Voltage-gated K ؉ channels control excitability in neuronal and various other tissues. We identified three unique ␣-subunits of voltage-gated K ؉ -channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K ؉ channel subunits, they did not produce K ؉ currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with ''silent subunits'' to diversify its function. Including the subunits described here, the ''silent subunits'' represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.electrically silent subunits ͉ ER retention ͉ heterotetrameric assembly ͉ KCNG3

We have cloned HERG USO , a C-terminal splice variant of the human ether-à -go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (I Kr ), from human heart, and we find that its mRNA is ϳ2-fold more abundant than that for HERG 1 (the originally described cDNA). After transfection of HERG USO in Ltk ؊ cells, no current was observed. However, coexpression of HERG USO with HERG 1 modified I Kr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERG USO , HERG ⌬C (a HERG 1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, I Kr was rescued by ligation of 104 amino acids from the C terminus of HERG 1 to the C terminus of HERG ⌬C , indicating that the C terminus of HERG 1 includes a domain (<104 amino acids) that is critical for faithful recapitulation of I Kr . The lack of this C-terminal domain not only explains the finding that HERG USO does not generate I Kr but also indicates a similar mechanism for hitherto-uncharacterized long QT syndrome HERG mutations that disrupt the splice site or the C-terminal. We suggest that the amplitude and gating of cardiac I Kr depends on expression of both HERG 1 and HERG USO .The long QT syndrome is a disorder characterized by prolongation of the QT interval as a result of unusually slow repolarization of the cardiac action potential (1, 2). One variant, LQT2, 1 is caused by mutations in the human ether-à -go-gorelated gene (HERG). Heterologous expression of HERG results in a current with the physiologic and pharmacologic properties of I Kr (3, 4). These properties include strong inward rectification that is now recognized to be determined by the ultra-rapid inactivation that the channel undergoes with depolarization (4 -8) and by sensitivity to I Kr -specific methanesulfonanilide blockers, such as dofetilide (4, 9). Importantly, HERG mRNA is detected at high abundance in human heart (10), and I Kr has been recorded in human atrial and ventricular myocytes (11,12).LQT2-associated HERG mutations in the pore of the channel display reduced or no I Kr (13) and, when expressed with wildtype cRNA, a dominant negative effect has been observed, implying that heteromeric (wild-type ϩ mutant) channel assembly occurs. Heteromeric assembly of physiologically occurring splice variants has also been suggested by the finding that an N-terminal splice variant of the human gene (or of the murine homolog mERG) itself resulted in no current in Xenopus oocytes, but when coexpressed with wild-type cRNA, I Kr with rapid activation and deactivation (typical of that observed in heart) was observed (14, 15).The initial identification of HERG mutations as a cause of LQT2 included a mutation in the 3Ј-region of the gene, which is predicted to result in disruption of a splice site and truncation of the C-terminal region of the protein (10). However, it is not clear how this mutation disrupts I Kr function because the membrane-spanning seg...

The recent crystallization of a voltage-gated K ؉ channel has given insight into the structure of these channels but has not resolved the issues of the location and the operation of the gate. The conserved PXP motif in the S6 segment of Shaker channels has been proposed to contribute to the intracellular gating structure. To investigate the role of this motif in the destabilization of the ␣-helix, both prolines were replaced to promote an ␣-helix (alanine) or to allow a flexible configuration (glycine). These substitutions were nonfunctional or resulted in drastically altered channel gating, highlighting an important role of these prolines. Combining these mutations with a proline substitution scan demonstrated that proline residues in the midsection of S6 are required for functionality, but not necessarily at the positions conserved throughout evolution. These results indicate that the destabilization or bending of the S6 ␣-helix caused by the PXP motif apparently creates a flexible "hinge" that allows movement of the lower S6 segment during channel gating and opening.

The congenital long QT syndrome is a cardiac disease characterized by an increased susceptibility to ventricular arrhythmias. The clinical hallmark is a prolongation of the QT interval, which reflects a delay in repolarization caused by mutations in cardiac ion channel genes. Mutations in the HERG (human ether-à -go-go-related gene KCNH2 can cause a reduction in I Kr , one of the currents responsible for cardiac repolarization. We describe the identification and characterization of a novel missense mutation T65P in the PAS (Per-Arnt-Sim) domain of HERG, resulting in defective trafficking of the protein to the cell membrane. Defective folding of the mutant protein could be restored by decreased cell incubation temperature and pharmacologically by cisapride and E-4031. When trafficking was restored by growing cells at 27°C, the kinetics of the mutated channel resembled that of wildtype channels although the rate of activation, deactivation, and recovery from inactivation were accelerated. No positive evidence for the formation of heterotetramers was obtained by co-expression of wild-type with mutant subunits at 37°C. As a consequence the clinical symptoms may be explained rather by haploinsufficiency than by dominant negative effects. This study is the first to relate a PAS domain mutation in HERG to a trafficking deficiency at body temperature, apart from effects on channel deactivation.

Noise-induced hearing loss (NIHL) is one of the most important occupational diseases and, after presbyacusis, the most frequent cause of hearing loss. NIHL is a complex disease caused by an interaction between environmental and genetic factors. The various environmental factors involved in NIHL have been relatively extensively studied. On the other hand, little research has been performed on the genetic factors responsible for NIHL. To test whether the variation in genes involved in coupling of cells and potassium recycling in the inner ear might partly explain the variability in susceptibility to noise, we performed a case-control association study using 35 SNPs selected in 10 candidate genes on a total of 218 samples selected from a population of 1,261 Swedish male noise-exposed workers. We have obtained significant differences between susceptible and resistant individuals for the allele, genotype, and haplotype frequencies for three SNPs of the KCNE1 gene, and for the allele frequencies for one SNP of KCNQ1 and one SNP of KCNQ4. Patch-clamp experiments in high K+-concentrations using a Chinese hamster ovary (CHO) cell model were performed to investigate the possibility that the KCNE1-p.85N variant (NT_011512.10:g.21483550G>A; NP_00210.2:p.Asp85Asn) was causative for high noise susceptibility. The normalized current density generated by KCNQ1/KCNE1-p.85N channels, thus containing the susceptibility variant, differed significantly from that from wild-type channels. Furthermore, the midpoint potential of KCNQ1/KCNE1-p.85N channels (i.e., the voltage at which 50% of the channels are open) differed from that of wild-type channels. Further genetic and physiological studies will be necessary to confirm these findings.