did not result in inactivation. The inability of alanine to support inactivation might result from several reasons. Alanine strongly stabilizes -helices relative to a random coil arrangement when introduced into artificial peptides (O'Neil and DeGrado, 1990), which might result in a much shorter average length of the alanine chain. However, alanine may also simply prefer a hydrophobic environment, such that the inactivation epitope remains anchored in a position unsuitable for producing inactivation.

For comparison to results with artificial NH₂-terminal linkers, the properties of native NH₂ termini with deletions (Fig. 11, D–F) were also plotted as a function of linker length. For _on (Fig. 11 D) and _off (Fig. 11 E), no clear trend with linker length can be discerned, although there is some suggestion, on average, of a faster _off with shorter chain lengths. However, ln[K*_mut/K*_ß2] (Fig. 11 F) varied qualitatively with chain length in a fashion somewhat similar to that of the poly-Q and poly-P chains with the apparent affinity of the inactivation process reduced at shorter chain lengths.

Mutations that Decrease Net Positive Charge Generally Have Little Effect or Increase the Rate of Current Inactivation
Results above indicate that the necessary elements required to make an inactivation-competent NH₂-terminal segment are a triplet of hydrophobic residues at the NH₂ terminus and a simple linker connecting the FIW triplet to TM1. Yet, in Kv channels charge on the linker is considered to be fundamentally important in the inactivation process, either in guiding interactions of a presumed "ball" domain with a binding site (Murrell-La