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Results for “HRK1”

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PubMed1994

Cloning and expression of a novel human brain inward rectifier potassium channel.

A complementary DNA encoding an inward rectifier K+ channel (HRK1) was isolated from human hippocampus using a 392-base pair cDNA (HHCMD37) as a probe. HRK1 shows sequence similarity to three recently cloned inwardly rectifying potassium channels (IRK1, GIRK1, and ROMK1, 60, 42, and 37%, respectively) and has a similar proposed topology of two membrane spanning domains that correspond to the inner core structure of voltage gated K+ channels. When HRK1 was expressed in Xenopus oocytes, large inward K+ currents were observed below the K+ reversal potential but very little outward K+ current was observed. In on-cell membrane patches, single channel conductance (g) was estimated to be 10 picosiemens by both direct measurement and noise analysis, in 102 mM external [K+]. HRK1 currents were blocked by external Ba2+ and Cs+ (K(0) = 183 microM, and K(-130) = 30 microM, respectively), and internal tetraethylammonium ion (K(0) = 62 microM), but were insensitive to external tetraethylammonium ion. The functional properties of HRK1 are very similar to those of glial cell inward rectifier K+ channels and HRK1 may represent a glial cell inward rectifier.

Amino Acid Sequence↗

PubMed1995

The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines.

The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time-dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.

Animals↗

PubMed2026

Acetic acid-induced translational repression involves eIF2B body formation and Ded1 sequestration into stress granules in yeast.

Elucidating the physiological impact of acetic acid stress and the corresponding yeast responses is essential for advancing fundamental biology and improving industrial alcoholic fermentation. Despite numerous genome-wide studies, information on the effects of acetic acid stress on yeast translational regulation remains limited. We found that a sublethal concentration of acetic acid (35 mM, 0.2% v/v) causes translational repression, accompanied by the formation of eIF2B bodies and the phosphorylation of eIF2α, both of which are involved in the regulation of translation initiation. Acetic acid also caused the sequestration of Ded1, a DEAD-box RNA helicase crucial for translation initiation, into stress granules. Removal of acetic acid restored translational activity and the proper localization of eIF2B and Ded1, indicating the reversibility of acetic acid-induced translational repression. Furthermore, when yeast cells were pretreated with 0.05% acetic acid, translational repression under subsequent 0.2% acetic acid stress was attenuated in wild-type cells but not in hrk1Δ cells. This indicates that Hrk1, a Pma1 activator, is required to sufficiently enhance tolerance to acetic acid-induced translational repression. These findings provide novel insights into the physiological effects of acetic acid stress on translational activity and translation-related factors in yeast cells.

Saccharomyces cerevisiae↗

PubMed1996

Spermidine release from xenopus oocytes. Electrodiffusion through a membrane channel.

The mechanism of spermidine release from Xenopus oocytes was examined by measuring release of radioactive [3H]spermidine under different ionic conditions, and under voltage-clamp. In normal solution (2 mM K+), the efflux rate is less than 1% per hour, and is stimulated approximately 2-fold by inclusion of Ca2+ (1 mm) in the incubation medium. Spermidine efflux is stimulated approximately 10-fold in high [K+] (KD98) solution. In KD98 solution, efflux is strongly inhibited by divalent cations (Ki for Ba2+ block of spermidine efflux is approximately 0.1 mM), but not by tetraethylammonium ions or verapamil. Spermidine efflux rates were not different between control oocytes and those expressing HRK1 inward rectifier K+ (Kir) channels. When the membrane potential was clamped, either by changing external [K+] in oocytes expressing HRK1, or by 2-microelectrode voltage-clamp, spermidine efflux was shown to be strongly dependent on voltage, as expected for a simple electrodiffusive process, where spermidine3+ is the effluxing species. This result argues against spermidine diffusing out as an uncharged species, or in exchange for similarly charged counterions. These results are the first conclusive demonstration of a simple electrodiffusive pathway for spermidine efflux from cells.

Animals↗

PubMed2000

Regulation of yeast H(+)-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters.

The regulation of electrical membrane potential is a fundamental property of living cells. This biophysical parameter determines nutrient uptake, intracellular potassium and turgor, uptake of toxic cations, and stress responses. In fungi and plants, an important determinant of membrane potential is the electrogenic proton-pumping ATPase, but the systems that modulate its activity remain largely unknown. We have characterized two genes from Saccharomyces cerevisiae, PTK2 and HRK1 (YOR267c), that encode protein kinases implicated in activation of the yeast plasma membrane H(+)-ATPase (Pma1) in response to glucose metabolism. These kinases mediate, directly or indirectly, an increase in affinity of Pma1 for ATP, which probably involves Ser-899 phosphorylation. Ptk2 has the strongest effect on Pma1, and ptk2 mutants exhibit a pleiotropic phenotype of tolerance to toxic cations, including sodium, lithium, manganese, tetramethylammonium, hygromycin B, and norspermidine. A plausible interpretation is that ptk2 mutants have a decreased membrane potential and that diverse cation transporters are voltage dependent. Accordingly, ptk2 mutants exhibited reduced uptake of lithium and methylammonium. Ptk2 and Hrk1 belong to a subgroup of yeast protein kinases dedicated to the regulation of plasma membrane transporters, which include Npr1 (regulator of Gap1 and Tat2 amino acid transporters) and Hal4 and Hal5 (regulators of Trk1 and Trk2 potassium transporters).

Adenosine Triphosphate↗