Changes in internal ionized Ca2+ and H+ in voltage clamped squid axons.
Squid giant axons were injected with aequorin and tetraethylammonium and were impaled with hydrogen ion sensitive, current and voltage electrodes. A newly designed horizontal microinjector was used to introduce the aequorin. It also served, simultaneously, as the current and voltage electrode for voltage clamping and as the reference for ion-sensitive microelectrode measurements. The axons were usually bathed in a solution containing 150 mM each of Na+, K+, and some inert cation, at either physiological or zero bath Ca2+ concentration [( Ca2+]o), and had ionic currents pharmacologically blocked. Voltage clamp pulses were repeatedly delivered to the extent necessary to induce a change in the aequorin light emission, a measure of axoplasmic ionized Ca2+ level, [( Ca2+]i). Alternatively, membrane potential was steadily held at values that represented deviations from the resting membrane potential observed at 150 mM [K+]o (i.e. approximately -15 mV). In the absence of [Ca2+]o a significant steady depolarization brought about by current flow increased [Ca2+]i (and acidified the axoplasm). Changes in internal hydrogen activity, [H+]i, induced by current flow from the internal Pt wire limited the extent to which valid measurements of [Ca2+]i could be made. However, there are effects on [Ca2+]i that can be ascribed to membrane potential. Thus, in the absence of [Ca2+]o, hyperpolarization can reduce [Ca2+]i, implying that a Ca2+ efflux mechanism is enhanced. It is also observed that [Ca2+]i is increased by depolarization. These results are consistent with the operation of an electrogenic mechanism that exchanges Na+ for Ca2+ in squid giant axon.
Department of Biophysics, University of Maryland School of Medicine, Baltimore.
L J
LJ
Mullins
J
J
Whittembury
J
J
Requena
D000331:Aequorin / Q000494:pharmacology; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D049832:Decapodiformes; D004553:Electric Conductivity; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000494:pharmacology
D013757:Tetraethylammonium Compounds; D000331:Aequorin; D019789:Tetraethylammonium; D012964:Sodium; D011188:Potassium; D002118:Calcium