The function of the human cardiac voltage-gated sodium channel Na V1.5 (hH1) is regulated in part by binding of calcium to an EF hand in the C-terminal cytoplasmic domain. hH1 is also regulated via an extrinsic calcium-sensing pathway mediated by calmodulin (CaM) via binding to an IQ motif immediately adjacent to the EF-hand domain. The intrinsic EF-hand domain is shown here to interact with the IQ motif, which controls calcium affinity. Remarkably, mutation of the IQ residues has only a minor effect on CaM affinity but drastically reduces calcium affinity of the EF-hand domain, whereas the Brugada mutation A1924T significantly reduces CaM affinity but has no effect on calcium affinity of the EF-hand domain. Moreover, the differences in the biochemical effects of the mutations directly correlate with contrasting effects on channel electrophysiology. A comprehensive model is proposed in which the hH1 IQ motif serves as a molecular switch, coupling the intrinsic and extrinsic calcium sensors.utations in the ␣-subunit of voltage-gated sodium channels (VGSCs) result in a wide variety of genetic diseases (1-3). Naturally occurring mutations in the cardiac VGSC hH1 lead to life-threatening arrhythmia syndromes (4). Some of these mutations cluster in and around an EF-hand calcium-binding site in the C-terminal region of the ␣-subunit, first noticed by Babitch (5). Experiments have uncovered a critical role for calcium in the regulation of channel function (6, 7). The ␤-subunit does not play a role in this calcium-dependent behavior (6). The long QT (LQT3) mutation D1790G (8, 9) disrupts binding of calcium to the EF hand and attenuates calcium-dependent changes to voltage-dependent steady-state channel availability (6). An IQtype calmodulin (CaM) binding motif (10, 11) has also been identified in the hH1 C terminus just downstream of the EF hand, in the region R1897-S1925 that also encompasses the Brugada syndrome mutation A1924T. CaM has been reported to bind to this IQ motif in a calcium-independent manner (10, 11), but the functional effect of CaM binding remains debated. The initial report described an enhancement of slow inactivation by the binding of Ca 2ϩ -CaM to the hH1 IQ motif (10), but others have found no effect of CaM on hH1, although it may modulate other isoforms of the VGSC family (12, 13). Further complicating the picture is a recent report suggesting that all calciumdependent changes experienced by hH1 are mediated by the extrinsic sensor (CaM) and that calcium does not bind directly to the EF-hand domain (14). ResultsThe intrinsic EF-hand calcium sensor was first demonstrated (6) in the construct E1773-S1920 [hH1-C-terminal domain (CTD)148, Fig. 1a]. To identify which residues constitute the folded core of the EF-hand calcium-binding domain, two significantly truncated constructs were generated (Fig. 1a): E1773-I1892 (hH1-CTD120) and E1773-S1865 (hH1-CTD93). The truncations were based on previously published secondary structure predictions (15) and multiple sequence alignment, which predict six helice...

Sodium channels initiate the electrical cascade responsible for cardiac rhythm, and certain life-threatening arrhythmias arise from Na(+) channel dysfunction. We propose a novel mechanism for modulation of Na(+) channel function whereby calcium ions bind directly to the human cardiac Na(+) channel (hH1) via an EF-hand motif in the C-terminal domain. A functional role for Ca(2+) binding was identified electrophysiologically, by measuring Ca(2+)-induced modulation of hH1. A small hH1 fragment containing the EF-hand motif was shown to form a structured domain and to bind Ca(2+) with affinity characteristic of calcium sensor proteins. Mutations in this domain reduce Ca(2+) affinity in vitro and the inactivation gating effects of Ca(2+) in electrophysiology experiments. These studies reveal the molecular basis for certain forms of long QT syndrome and other arrhythmia-producing syndromes, and suggest a potential pharmacological target for antiarrhythmic drug design.

This study lacked sufficient evidence to claim an association between a positive RVP and LOS in pediatric patients, adjusting for their underlying diagnosis. However, we found that a positive RVP was associated with a shorter duration of intravenous antibiotic administration in certain groups of patients and those with some common respiratory diagnoses. These findings help clarify the utility of rapid viral testing in the management of hospitalized pediatric patients.

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Background: A common mechanism for block of ion channels is current inhibition by drug binding to residues - notably in S6 - within the permeating pore. Open state block is commonly observed. Methods and Results: In this study, we used a combination of alanine/cysteine mutagenesis, patch clamp, and homology modeling to identify a novel allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1 (the α-subunit encoding IKs). The initial mutagenesis experiments studied a total of 45 amino acid residues in the S6 segment (30 residues), and the proximal C-terminus (15 residues) of KCNQ1. Residues required for drug block were identified as alanine/cysteine mutants displaying <50% block of expressed current (n=4 – 6 each) upon exposure to drug concentrations inhibiting wild-type current by ≥90% (quinidine 100 μm, clofilium 30 μm and L-7 10 μm). Among the 30 residues in S6, there were five (F332, F335, S338, A341 and G348) displaying such reduced block and in the proximal C-terminus, 5/15 were less sensitive, including F351 which was near-totally resistant (current decrease = 4±3%). Homology modeling of KCNQ1 based on the Kv1.2 structure unexpectedly suggested that F351 faces away from the permeating pore, arguing against pore block as the mechanism for drug inhibition of KCNQ1 current. In the open channel model, F351 lines a pocket that also includes L251 and V254 in S4–S5. The model allows drug access to this pocket, and alanine substitutions at both sites yielded potassium currents that were resistant to drug block (↓34±4% for L251A and ↓40±5% for V254A). Conclusion: These data strongly support a model in which open state block of this channel occurs not via binding to a site in the pore but rather by a novel allosteric mechanism, drug access to a side pocket generated in the open channel configuration and lined by S6 and S4/S5 residues.