Abstract-The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system.In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a ␤ subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a ␤ subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;88:e84-e87.)Key Words: HCN family Ⅲ MiRP1 Ⅲ KCNE family Ⅲ ␤ subunit T he HCN (hyperpolarization-activated cyclic nucleotidegated) family of ion channel subunits has been identified as the molecular correlate of the currents I f in heart and I h and I q in neurons. [1][2][3] However, several ion channels are heteromultimers of a large ␣ subunit (like the HCN family members) and smaller ␤ subunits. The cardiac delayed rectifiers I Kr 4 and I Ks 5 are examples of this basic principle. Their ␣ subunits derive from the ERG and KCNQ families, respectively, but both also contain ␤ subunits from the KCNE family of single transmembranespanning proteins called minK and minK-related peptides (MiRPs). In this study, we report that MiRP1 enhances the expression and speeds the kinetics of activation of the HCN family of channel subunits. From immunoprecipitation experiments, we show that it most probably forms a complex with HCN1. Using RNase protection assays (RPAs), we demonstrate that MiRP1 mRNA is prevalent in the primary cardiac pacemaking region, the sinoatrial (SA) node, and barely detectable in ventricle. Cardiac pacemaker activity is generated by a narrow balance of inward (I f ) and outward (I Kr ) currents. Our results demonstrate for the first time the potential importance of a single ␤ subunit in simultaneously regulating both the expression and gating of both inward and outward cardiac pacemaker channels. Materials and Methods Heterologous Expression in Xenopus OocytescRNA encoding mouse HCN1 or HCN2, rat MiRP1 with or without an HA tag at the carboxy-terminal, and rat minK were transcribed using the mMessage mMachine kit (Ambion). Xenopus laevis oocytes were isolated, injected with 2 to 5 ng (50 to 100 nL) of cRNA, and maintained in Barth medium at 18°C for 1 to 3 days. For experiments using both HCN1 or HCN2 and MiRP1 or minK, the respective cRNAs were injected in a 1:0.04 to 1 ratio. Electrophysiological studies on oocytes used the 2-microelectrode voltage clamp. The extracellular recording solution (OR2) contained, in mmol/L, NaCl 80, KCl 2, MgCl 2 1, and Na-HEPES 5 (pH 7.6). Group data are presented as meanϮSEM. Tests of statistical significance for midpoint and slope of activation curves were performed using unpaired Student's t tests. PϽ0.05 is considered significant. RNase Protection AssaysThe procedures for the preparation of total RNA from rabbit he...

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Osmotically driven water flow, u (cm/s), between two solutions of identical osmolarity, c(o) (300 mM: in mammals), has a theoretical isotonic maximum given by u = j/c(o), where j (moles/cm(2)/s) is the rate of salt transport. In many experimental studies, transport was found to be indistinguishable from isotonic. The purpose of this work is to investigate the conditions for u to approach isotonic. A necessary condition is that the membrane salt/water permeability ratio, epsilon, must be small: typical physiological values are epsilon = 10(-3) to 10(-5), so epsilon is generally small but this is not sufficient to guarantee near-isotonic transport. If we consider the simplest model of two series membranes, which secrete a tear or drop of sweat (i.e., there are no externally-imposed boundary conditions on the secretion), diffusion is negligible and the predicted osmolarities are: basal = c(o), intracellular approximately (1 + epsilon)c(o), secretion approximately (1 + 2epsilon)c(o), and u approximately (1 - 2epsilon)j/c(o). Note that this model is also appropriate when the transported solution is experimentally collected. Thus, in the absence of external boundary conditions, transport is experimentally indistinguishable from isotonic. However, if external boundary conditions set salt concentrations to c(o) on both sides of the epithelium, then fluid transport depends on distributed osmotic gradients in lateral spaces. If lateral spaces are too short and wide, diffusion dominates convection, reduces osmotic gradients and fluid flow is significantly less than isotonic. Moreover, because apical and basolateral membrane water fluxes are linked by the intracellular osmolarity, water flow is maximum when the total water permeability of basolateral membranes equals that of apical membranes. In the context of the renal proximal tubule, data suggest it is transporting at near optimal conditions. Nevertheless, typical physiological values suggest the newly filtered fluid is reabsorbed at a rate u approximately 0.86 j/c(o), so a hypertonic solution is being reabsorbed. The osmolarity of the filtrate c(F) (M) will therefore diminish with distance from the site of filtration (the glomerulus) until the solution being transported is isotonic with the filtrate, u = j/c(F).With this steady-state condition, the distributed model becomes approximately equivalent to two membranes in series. The osmolarities are now: c(F) approximately (1 - 2epsilon)j/c(o), intracellular approximately (1 - epsilon)c(o), lateral spaces approximately c(o), and u approximately (1 + 2epsilon)j/c(o). The change in c(F) is predicted to occur with a length constant of about 0.3 cm. Thus, membrane transport tends to adjust transmembrane osmotic gradients toward epsilonc(o), which induces water flow that is isotonic to within order epsilon. These findings provide a plausible hypothesis on how the proximal tubule or other epithelia appear to transport an isotonic solution.

The misorientations of over 200 pairs of adjacent grains separated by grain boundaries in textured YBa2Cu3O7−δ were measured using a transmission electron microscopy technique. The results indicate that there exist discrete preferred rotation angles and rotation axes. The existence of low-energy boundaries is inferred. The results are analyzed based on the Constrained Coincidence Site Lattice (CCSL) and O2-lattice theories and imply the applicability of such theories for the case of large-angle grain boundaries in a complex crystal structure such as YBa2Cu3O7−δ. The results of analysis also show that some boundaries are likely to be reduced in oxygen near the boundary to satisfy the constraint of the coincidence site lattice.