Spontaneous rhythmic activity in mammalian heart and brain depends on pacemaker currents (I h ), which are produced by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here, we report that the mouse HCN2 pacemaker channel isoform also produced a large instantaneous current (I inst(HCN2) ) in addition to the well characterized, slowly activating I h . I inst(HCN2) was specific to expression of HCN2 on the plasma membrane and its amplitude was correlated with that of I h . The two currents had similar reversal potentials, and both were modulated by changes in intracellular Cl

SummaryIn addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

Background Genome-wide association studies (GWAS) have shown that the common single nucleotide polymorphism (SNP) rs6800541 located in SCN10A, encoding the voltage-gated Nav1.8 sodium channel, is associated with PR–interval prolongation and atrial fibrillation (AF). SNP rs6800541 is in high linkage disequilibrium with the non-synonymous variant in SCN10A, rs6795970 (V1073A, r2=0.933). We therefore sought to determine whether common and rare SCN10A variants are associated with early onset AF. Methods and Results SCN10A was sequenced in 225 AF patients in whom there was no evidence of other cardiovascular disease or dysfunction (lone AF). In an association study of the rs6795970 SNP variant, we included 515 AF patients, and two control cohorts of 730 individuals free of AF and 6,161 randomly sampled individuals. Functional characterization of SCN10A variants was performed by whole-cell patch-clamping. In the lone AF cohort, nine rare missense variants and one splice site donor variant were detected. Interestingly, AF patients were found to have higher G allele frequency of rs6795970 which encodes the alanine variant at position 1073 (described from here on as A1073, odds ratio = 1.35 [1.16–1.54]; p=2.3×10−05). Both of the common variants, A1073 andP1092, induced a gain-of-channel function, while the rare missense variants, V94G and R1588Q, resulted in a loss-of-channel function. Conclusions The common variant A1073 is associated with increased susceptibility to AF. Both rare and common variants have impact on the function of the channel, indicating that these variants influence susceptibility to AF. Hence, our study suggests that SCN10A variations are involved in the genesis of AF.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (I f ) and an instantaneous voltage-independent current (I inst ) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs ؉ like I f and unlike I inst . Together with a concomitant reduction in I f , the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "I f -like," and not an "I inst -like," state. The "I f -like" instantaneous current represented ϳ70% of total I f over voltages ranging from ؉20 to ؊150 mV in high K ؉ solutions. I f activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K ؉ /Na ؉ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing I inst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for I inst . The data are consistent with I inst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits. Hyperpolarization-activated cyclic nucleotide-gated (HCN)1 subunits produce a slowly activating current in response to hyperpolarization known as I f , I h , or I q (1) and are thus involved in regulating membrane potential and spontaneous activity in a variety of excitable cells (2-4). In addition to I f , instantaneous currents were noted or are apparent in experiments describing the expression of wild-type HCN subunits in mammalian cells (5-9) and Xenopus oocytes (10). We recently described a mixed cation instantaneous current, which we refer to as "I inst ," that appeared in addition to I f when the HCN2 isoform was expressed in Chinese hamster ovary (CHO) cells (11). Mutations that reduced or eliminated the trafficking of HCN2 to the plasma membrane also reduced or eliminated I inst associated with HCN2 expression (12, 13). Furthermore, the overexpression of a protein, with a single transmembrane segment and found on the plasma membrane, did not produce I inst . Finally, we found that the amplitudes of I inst and I f were directly correlated. Thus, I inst was not simply the result of overexpression of protein but was correlated specifically with the amount of HCN protein expressed on the plasma membrane.We subsequently examined the role of the selectivity filter and the positively charged S4 region in producing I inst . This current was not affected ...

Background Atrial fibrillation (AF) is the most common arrhythmia, and a recent genome-wide association study identified HCN4 as a novel AF susceptibility locus. HCN4 encodes for the cardiac pacemaker channel and HCN4 mutations are associated with familial sinus bradycardia and AF. Objective To determine whether novel variants in the coding region of HCN4 contribute to the susceptibility for AF. Methods We sequenced the coding region of HCN4 for novel variants from 527 cases with early-onset AF from the Massachusetts General Hospital AF Study and 443 referents from the Framingham Heart Study. We used site-directed mutagenesis, cellular electrophysiology, immunocytochemistry and confocal microscopy to functionally characterize novel variants. Results We found the frequency of novel coding HCN4 variants was 2-fold greater for individuals with AF (seven variants) compared to the referents (three variants). We determined that one, (p.Pro257Ser, located in the amino-terminus adjacent to the first transmembrane spanning domain) of the seven novel HCN4 variants in our AF cases did not traffick to cell membrane while the remaining six were not functionally different from wild type. Also, the three novel variants in our referents did not alter function compared to wild type. Co-expression studies showed that the p.Pro257Ser mutant channel failed to co-localize with the wild type HCN4 channel on the cell membrane. Conclusion Our findings are consistent with HCN4 haploinsufficiency as the likely mechanism for early-onset AF in the p.Pro257Ser carrier.