Vicia faba TPC1, a genetically encoded variant of the vacuole Two Pore Channel 1, is hyperexcitable

To fire action-potential-like electrical signals, the vacuole membrane requires the depolarization-activated two-pore channel TPC1, also called Slowly activating Vacuolar SV channel. The TPC1/SV channel, encoded by the TPC1 gene, functions as a voltage-dependent and Ca2+-regulated potassium channel. TPC1 currents are activated by a rise in cytoplasmic Ca2+ but blocked by luminal Ca2+. In search for species-dependent functional TPC1 channel variants, we studied polymorphic amino acids contributing to luminal Ca2+ sensitivity. We found that the acidic residues Glu457, Glu605 and Asp606 of the Ca2+-sensitive Arabidopsis AtTPC1 channel were neutralized by either asparagine or alanine in Vicia faba and many other Fabaceae as well. When expressed in the Arabidopsis loss-of-AtTPC1 function background, the wild type VfTPC1 was hypersensitive to vacuole depolarization and insensitive to blocking luminal Ca2+. When AtTPC1 was mutated for the three VfTPC1-homologous polymorphic site residues, the Arabidopsis At-VfTPC1 channel mutant gained VfTPC1-like voltage and luminal Ca2+ insensitivity that together made vacuoles hyperexcitable. These findings indicate that natural TPC1 channel variants in plant families exist which differ in vacuole excitability and very likely respond to changes in environmental settings of their ecological niche.

Abstract 247: Dose-dependent Role of Inwardly Rectifying Potassium Current on Cardiac Automaticity of Human Embryonic Stem Cell-derived Cardiomyocytes

The inwardly rectifying potassium current (IK1), encode by Kir2 family, is responsible for maintaining the negative resting potential, and contributes to phase 3 repolarization of the cardiac action potential. IK1 was generally thought to suppress cardiac automaticity, while the suppression of IK1 in adult ventricular cardiomyocytes (CMs) could engineer bio-artificial pacemaker-like cells to spontaneously fire action potential. Our studies also showed that overexpressed the gene of Kir2.1 could facilitate the electrophysiological maturing of mouse and human embryonic stem cell-differentiated CMs (ESC-CMs), which have the high degree of automaticity with nearly 50% of cells that can spontaneously fire action potential. In this study, we extensively analyzed the electrophysiology of mouse and human ESC-CMs, and found that the maximum diastolic potential in spontaneously firing ESC-CMs, -72.1±1.3 mV in atrial cells and -75.0±2.1 mV in ventricular cells, were significantly more hyperpolarized than that in quiescent ESC-CMs (-64.4±2.1 mV in atrial cells and -67.1±3.2 mV in ventricular cells). Applying a small amount of IK1 to hyperpolarize the membrane potential could enable those quiescent ESC-CMs to spontaneously fire action potential, indicating the enhancement of cardiac automaticity, while a large amount of IK1 could quiet those spontaneously firing cells down. By combining computational and experimental analyses, we confirmed that the synergistic interaction of IK1 and pacemaker current (If) could efficiently regulate cardiac automaticity during the differentiation. Our studies disclosed a dose-dependent role of IK1 on cardiac automaticity that a small amount of IK1 enhances and a large amount of IK1 suppresses cardiac automaticity in ESC-CMs during differentiation.

Muscarinic Receptor Activation Induces Depolarizing Plateau Potentials in Bursting Neurons of the Rat Subiculum

Acetylcholine functions as a neuromodulator in the mammalian brain by binding to specific receptors and thus bringing about profound changes in neuronal excitability. Activation of muscarinic receptors often results in an increased excitability of cortical cells. It is, however, unknown whether such an action is present in the subiculum, a limbic structure that may be involved in cognitive processes as well as in seizure propagation. Most rat subicular neurons are endowed of intrinsic membrane properties that make them fire action potential bursts. Using intracellular recordings from these bursting cells in a slice preparation, we report here that application of the cholinergic agonist carbachol (CCh, 30–100 μM) to medium containing ionotropic excitatory amino acid receptor antagonists reduces burst-afterhyperpolarizations (burst-AHPs) and discloses depolarizing plateau potentials that outlast the triggering current pulses by 140–2,800 ms. These plateau potentials appear with CCh concentrations >50 μM and are dependent on the resting membrane potential and on the intensity/duration of the triggering pulse; are recorded during application of tetrodotoxin (1 μM, n = 5 neurons); but are markedly reduced by replacing 82% of extracellular Na+with equimolar choline ( n = 6). Plateau potentials also are abolished by Co2+ (2 mM; n = 5) or Cd2+ (1 mM; n = 2) application and by recording with electrodes containing the Ca2+chelator bis(2-aminophenoxy)ethane- N, N,N′,N′-tetraacetic acid (0.2 M; n = 6). CCh-induced burst-AHP reduction and plateau potentials are reversed by the muscarinic antagonist atropine (0.5 μM, n = 7). In conclusion, our findings demonstrate a powerful muscarinic modulation of the intrinsic excitability of subicular bursting cells that is predominated by the appearance of plateau potentials. These changes in excitability may contribute to physiological processes such as learning or memory and play a role in the generation of epileptiform depolarizations. We propose that, as in other limbic structures, muscarinic plateau potentials in the subiculum are mainly due to a Ca2+-dependent nonselective cationic conductance.