RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression. We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus. Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization. Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter. Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity. Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry. Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm. Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF. It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

I f , encoded by the hyperpolarization-activated cyclic nucleotide-modulated channel family (HCN1-4), contributes significantly to neuronal and cardiac pacing. Recently, we reported that the S3-S4 residue Glu-235 of HCN1 influences activation by acting as a surface charge. However, it is uncertain whether other residues of the external S3-S4 linker are also involved in gating. Furthermore, the secondary conformation of the linker is not known. Here we probed the structural and functional role of the HCN1 S3-S4 linker by introducing systematic mutations into the entire linker (defined as 229 -237) and studying their effects. We found that the mutations K230A (؊62.2 ؎ 3.4 mV versus ؊72.2 ؎ 1.7 mV of wild type (WT)), G231A (؊64.4 ؎ 1.3 mV), M232A (V1 ⁄2 ‫؍‬ ؊63.1 ؎ 1.1 mV), and E235G (؊65.4 ؎ 1.5 mV) produced depolarizing activation shifts. Although E229A and M232A decelerated gating kinetics (<13-and 3-fold, respectively), K230A and G231A accelerated both activation and deactivation (<ϳ2-3-fold). D233A, S234A, V236A, and Y237A channels exhibited WT properties (p > 0.05). Shortening the linker (EVY235-237⌬⌬⌬) caused depolarizing activation shift and slowed kinetics that could not be explained by removing the charge at position 235 alone. Secondary structural predictions by the modeling algorithms SSpro2 and PROF, along with refinements by our experimental data, suggest that part of the S3-S4 linker conforms a helical structure with the functionally important residues Met-232, Glu-235, and Gly-231 (ͦ⌬⌬Gͦ>1 kcal/mol) clustered on one side.I f or I h , commonly referred to as pacemaker current, is encoded by the hyperpolarization-activated cyclic nucleotidemodulated (HCN) 1 channel gene family (1-9). I f plays an important role in the pacing activity of cardiac and neuronal cells by modulating their rhythmic oscillation frequency via the regulation of the rate of cellular depolarization at the end of each excitation cycle. To date, four isoforms, namely HCN1-4, whose tissue distribution, gene expression, and functional properties differ significantly from each other, have been identified and described (8 -13). Different HCN isoforms can heteromerize (14 -16) in vivo, greatly increasing the molecular diversity of the native currents in different tissues.Although HCN-encoded pacemaker channels are functionally distinctive from voltage-gated K ϩ (Kv) channels in their unique opening upon hyperpolarization rather than depolarization, the two classes of channel proteins are structurally homologous to each other. For instance, both HCN and Kv channels are tetrameric with fundamental building blocks consisting of six transmembrane segments, namely S1-S6 (7-9, 16). By analogy, it is possible that regions of HCN channels that are homologous to those critical for Kv gating also contribute to their own gating. Indeed, not only is the S4 voltagesensing domain of HCN channels a prominent player of their "backward" gating behavior (17-19), the HCN pore is also coupled to gating in a manner similar to that of Kv channels (20, 21). H...

I f or I h , a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229 EKGMDSEVY 237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K ؉ channels, complete deletion of the S3-S4 linker (⌬229 -237) did not produce functional channels. Similarly, the deletions ⌬229 -234, ⌬232-234, and ⌬232-237 also abolished normal current activity. Interestingly, ⌬229 -231, ⌬233-237, ⌬234 -237, ⌬235-237, ⌬229 -231/⌬233-237, ⌬229 -231/⌬234 -237, and ⌬229 -231/⌬235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met 232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. ⌬V1 ⁄2 of ⌬229-231, ⌬233-237, ⌬235-237 >؉10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (؊35 mV < ⌬V1 ⁄2 < ؊4 mV). Based on these results, we conclude that only Met 232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K ؉ channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene-and cell-based therapies.I f or I h , whose encoding genes are collectively known as the hyperpolarization-activated cyclic nucleotide-modulated (HCN) 1 or the so-called pacemaker channel family, plays an important role in the spontaneous rhythmic activity in cardiac, neuronal, and insulin-secreting cells (1-7). Although classical depolarization-activated voltage-gated K ϩ (K v ) and HCN channels are structurally homologous to each other, the latter are uniquely distinctive from the K v counterparts by their signature "backward" gating (i.e. activation upon hyperpolarization rather than depolarization). The basis of HCN gating is largely unknown. Structural and functional comparison of HCN channels with the much better studied K v channels to reveal their evolutionary similarities and differences should shed invaluable insights into the basic biology of HCN channels.Recent evidence suggested that the voltage-sensing mechanisms of HCN and K v channels are conserved despite their opposite gating behaviors (i.e. the HCN S4 also moves outwar...

No abstract