Takao Shioya

Altered KCNQ3 Potassium Channel Function Caused by the W309R Pore-Helix Mutation Found in Human Epilepsy

The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K+ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K+ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.

Multistep Ion Channel Remodeling and Lethal Arrhythmia Precede Heart Failure in a Mouse Model of Inherited Dilated Cardiomyopathy

Background Patients with inherited dilated cardiomyopathy (DCM) frequently die with severe heart failure (HF) or die suddenly with arrhythmias, although these symptoms are not always observed at birth. It remains unclear how and when HF and arrhythmogenic changes develop in these DCM mutation carriers. In order to address this issue, properties of the myocardium and underlying gene expressions were studied using a knock-in mouse model of human inherited DCM caused by a deletion mutation ΔK210 in cardiac troponinT. Methodology/Principal Findings By 1 month, DCM mice had already enlarged hearts, but showed no symptoms of HF and a much lower mortality than at 2 months or later. At around 2 months, some would die suddenly with no clear symptoms of HF, whereas at 3 months, many of the survivors showed evident symptoms of HF. In isolated left ventricular myocardium (LV) from 2 month-mice, spontaneous activity frequently occurred and action potential duration (APD) was prolonged. Transient outward (Ito) and ultrarapid delayed rectifier K+ (IKur) currents were significantly reduced in DCM myocytes. Correspondingly, down-regulation of Kv4.2, Kv1.5 and KChIP2 was evident in mRNA and protein levels. In LVs at 3-months, more frequent spontaneous activity, greater prolongation of APD and further down-regulation in above K+ channels were observed. At 1 month, in contrast, infrequent spontaneous activity and down-regulation of Kv4.2, but not Kv1.5 or KChIP2, were observed. Conclusions/Significance Our results suggest that at least three steps of electrical remodeling occur in the hearts of DCM model mice, and that the combined down-regulation of Kv4.2, Kv1.5 and KChIP2 prior to the onset of HF may play an important role in the premature sudden death in this DCM model. DCM mice at 1 month or before, on the contrary, are associated with low risk of death in spite of inborn disorder and enlarged heart.

Causes of Abnormal Ca2+ Transients in Guinea Pig Pathophysiological Ventricular Muscle Revealed by Ca2+ and Action Potential Imaging at Cellular Level

Background Abnormal Ca2+ transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca2+ transients can be attributed to abnormal action potential generation or abnormal Ca2+ handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca2+ and action potential signals at the cellular level in isolated heart tissues. Methodology/Principal Findings After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca2+ transients. For the failed and alternating Ca2+ transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca2+ transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na+ channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na+ channel inactivation. Conclusions/Significance Our results indicate that there are multiple patterns for the causes of abnormal Ca2+ signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.

Effects of Candesartan on Electrical Remodeling in the Hearts of Inherited Dilated Cardiomyopathy Model Mice

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (Ito channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (IKur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased Ito and IKur and enlarged cells with greatly reduced K+ currents (Ito, IKur IK1 and Iss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the Ito, and IKur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.

Different cation sensitivities and binding site domains of Na+–Ca2+–K+ and Na+–Ca2+ exchangers

We examined inhibitory effects of external multivalent cations Ni2+, Co2+, Cd2+, La3+, Mg2+, and Mn2+ on reverse‐mode exchange of the K+‐dependent Na+/Ca2+ exchanger NCKX2 and the K+‐independent exchanger NCX1 expressed in CCL‐39 cells by measuring the rate of Ca2+ uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca2+ uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li+ stimulated reverse‐mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K+. Thus, both exchangers exhibited differential sensitivities to not only K+ but also many other external cations. We attempted to locate the putative binding sites within the α motifs for multivalent cations by site‐directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the α‐1 motif, but not by mutations in the α‐2 motif. These results contrast with those for NCX1 where mutations in both α‐1 and α‐2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the α‐1 and α‐2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular‐facing α‐1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both α‐1 and α‐2 motifs together form the binding sites in NCX1.