John A. Drewe

Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.

Alteration and restoration of K+ channel function by deletions at the N- and C-termini

Voltage-dependent ion channels are thought to consist of a highly conserved repeated core of six transmembrane segments, flanked by more variable cytoplasmic domains. Significant functional differences exist among related types of K+ channels. These differences have been attributed to the variable domains, most prominently the N- and C-termini. We have therefore investigated the functional importance of both termini for the delayed rectifier K+ channel from rat brain encoded by the drk1 gene. This channel has an unusually long C-terminus. Deletions in either terminus affected both activation and inactivation, in some cases profoundly. Unexpectedly, more extensive deletions in both termini restored gating. We could therefore define a core region only slightly longer than the six transmembrane segments that is sufficient for the formation of channels with the kinetics of a delayed rectifier.

Inactivation determined by a single site in K+ pores

An N-terminus peptide or a C-terminus mechanism involving a single residue in transmembrane segment 6 produces inactivation in voltage-dependent K+ channels. Here we show that a single position in the pore of K+ channels can produce inactivation having characteristics distinct from either N- or C-type inactivation. In a chimeric K+ channel (CHM), the point reversion CHM V 369I produced fast inactivation and CHM V 369S had the additional effect of halving K+ conductance consistent with a position in the pore. The result was not restricted to CHM; mutating position 369 in the naturally occurring channel Kv2.1 also produced fast inactivation. Like N- and C-types of inactivation, pore or P-type inactivation was characterized by short bursts terminated by rapid entry into the inactivated state. Unlike C-type inactivation, in which external tetraethylammonium (TEA) produced a simple blockade that slowed inactivation and reduced currents, in P-type inactivation external TEA increased currents. Unlike N-type inactivation, internal TEA produced a simple reduction in current and K+ occupancy of the pore had no effect. External TEA was not the only cation to increase current; external K+ enhanced channel availability and recovery from inactivation. Additional features of P-type inactivation were residue-specific effects on the extent of inactivation and removal of inactivation by a point reversion at position 374, which also regulates conductance. The demonstration of P-type inactivation indicates that pore residues in K+ channels may be part of the inactivation gating machinery.

Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores

4-Aminopyridine (4AP) blocks the intracellular mouth of voltage-gated K+ channels. We identified critical regions for 4AP binding with chimeric channels in which segments of a low affinity clone (Kv2.1, IC50 = 18 mM) were replaced with those of a high affinity clone (Kv3.1, IC50 = 0.1 mM). 4AP sensitivity was not transferred with the S5-S6 linker (pore or P region). Instead, a chimera of the cytoplasmic half of S6 increased block 20-fold, without affecting gating. A double chimera of the cytoplasmic halves of S5 and S6 fully transferred 4AP sensitivity. Because 4AP block was inhibited by tetrapentylammonium, we conclude that determinants of 4AP binding lie in the S6 segment that forms the cytoplasmic vestibule of the pore and that this site may overlap a quaternary ammonium site.

Differences between the deep pores of K+ channels determined by an interacting pair of nonpolar amino acids

The pore of a chimeric K+ channel, CHM, differed from its parental host channel, Kv2.1, by 9 amino acids. Four were located in a putative deep region and 5 in a nearby outer mouth. Point reversions were without restorative effects, and reversions V369I or L374V in the deep pore produced novel phenotypes. Among double mutations, only V369I and L374V were effective in restoring the Kv2.1 pore phenotype. Adding a change in charge at Q382K in the outer pore fully restored the parental phenotype. Thus, the pore appears to have an inner, deep region where ions such as K+ and TEA+ may be regulated by nonpolar residues and an outer region where ions may be regulated by charged residues.

A single nonpolar residue in the deep pore of related K+ channels acts as a K+:Rb+ conductance switch.

K+ and Rb+ conductances (GK+ and GRb+) were investigated in two delayed rectifier K+ channels (Kv2.1 and Kv3.1) cloned from rat brain and a chimera (CHM) of the two channels formed by replacing the putative pore region of Kv2.1 with that of Kv3.1. CHM displayed ion conduction properties which resembled Kv3.1. In CHM, GK+ was three times greater than that of Kv2.1 and GRb+/GK+ = 0.3 (compared with 1.5 and 0.7, respectively, in Kv2.1 and Kv3.1). A point mutation in CHM L374V, which restored 374 to its Kv2.1 identity, switched the K+/Rb+ conductance profiles so that GK+ was reduced fourfold, GRb+ was increased twofold, and GRb+/GK+ = 2.8. Quantitative restoration of the Kv2.1 K+/Rb+ profiles, however, required simultaneous point mutations at three nonadjacent residues suggesting the possibility of interactions between residues within the pore. The importance of leucine at position 374 was verified when reciprocal changes in K+/Rb+ conductances were produced by the mutation of V374L in Kv2.1 (GK+ was increased threefold, GRb+ was decreased threefold, and GRb+/GK+ = 0.2). We conclude that position 374 is responsible for differences in GK+ and GRb+ between Kv2.1 and Kv3.1 and, given its location near residues critical for block by internal tetraethylammonium, may be part of a cation binding site deep within the pore.

