Abnormal cardiac Na(+) channel properties and QT heart rate adaptation in neonatal ankyrin(B) knockout mice.

The cytoskeleton of the cardiomyocyte has been shown to modulate ion channel function. Cytoskeletal disruption in vitro alters Na(+) channel kinetics, producing a late Na(+) current that can prolong repolarization. This study describes the properties of the cardiac Na(+) channel and cardiac repolarization in neonatal mice lacking ankyrin(B), a cytoskeletal "adaptor" protein. Using whole-cell voltage clamp techniques, I(Na) density was lower in ankyrin(B)(-/-) ventricular myocytes than in wild-type (WT) myocytes (-307+/-26 versus -444+/-39 pA/pF, P<0.01). Ankyrin(B)(-/-) myocytes exhibited a hyperpolarizing shift in activation and inactivation kinetics compared with WT. Slower recovery from inactivation contributed to the negative shift in steady-state inactivation in ankyrin(B)(-/-). Single Na(+) channel mean open time was longer in ankyrin(B)(-/-) versus WT at test potentials (V(t)) of -40 mV (1.0+/-0.1 versus 0. 61+/-0.04 ms, P<0.05) and -50 mV (0.8+/-0.1 versus 0.39+/-0.05 ms, P<0.05). Ankyrin(B)(-/-) exhibited late single-channel openings at V(t) -40 and -50 mV, which were not seen in WT. Late I(Na) contributed to longer action potential durations measured at 90% repolarization (APD(90)) at 1 Hz stimulation in ankyrin(B)(-/-) compared with WT (354+/-26 versus 274+/-22 ms, P<0.05). From ECG recordings of neonatal mice, heart rates were slower in ankyrin(B)(-/-) than in WT (380+/-14 versus 434+/-13 bpm, P<0.01). Although the QT interval was similar in ankyrin(B)(-/-) and WT at physiological heart rates, QT-interval prolongation in response to heart rate deceleration was greater in ankyrin(B)(-/-). In conclusion, Na(+) channels in ankyrin(B)(-/-) display reduced I(Na) density and abnormal kinetics at the whole-cell and single-channel level that contribute to prolonged APD(90) and abnormal QT-rate adaptation.

Ankyrin-B is required for intracellular sorting of structurally diverse Ca2+ homeostasis proteins.

This report describes a congenital myopathy and major loss of thymic lymphocytes in ankyrin-B (-/-) mice as well as dramatic alterations in intracellular localization of key components of the Ca(2+) homeostasis machinery in ankyrin-B (-/-) striated muscle and thymus. The sarcoplasmic reticulum (SR) and SR/T-tubule junctions are apparently preserved in a normal distribution in ankyrin-B (-/-) skeletal muscle based on electron microscopy and the presence of a normal pattern of triadin and dihydropyridine receptor. Therefore, the abnormal localization of SR/ER Ca ATPase (SERCA) and ryanodine receptors represents a defect in intracellular sorting of these proteins in skeletal muscle. Extrapolation of these observations suggests defective targeting as the basis for abnormal localization of ryanodine receptors, IP3 receptors and SERCA in heart, and of IP3 receptors in the thymus of ankyrin-B (-/-) mice. Mis-sorting of SERCA 2 and ryanodine receptor 2 in ankyrin-B (-/-) cardiomyocytes is rescued by expression of 220-kD ankyrin-B, demonstrating that lack of the 220-kD ankyrin-B polypeptide is the primary defect in these cells. Ankyrin-B is associated with intracellular vesicles, but is not colocalized with the bulk of SERCA 1 or ryanodine receptor type 1 in skeletal muscle. These data provide the first evidence of a physiological requirement for ankyrin-B in intracellular targeting of the calcium homeostasis machinery of striated muscle and immune system, and moreover, support a catalytic role that does not involve permanent stoichiometric complexes between ankyrin-B and targeted proteins. Ankyrin-B is a member of a family of adapter proteins implicated in restriction of diverse proteins to specialized plasma membrane domains. Similar mechanisms involving ankyrins may be essential for segregation of functionally defined proteins within specialized regions of the plasma membrane and within the Ca(2+) homeostasis compartment of the ER.