Proceedings of The Physiological Society
Kings College London (2005) J Physiol 565P, C171
Na/K ATPase pump current is increased in ventricular myocytes isolated from phospholemman knockout mice
Berry, Roger G; Fuller, Will ; Tucker, Amy L; Shattock, Micheal J;
1. Cardiovascular Division, King's College London, London, United Kingdom. 2. University of Virginia, Charlottesville, VA, USA.
Phospholemman (PLM) is a member of the FXYD family of small single transmembrane domain proteins that have been shown to be tissue-specific regulators of Na/K ATPase. We have previously shown that PLM forms an integral part of the cardiac Na/K ATPase complex (Fuller et al 2004) and may provide the link between adrenergic stimulation and pump activation (Silverman et al 2004). We have therefore investigated the effects of PLM knockout on Na/K pump function in myocytes isolated from PLM knockout (-/-) (KO) and wildtype (+/+) (WT) mice. Mice were anaesthetised using pentabarbitone (60mg/kg ip), hearts excised and ventricular myocytes isolated using standard enzymatic digestion techniques. Whole-cell Na+/K+ ATPase pump current (Ip) was recorded using the perforated patch technique during the descending phase of a voltage ramp (+40 to -80mV) and was defined as that sensitive to extracellular K+ ([K+]o). Pipette and extracellular solutions were designed to prevent activation of other endogenous currents. In 5mM [K+]o, Ip at 0mV was increased from 2.90±0.19pA/pF (n=19 from 8 animals: mean±sem) to 3.91±0.14pA/pF (n=15 from 5 animals) in myocytes isolated from WT and KO animals respectively (P〈0.05 Student's t test). Normalised I-V relationships from KO and WT myocytes were identical indicating the voltage-dependence of Ip was unaffected by PLM deletion. The relationship between [K+]o (1-20mM) and Ip at 0mV was investigated in KO and WT myocytes. In both groups, mean data were best fitted by a single exponential association. Maximally activated Ip (Vmax), estimated from each curve fit, was increased by 28.3% in PLM KO cf WT controls. The absence of PLM had no effect on the K+ affinity of the pump at 0mV. The respective K1/2s for K+ activation, measured at 0mV, were 1.31 and 1.28mM in KO and WT myocytes. However, K+ affinity was decreased at more negative potentials in KO animals (for example, at -60mV K1/2 = 1.43 and 1.06mM in KO and WT respectively). In conclusion, PLM appears to modulate cardiac Na/K ATPase by applying a tonic inhibition that, when relieved by PLM knockout, results in an increase in Vmax without a change in K+ sensitivity assessed at 0mV. At more negative potentials, PLM deletion slightly decreases K-sensitivity. This effect may, however, have little physiological significance as at normal [K+]o (ie ~5mM) Ip is saturated at 93.3% and 92.9% of maximum current in WT and KO myocytes respectively.
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