Biomedical subjects

Eric A Accili

Publications and source records attributed to Eric A Accili.

6 recordsLinked to original sources

PubMed2007

Evolution and structural diversification of hyperpolarization-activated cyclic nucleotide-gated channel genes.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated channel superfamily and play a critical role in cellular pace-making. Overall sequence conservation is high throughout the family, and channel functions are similar but not identical. Phylogenetic analyses are imperative to understand how these genes have evolved and to make informed comparisons of HCN structure and function. These have been previously limited, however, by the small number of available sequences, from a minimal number of species unevenly distributed over evolutionary time. We have now identified and annotated 31 novel genes from invertebrates, urochordates, fish, amphibians, birds, and mammals. With increased sequence numbers and a broader species representation, a more precise sequence comparison was performed and an evolutionary history for these genes was constructed. Our data confirm the existence of at least four vertebrate paralogs and suggest that these arose via three duplication and diversification events from a single ancestral gene. Additional lineage-specific duplications appear to have occurred in urochordate and fish genomes. Based on exon boundary conservation and phylogenetic analyses, we hypothesize that mammalian gene structure was established, and duplication events occurred, after the divergence of urochordates and before the divergence of fish from the tetrapod lineage. In addition, we identified highly conserved sequence regions that are likely important for general HCN functions, as well as regions with differences conserved among each of the individual paralogs. The latter may underlie more subtle isoform-specific properties that are otherwise masked by the high identity among mammalian orthologs and/or inaccurate alignments between paralogs.

Amino Acid Sequence↗

PubMed2004

Structural elements of instantaneous and slow gating in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (If) and an instantaneous voltage-independent current (Iinst) 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 If and unlike Iinst. Together with a concomitant reduction in If, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "If-like," and not an "Iinst-like," state. The "If-like" instantaneous current represented approximately 70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If 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 Iinst 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 Iinst. The data are consistent with Iinst 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.

Amino Acid Sequence↗

PubMed2002

A conserved domain in the NH2 terminus important for assembly and functional expression of pacemaker channels.

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.

Amino Acid Sequence↗

PubMed2002

Separable gating mechanisms in a Mammalian pacemaker channel.

Despite permeability to both K(+) and Na(+), hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K(+) channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (I(f)) but, surprisingly, did not affect the instantaneous current (I(inst)). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of I(f) deactivation and I(f) amplitude had a parallel dependence on the ratio of K(+)/Na(+) driving forces. In addition, the amplitude of fully activated I(f), unlike I(inst), was not well predicted by equal and independent flow of K(+) and Na(+). The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (I(f)) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (I(inst)) that is insensitive to these influences.

Amino Acid Sequence↗

PubMed2002

Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels.

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.

Animals↗

PubMed2001

Pacemaker channels produce an instantaneous current.

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(-) and cAMP. A mutation in the S4 domain of HCN2 (S306Q) decreased I(h) but did not alter I(inst(HCN2)), and instantaneous currents in cells expressing either wild type HCN2 or mutant S306Q channels were insensitive to block by Cs(+). Co-expression of HCN2 with the accessory subunit, MiRP1, decreased I(h) and increased I(inst(HCN2)), suggesting a mechanism for modulation of both currents in vivo. These data suggest that expression of HCN channels may be accompanied by a background conductance in native tissues and are consistent with at least two open states of HCN channels: I(inst(HCN2)) is produced by a Cs(+)-open state; hyperpolarization produces an additional Cs(+)-sensitive open state, which results in I(h).

Animals↗