Histidine substitution identifies a surface position and confers Cs+ selectivity on a K+ pore.

The amino acid located at position 369 is a key determinant of the ion conduction pathway or pore of the voltage-gated K+ channels, Kv2.1 and a chimeric channel, CHM, constructed by replacing the pore region of Kv2.1 with that of Kv3.1. To determine the orientation of residue 369 with respect to the aqueous lumen of the pore, the nonpolar Ile at 369 in Kv2.1 was replaced with a basic His. This substitution produced a Cs(+)-selective channel with Cs+:K+ permeability ratio of 4 compared to 0.1 in the wild type. Block by external tetraethylammonium (TEA) was reduced about 20-fold, while block by internal TEA was unaffected. External protons and Zn2+, that are known to interact with the imidazole ring of His, blocked the mutant channel much more effectively than the wild type channel. The blockade by Zn2+ and protons was voltage-independent, and the proton blockade had a pKa of about 6.5, consistent with the pKa for His in solution. The histidyl-specific reagent diethylpyrocarbonate produced greatly exaggerated blockade of the mutated channel compared to the wild type. The residue at position 369 appears to form part of the binding site for external TEA and to influence the selectivity for monovalent cations. We suggest that the imidazole side-chain of His369 is exposed to the aqueous lumen at a surface position near the external mouth of the pore.

Electrophysiological characterization of a new member of the RCK family of rat brain K+ channels

A novel member of the RCK family of rat brain K+ channels, called RCK2, has been sequenced and expressed inXenopus oocytes. The K+ currents were voltage‐dependent, activated within 20 ms (at 0 mV), did not inactivate in 5 s, and had a single channel conductance in frog Ringers of 8.2 pS. Compared to other members of the RCK family the pharmacological profile of RCK2 was unique in that the channel was resistant to block (IC50 = 3.3 μM) by charybdotoxin [(1988) Proc. Natl. Acad. Sci. USA 85, 3329–3333] but relatively sensitive to 4‐aminopyridine (0.3 mM), tetraethylammonium (1.7 mM), α‐dendrotoxin (25 nM), noxiustoxin (200 nM), and mast cell degranulating peptide (200 nM). Thus, RCK2 is a non‐inactivating delayed rectifier K+ channel with interesting pharmacological properties.

Regulation of K+/Rb+ selectivity and internal TEA blockade by mutations at a single site in K+ pores

A conservative reversion at position 374 in a chimeric K+ pore, CHM, switched the preferred ionic conductance from K+ to Rb+. To understand how selectivity was switched, codons for 18 different amino acids were substituted at position 374 in each of two different K+ channels CHM and Kv2.1, the host channel for CHM. After injection of cRNA into Xenopus oocytes, less than half of the substituted mutants expressed functional channels. In both CHM and Kv2.1, channels with the substituted hydrophobic residues Val or Ile expressed Rb+-preferring pores while channels with the substituted polar residues Thr or Ser expressed K+-preferring pores. Val or Ile stabilized while Thr or Ser destabilized blockade by internal tetraethylammonium (TEA) confirming the importance of hydrophobic interactions for blockade. TEA blockade was dependent upon the charge carrier and was more effective in the presence of the ion having the larger conductance. The results are consistent with a model in which the side chains at position 374 form a filter for K+ and Rb+ ions and a site for blockade by internal TEA.

Potassium Channels in Mammalian Brain: A Molecular Approach

Publisher Summary This chapter focuses on potassium channels in the mammalian brain and describes some of the molecular techniques used in the structure–function analysis of rat brain K + channels. The coding region of Kv2.1, a typical representative of rat brain K + channels, contains six transmembrane domains (S1–S6) flanked by long C- and N-termini. The chapter describes two techniques to make deletions in the amino and carboxy ends that define the minimal core protein necessary for a functional channel. The first method uses restriction endonuclease sites, and the second technique uses exonucleases to achieve nested deletions. Deletions in both the amino and carboxy termini, beginning just upstream of the S1 and just downstream of the S6 transmembrane regions, are made using exonuclease III followed by treatment with S1 nuclease and blunt ending with T4 DNA polymerase. The donor DNA cassette is prepared using PCR methods. The K + -channel cDNA clones are propagated in either pSP72 or pBluescript SK plasmid vectors. These clones have different sequences corresponding to the three common phage RNA polymerase recognition sites: (1) T7, (2) T3, and (3) SP6. In vitro translation is optimized for a high-quality yield of cRNA using either T7 or T3 RNA polymerase.