Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: activation by photolysis of Ca2+.
The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mM NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mM NaCl in the external medium with 160 mM KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DM-Nitrophen-Ca2+ ("caged Ca2+") was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations > 25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with half-maximal activation (KM(Ca)) at < 10 microM Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysis-evoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Ca-dependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 microM DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electro-physiological methods.
Eisenrauch
A
A
Laboratory of Biophysical Chemistry, Max-Planck-Institute für Biophysik, Frankfurt/M, Germany.
Juhaszova
M
M
Ellis-Davies
G C
GC
Kaplan
J H
JH
Bamberg
E
E
Blaustein
M P
MP
D000085:Acetates / Q000528:radiation effects*; D000327:Adsorption; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000502:physiology*; D002413:Cations, Divalent / Q000494:pharmacology; D005029:Ethylenediamines / Q000528:radiation effects*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D008051:Lipid Bilayers; D008564:Membrane Potentials / Q000528:radiation effects; D009124:Muscle Proteins / Q000302:isolation & purification / Q000502:physiology*; D008121:Nephropidae / Q000378:metabolism*; D010713:Phosphatidylcholines; D010743:Phospholipids; D010782:Photolysis; D011189:Potassium Chloride / Q000378:metabolism; D011510:Proteolipids; D012965:Sodium Chloride / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D014466:Ultraviolet Rays
D000085:Acetates; D002352:Carrier Proteins; D002413:Cations, Divalent; D005029:Ethylenediamines; D008051:Lipid Bilayers; D009124:Muscle Proteins; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; D019831:Sodium-Calcium Exchanger; C020485:proteoliposomes; C057014:DM-nitrophen; D012965:Sodium Chloride; D011189:Potassium Chloride; C024990:asolectin; D007811:Lanthanum; D002118:Calcium