A. M. Brown

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.

Identity of a novel delayed rectifier current from human heart with a cloned K+ channel current.

In human myocardium, the nature of the K+ currents mediating repolarization of the action potential is still speculative. Delayed rectifier channels have recently been cloned from human myocardium, but it is unclear whether or not these currents are involved in the termination of the cardiac action potential plateau. In intact human atrial myocytes, we have identified a rapid delayed rectifier K+ current with properties and kinetics identical to those expressed by a K+ channel clone (fHK) isolated from human heart and stably incorporated into a human cell line for the first time. The myocyte current amplitude was 3.6 +/- 0.2 pA/pF (at +20 mV, n = 15) and activated with a time constant of 13.1 +/- 2 milliseconds at 0 mV (n = 15). The half-activation potential (V0.5) was -6 +/- 2.5 mV (n = 10) with a slope factor (k) of 8.6 +/- 2.2 (n = 10). The heterologously expressed fHK current amplitude was 136 pA/pF (at +20 mV, n = 9) with an activation time constant of 11.8 +/- 4.6 milliseconds at 0 mV; V0.5 was 4.1 +/- 2.4 mV (mean +/- SEM, n = 8); and k was 7.0. The conductance of single fHK channels was 16.9 picosiemens in 5 mM bath K+. Both native and cloned channel currents inactivated partially during sustained depolarizing pulses. Both currents were blocked by micromolar concentrations of 4-aminopyridine and were relatively insensitive to tetraethylammonium ions and class III antiarrhythmic agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of III-IV linker mutations on human heart Na+ channel inactivation gating.

Na+ channel inactivation, a critical determinant of refractoriness, differs in cardiomyocytes and neurons. In rat brain type IIa (rB2a) Na+ channels, a critical residue in the cytoplasmic linker between domains III and IV regulates fast inactivation such that a Phe-->Gln substitution (F1489Q) inhibits inactivation by at least 85%. Since this residue is conserved in voltage-gated Na+ channels, we tested whether F1485Q, the analogous mutation in human heart (hH1a) Na+ channels, has a similar functional effect. We found that fast inactivation in wild-type (WT) channels expressed in Xenopus oocytes was complete within 15 milliseconds at a test potential of 0 mV, and its time course was biexponential with time constants of 0.4 and 2 milliseconds. But in contrast to rB2a, the FQ mutation inhibited inactivation by < 50% and increased mean single-channel open time by only twofold. Residual fast inactivation was monoexponential, with a time constant similar to that of the slower phase of normal inactivation (2 milliseconds). In the mutant channels, unlike WT, null tracings were absent at holding potentials in the range of -140 to -120 mV, and the voltage range of steady-state inactivation coincided exactly with that of activation, suggesting that residual inactivation was tightly coupled to the open state. As in rB2a, simultaneous mutations of I1484Q and M1486Q, in addition to mutation F1485Q, completely inhibited fast inactivation. Our results show that in heart Na+ channels, the IFM cluster controls the stability of both open- and closed-channel inactivation in a manner qualitatively similar to that in the brain. Structural differences in the putative inactivation receptor may explain the distinct gating patterns in channel subtypes.

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.

T-type and N-type calcium channels of Xenopus oocytes: evidence for specific interactions with beta subunits.

We used amplifying effects of calcium channel beta subunits to identify endogenous calcium channels in Xenopus oocytes. Expression of rat brain beta 4 increased macroscopic endogenous current magnitude with a small effect on kinetics. In contrast, expression of rat brain/cardiac beta 2 produced a much larger increase in current magnitude and dramatically slowed current decay. Low concentrations of omega-conotoxin GVIA irreversibly blocked currents in both uninjected and beta 2-injected oocytes. Single channel recordings revealed both T- and N-type calcium channels with conductances of 9 and 18 pS, respectively, in uninjected oocytes and in oocytes expressing either beta subunit. Expression of either beta subunit slowed average current decay of T-type single channels. Slowing of T-type current decay by expression of beta 2 was due to reopening of the channels. N-type single channel average current decay showed little change with expression of beta 4, whereas expression of beta 2 slowed average current decay.

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.

Multiple residues specify external tetraethylammonium blockade in voltage-gated potassium channels.

We have mapped residues in the carboxyl half of the P region of a voltage-gated K+ channel that influence external tetraethylammonium (TEA) block. Fifteen amino acids were substituted with cysteine and expressed in oocytes from monomeric or heterodimeric cRNAs. From a total of six mutant channels with altered TEA sensitivity, three were susceptible to modification by extracellularly applied charged methanethiosulfonates (MTSX). Another residue did not affect TEA block but was protected from MTSX by TEA. MTSX modification of position Y380C, thought to form the TEA binding site, affected TEA affinity only moderately, and this effect could be reversed by additional charge transfer from an oppositely charged MTSX analog. The results show that TEA block is modulated from multiple sites, including residues located deep in the pore and that several side chains besides that of Y380 are exposed at the TEA receptor.

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.

Cloned Human Inward Rectifier K+ Channel as a Target for Class III Methanesulfonanilides

Methanesulfonanilide derivatives such as dofetilide are members of the widely used Class III group of cardiac antiarrhythmic drugs. A methanesulfonanilide-sensitive cardiac current has been identified as IKr, the rapidly activating component of the repolarizing outward cardiac K+ current, IK. IKr may be encoded by the human ether-related gene (hERG), which belongs to the family of voltage-dependent K+ (Kv) channels having six putative transmembrane segments. The hERG also expresses an inwardly rectifying, methanesulfonanilide-sensitive K+ current. Here we show that hIRK, a member of the two-transmembrane-segment family of inward K+ rectifiers that we have cloned from human heart, is a target for dofetilide. hIRK currents, expressed heterologously in Xenopus oocytes, are blocked by dofetilide at submicromolar concentrations (IC50 = 533 nmol/L at 40 mV and 20 degrees C). The drug has no significant blocking effect on the human cardiac Kv channels hKv1.2, hKv1.4, hKv1.5, or hKv2.1. The block is voltage dependent, use dependent, and shortens open times in a manner consistent with open-channel block. While steady state block is strongest at depolarized potentials, recovery from block is very slow even at hyperpolarized potentials (tau = 1.17 seconds at -80 mV). Thus, block of hIRK may persist during diastole and might thereby affect cardiac excitability.