Tissue extract recovers cardiac calcium channels from ‘run-down’

Abstract

Effects of cardiac-tissue extract on the activity of L-type Ca2+ channels were investigated in guinea-pig ventricular myocytes with the patch-clamp method. In most patches, Ca2+-channel current recorded with a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644 ran down within 5 min after excision of the patches into a solution containing EGTA. This run-down of Ca2+ channels was prevented when patches were excised into a solution containing a supernatant fraction of homogenate of guinea-pig or bovine heart. Furthermore, this tissue extract was able to restore channel activity after run-down. This channel-activating effect of the extract was abolished by heat treatment or trypsin digestion. Fractionation of the extract by gel filtration suggested that the channel-activating factor(s) had an apparent molecular weight of 2-3 x 10(5). These results suggest that some cytoplasmic protein(s) maintains the activity of the cardiac L-type Ca2+ channel.

Differential Effects of Endothelin‐1 on Basal and Isoprenaline‐Enhanced Ca2+ Current in Guinea‐Pig Ventricular Myocytes

“…Consistent with the above finding, under perforated patch configuration, isoprenaline (500 nÒ) induced a 3-to 5-fold increase in ICa,L. Under perforated patch configuration, isoprenaline responses to ICa,L are maintained for longer periods of time compared with the conventional whole cell configuration (Kameyama, Kameyama, Nakayama & Kaibara, 1988;Belles, Malecot, Hescheler & Trautwein, 1988). In the present experiments, the response of ICa,L to the â_agonist isoprenaline remained largely unaltered for at least 20 min in control experiments.…”

Calpastatin Domain L Is Involved in the Regulation of L-Type Ca2+ Channels in Guinea Pig Cardiac Myocytes

“…Therefore, CS or its domain L may not be unique player to maintain the channel activity in the intact cell, as suggested previously (3)(4)(5)(6)(7)(8). In our previous reports, Ca 2ϩ channel activity was restored to about 64 -86% of the activity in cell-attached condition by crude cytoplasm or a solution containing ATP, CS, and H fraction, a chromatographically-separated high molecular fraction from bovine heart cytoplasm (8,21).…”

“…Figure 2B shows an example of the effect of domain 1. The chan- 3.3% of that in the cell-attached mode after the patch was excised. When 25 ⌴ domain 1, supplemented with 3 mM ATP, was applied, further run-down was not prevented, unlike full-length CS.…”

“…Recently, it has been suggested that CS also regulates membrane Ca 2ϩ channels (3)(4)(5)(6)(7)(8)(9)(10), based on the following findings: (i) Run-down of L-type channels in guinea pig cardiac myocytes is prevented and/or reversed by cytoplasm extracted from bovine heart (3,5,6,8,9), in which one of the effective components is known to be CS (3,6,8,9); (ii) CS, in the presence of ATP, partially reversed Ca 2ϩ channel run-down (7,8); (iii) The effect of CS was not mimicked by synthetic calpain inhibitors (10). However, the molecular basis of the new function of CS has not been determined.…”

Calmodulin Kinase II Activation Is Required for the Maintenance of Basal Activity of L-Type Ca2+ Channels in Guinea-Pig Ventricular Myocytes

“…Recent work also shows that phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can stabilize the activity of P / Q-type Ca 2+ channels (5,6). Alternatively, we have reported that a cytoplasmic extract from cardiac tissue recovers the Ca 2+ channel activity from run-down (3,7,8), suggesting that run-down of the Ca 2+ channel activity may be due to the washout of certain cytoplasmic factors. Subsequently the cytoplasmic factors were identified as calpastatin, ATP, and a yet unidentified factor in a high molecular mass fraction (H fraction) obtained from gel filtration of the cardiac tissue (3,9,10).…”