Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with ...febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel alpha-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is approximately 0.3 ms in the -30 to -10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.
Mutations in SCN1A, the gene encoding the brain voltage‐gated sodium channel α1 subunit (NaV1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures ...plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic–clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole‐cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non‐functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild‐type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance–voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady‐state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from ∼1–3% of peak current amplitude that was significantly greater than WT‐SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use‐dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild‐to‐moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype–phenotype correlations.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
We report a case of a woman who experienced intrauterine fetal death at full term pregnancy, and then died suddenly soon after learning about the death of her fetus. At autopsy, previously ...undiagnosed neurofibromatosis and an adrenal gland pheochromocytoma were discovered in the mother. Genetic screening also revealed a novel
KCNH2
mutation in both fetus and mother indicating type 2 congenital long-QT syndrome (LQTS). A catecholamine surge was suspected as the precipitating event of fetal cardiac arrhythmia and sudden fetal death, while the addition of emotional stress provoked a lethal cardiac event in the mother. This case illustrates the potential for lethal interactions between two occult diseases (pheochromocytoma, LQTS).
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
KCNE4 domains required for inhibition of KCNQ1 Manderfield, Lauren J.; Daniels, Melissa A.; Vanoye, Carlos G. ...
The Journal of physiology,
01/2009, Volume:
587, Issue:
2
Journal Article
Peer reviewed
Open access
Voltage-gated potassium (K v ) channels are modulated in distinct ways by members of the KCNE family of single transmembrane domain accessory subunits.
KCNE4 has a dramatic inhibitory effect on KCNQ1 ...that differs substantially from the activating effects of KCNE1 and KCNE3.
The structural features of KCNE4 that enable this behaviour are unknown. We exploited chimeras of KCNE1, KCNE3 and KCNE4 to
identify specific domains responsible for the inhibitory effects on heterologously expressed KCNQ1. Previous structureâfunction
analysis of KCNE1 and KCNE3 identified a critical tripeptide motif within the transmembrane domain that accounts for the differences
in KCNQ1 modulation evoked by these two KCNE proteins. Swapping the transmembrane tripeptide motif of KCNE4 with the corresponding
amino acid sequence of KCNE1 did not influence the behaviour of either protein. Similarly, exchanging the tripeptide regions
of KCNE3 and KCNE4 further demonstrated that this transmembrane motif does not explain the activity of KCNE4. Using a more
systematic approach, we demonstrated that the KCNE4 C-terminus was critical for KCNQ1 modulation. Replacement of the KCNE1
or KCNE3 C-termini with that of KCNE4 created chimeric proteins that strongly inhibited KCNQ1. Additional evidence supported
a cooperative role of the KCNE4 transmembrane domain. Although the C-terminus was necessary for KCNE4 activity, we demonstrated
that a surrogate transmembrane domain derived from the cytokine receptor CD8 did not enable inhibition of KCNQ1, indicating
that the KCNE4 C-terminus alone was not sufficient for KCNQ1 modulation. We further demonstrated that the KCNE4 C-terminus
interacts with KCNQ1. Our data reveal important structureâfunction relationships for KCNE4 that help advance our understanding
of potassium channel modulation by KCNE proteins.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Modulation of voltage-gated potassium (KV) channels by the KCNE family of single transmembrane proteins has physiological and pathophysiological importance. All five KCNE proteins (KCNE1-KCNE5) have ...been demonstrated to modulate heterologously expressed KCNQ1 (KV7.1) with diverse effects, making this channel a valuable experimental platform for elucidating structure-function relationships and mechanistic differences among members of this intriguing group of accessory subunits. Here, we specifically investigated the determinants of KCNQ1 inhibition by KCNE4, the least well-studied KCNE protein. In CHO-K1 cells, KCNQ1, but not KCNQ4, is strongly inhibited by coexpression with KCNE4. By studying KCNQ1-KCNQ4 chimeras, we identified two adjacent residues (K326 and T327) within the extracellular end of the KCNQ1 S6 segment that determine inhibition of KCNQ1 by KCNE4. This dipeptide motif is distinct from neighboring S6 sequences that enable modulation by KCNE1 and KCNE3. Conversely, S6 mutations (S338C and F340C) that alter KCNE1 and KCNE3 effects on KCNQ1 do not abrogate KCNE4 inhibition. Further, KCNQ1-KCNQ4 chimeras that exhibited resistance to the inhibitory effects of KCNE4 still interact biochemically with this protein, implying that accessory subunit binding alone is not sufficient for channel modulation. These observations indicate that the diverse functional effects observed for KCNE proteins depend, in part, on structures intrinsic to the pore-forming subunit, and that distinct S6 subdomains determine KCNQ1 responses to KCNE1, KCNE3, and KCNE4.
Abstract Life-threatening arrhythmias have been suspected as one cause of the sudden infant death syndrome (SIDS), and this hypothesis is supported by the observation that mutations in arrhythmia ...susceptibility genes occur in 5–10% of cases. However, the functional consequences of cardiac potassium channel gene mutations associated with SIDS and how these alleles might mechanistically predispose to sudden death are unknown. To address these questions, we studied four missense KCNH2 (encoding HERG) variants, one compound KCNH2 genotype, and a missense KCNQ1 mutation all previously identified in Norwegian SIDS cases. Three of the six variants exhibited functional impairments while three were biophysically similar to wild-type channels ( KCNH2 variants V279M, R885C, and S1040G). When co-expressed with WT-HERG, R273Q and K897T/R954C generated currents resembling the rapid component of the cardiac delayed rectifier current ( IKr ) but with significantly diminished amplitude. Action potential modeling demonstrated that this level of functional impairment was sufficient to evoke increased action potential duration and pause-dependent early afterdepolarizations. By contrast, KCNQ1 -I274V causes a gain-of-function in IKs characterized by increased current density, faster activation, and slower deactivation leading to accumulation of instantaneous current upon repeated stimulation. Action potential simulations using a Markov model of heterozygous I274V- IKs incorporated into the Luo–Rudy (LRd) ventricular cell model demonstrated marked rate-dependent shortening of action potential duration predicting a short QT phenotype. Our results indicate that certain potassium channel mutations associated with SIDS confer overt functional defects consistent with either LQTS or SQTS, and further emphasize the role of congenital arrhythmia susceptibility in this syndrome.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy ...in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying 1C channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs)--not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances--not antagonizes--the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.
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