A cytoplasmic factor, calpastatin and ATP together reverse run‐down of Ca²⁺ channel activity in guinea‐pig heart

Abstract

The cytoplasmic extract of bovine heart was separated into four fractions by gel filtration: H (molecular mass > 300 kDa), P (250‐300 kDa), L1 (180‐250 kDa) and L2 (< 180 kDa). The effects of these fractions on the run‐down of L‐type Ca2+ channel activity were investigated in guinea‐pig ventricular myocytes. After run‐down induced by inside‐out patch formation, Ca2+ channel activity was restored by P or H (+ 3 mM ATP) to 7·5 and 5·8 % of that in the cell‐attached mode, respectively, but to as high as 86 % by P + H + ATP. The reversal of run‐down brought about by the P fraction was mimicked by calpastatin. The restorative effect of calpastatin + ATP showed a biphasic time course: 38 % in the early transient phase and 11 % in the late phase. However, calpastatin + H + ATP showed a sustained effect: 66 % in the early transient phase, and 87 % in the late phase. The effective component of the H fraction showed a protein‐like nature: heat and trypsin sensitivity. The activities of cAMP‐dependent protein kinase, casein kinase I, casein kinase II, protein tyrosine kinase, protein serine/threonine or tyrosine phosphatases were measured. However, these kinases and phosphatases were not confirmed as the effective component of cytoplasm or the H fraction. Run‐down was not prevented by 2 μM phalloidin or 2 μM paclitexal, suggesting that neither actin filaments nor microtubules are directly involved in the run‐down. Our results support the view that the basal activity of the Ca2+ channel is maintained by at least three factors: a protein‐like factor in the H fraction, calpastatin, and ATP.

Calmodulin- and Ca2+-Dependent Facilitation and Inactivation of the CaV1.2 Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…Adult guinea-pig ventricular myocytes were dispersed by collagenase and protease dissociation as described previously (27). Briefly, a female guinea pig (weight, 300 -500 g) was anesthetized with pentobarbital sodium (30 mg/kg, i.p.…”

The Distinct Roles of Calmodulin and Calmodulin Kinase II in the Reversal of Run-Down of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…This phenomenon is referred to as rundown and can be considered as a failure of the mechanism by which the basal activity of the channel is maintained (1,2). So far, several mechanisms have been proposed for the run-down of Ca 2+ channels, including dephosphorylation of protein kinase A (PKA)-mediated phosphorylation (3) and proteolysis induced by calpain, a Ca…”

The Ca2+‐dependent interaction of calpastatin domain L with the C‐terminal tail of the Cav1.2 channel

“…A previous report suggested that CS-induced recovery of Ca 2+ channel activity after run-down is related to the C-terminal region of the Cav1.2 channel, which includes the PreIQ and IQ motifs [2][3][4]14]. Therefore, binding of CS L with IQ and PreIQ may be one of the mechanisms required to maintain the basal activity of the Ca 2+ channel.…”

“…Wash-out of regulatory factors has been suggested as the primary mechanism responsible for the run-down of the L-type Ca 2+ channel [2][3][4]. A previous report suggested that CS-induced recovery of Ca 2+ channel activity after run-down is related to the C-terminal region of the Cav1.2 channel, which includes the PreIQ and IQ motifs [2][3][4]14].…”

“…Recently, it has been suggested that CS L and its fragmental peptides can recover the activity of Cav1.2 in guinea pig cardiac myocytes dose-dependently after run-down in the inside-out configuration of the patch-clamp technique [3][4][5].…”