Ion channels: structural basis for function and disease.

Ion channels are ubiquitous proteins that mediate nervous and muscular function, rapid transmembrane signaling events, and ionic and fluid balance. The cloning of genes encoding ion channels has led to major strides in understanding the mechanistic basis for their function. These advances have shed light on the role of ion channels in normal physiology, clarified the molecular basis for an expanding number of diseases, and offered new direction to the development of rational therapeutic interventions

The conduction pore of a cardiac potassium channel.

Ion channels form transmembrane water-filled pores that allow ions to cross membranes in a rapid and selective fashion. The amino acid residues that line these pores have been sought to reveal the mechanisms of ion conduction and selectivity. The pore (P) loop is a stretch of residues that influences single-channel-current amplitude, selectivity among ions and open-channel blockade and is conserved in potassium-channel subunits previously recognized to contribute to pore formation. To date, potassium-channel pores have been shown to form by symmetrical alignment of four P loops around a central conduction pathway. Here we show that the selectivity-determining pore region of the voltage-gated potassium channel of human heart through which the I(Ks) current passes includes the transmembrane segment of the non-P-loop protein minK. Two adjacent residues in this segment of minK are exposed in the pore on either side of a short barrier that restricts the movement of sodium, cadmium and zinc ions across the membrane. Thus, potassium-selective pores are not restricted to P loops or a strict P-loop geometry

Potassium channel subunits encoded by the KCNE gene family: physiology and pathophysiology of the MinK-related peptides (MiRPs).

Voltage-gated potassium channels provide tightly Controlled, ion-specific pathways across membranes and are key to the normal function of nerves muscles. They arise from the assembly of four pore-forming proteins called alpha-subunits. To attain the properties of native currents, alpha-subunits interact with additional molecules such as the mink-related peptides (MiRPs), single-transmembrane subunits encoded by the KCNE genes. Significantly, mutations in KCNE 1, 2 and 3 have been linked either to life-threatening cardiac arrhythmia or a disorder of skeletal muscle, familial periodic paralysis. The capacity of MiRPs to partner with multiple alpha-subunits in experimental cells appears to reflect still undiscovered roles for the KCNE-encoded peptides in vivo. Here, we consider these unique peptides in health disease and discuss future research directions

T cell recognition of nonpolymorphic determinants on H-2 class I molecules.

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants

Determination of fluphenazine, related phenothiazine drugs and metabolites by combined high-performance liquid chromatography and radioimmunoassay.

Antibodies have been produced in rabbits immunized with a fluphenazine succinate-human serum albumin conjugate. By radioimmunoassay it is possible to quantify fluphenazine (FPZ), related phenothiazine drugs and several of their metabolites at the femtomole level. As little as 370 fmol (160 pg) of FPZ can be detected and up to 0.4 ml of plasma can be added to the incubation mixture (final volume = 1.1 ml). The phenothiazine heterocyclic nucleus is immunodominant and determines the specificity of the antiserum. When a parent drug cross-reacts significantly with antibody, its 7-hydroxide, N-oxide and N-10 side chain altered metabolites can also be determined by the assay. The 8-hydroxide, sulfoxide and 7-hydroxyglucuronide metabolites are not detectable unless present in large amounts. High-performance liquid chromatography was used to separate phenothiazine drugs and metabolites. Since the antiserum has broad specificity, a combined high-performance liquid chromatography and radioimmunoassay procedure permits the identification and quantification of a phenothiazine drug and its serologically reactive metabolites. Patterns of high-performance liquid chromatographic elution and extent of immunologic cross-reaction are characteristic for metabolites relative to the parent drug. This procedure offers distinct advantages in the analysis of this complex family of compounds. FPZ was quantitatively extracted from plasma samples obtained from patients receiving FPZ per os. Although large amounts of serological activity were present in the samples 2 to 6 hr after FPZ ingestion, only 2 to 23% was extractable. The major contributors to the serological activity at times greater than 6 hr were FPZ metabolites. In a preliminary application of the combined techniques, FPZ and a metabolite identified as N-[alpha-(trifluoromethylphenothiazinyl-10)propyl]perazine were quantified in the organic extract of one plasma sample

The charybdotoxin receptor of a Shaker K+ channel: peptide and channel residues mediating molecular recognition.

Charybdotoxin (CTX) is a peptide of known structure that inhibits Shaker K+ channels by a pore-blocking mechanism. Point mutagenesis of all 30 solvent-exposed residues identified the part of the CTX molecular surface making contact with the receptor in the K+ channel. All close-contact residues are clustered in a well-defined interaction surface; the shape of this surface implies that the outer opening of the Shaker channel conduction pore abruptly widens to a 25 x 35 A plateau. A mutagenic scan of the S5-S6 linker sequence of the Shaker K+ channel identified those channel residues influencing CTX binding affinity. The Shaker residues making the strongest contribution to toxin binding are located close to the pore-lining sequence, and more distant residues on both sides of this region influence CTX binding weakly, probably by an electrostatic mechanism. Complementary mutagenesis of both CTX and Shaker suggests that Shaker-F425 contacts a specific area near T8 and T9 on the CTX molecular surface. This contact point constrains Shaker-F425 to be located at a 20 A radial distance from the pore axis and 10-15 A above the "floor" of the CTX receptor

Block of Kcnk3 by protons. Evidence that 2-P-domain potassium channel subunits function as homodimers.

KCNK subunits have two pore-forming P domains and four predicted transmembrane segments. To assess the number of subunits in each pore, we studied external proton block of Kcnk3, a subunit prominent in rodent heart and brain. Consistent with a pore-blocking mechanism, inhibition was dependent on voltage, potassium concentration, and a histidine in the first P domain (P1H). Thus, at pH 6.8 with 20 mm potassium half the current passed by P1H channels was blocked (apparently via two sites approximately 10% into the electrical field) whereas channels with an asparagine substitution (P1N) were fully active. Furthermore, pore blockade by barium was sensitive to pH in P1H but not P1N channels. Although linking two Kcnk3 subunits in tandem to produce P1H-P1H and P1N-P1N channels bearing four P domains did not alter these attributes, the mixed tandems P1H-P1N and P1N-P1H were half-blocked at pH approximately 6.4, apparently via a single site. This implicates a dimeric structure for Kcnk3 channels with two (and only two) P1 domains in each pore and argues that P2 domains also contribute to pore formation

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block.

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein

A superfamily of small potassium channel subunits: form and function of the MinK-related peptides (MiRPs).

MinK and MinK-related peptide I (MiRPI) are integral membrane peptides with a single transmembrane span. These peptides are active only when co-assembled with pore-forming K+ channel subunits and yet their role in normal ion channel behaviour is obligatory. In the resultant complex the peptides establish key functional attributes: gating kinetics, single-channel conductance, ion selectivity, regulation and pharmacology. Co-assembly is required to reconstitute channel behaviours like those observed in native cells. Thus, MinK/KvLQT1 and MiRPI/HERG complexes reproduce the cardiac currents called I(Ks) and I(Kr), respectively. Inherited mutations in KCNEI (encoding MinK) and KCNE2(encoding MiRPI) are associated with lethal cardiac arrhythmias. How these mutations change ion channel behaviour has shed light on peptide structure and function. Recently, KCNE3 and KCNE4 were isolated. In this review, we consider what is known and what remains controversial about this emerging superfamily