Brain Research, 563 (1991) 321-324 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391249070 BRES 24907 321 Novel inward rectifier blocked by Cd ~+ in crayfish muscle Alfonso Araque and Washington Bufio Neurofisiologla, Instituto Cajal, CSIC, Madrid (Spain) (Accepted 30 July 1991) Key words: Inward rectifier; Anomalous rectification; Crayfish muscle; Cd2+-blockade; K+ current; Hyperpolarization-activated current The characteristics of a voltage- and time-dependent inward rectifying current were examined with voltage clamp techniques in crayfish muscle. The inward current, carried by K+, was activated by hyperpolarization. Although this inward current increased with the extracellular K÷ concentration ([K+]o), the voltage-dependence of the underlying conductance was independent of [K+]o. The current was unaffected by Cs+ and Ba 2+, but was blocked by low concentrations of Cd 2+. Therefore, this inward rectifier is different than previously described ones. A lower membrane resistance for inward than for out- ward current was first described in frog skeletal muscle by Katz 15. This phenomenon, called inward or anoma- lous rectification, accounts for the instantaneous increase of the membrane conductance with hyperpolarization. The underlying conductance is specific for K + and its voltage-dependence varies as a function of membrane potential (Vm) and extracellular K + concentration ([K+]o) 6'16. Similar rectification, which could also show a delayed component, was later found in cardiac mus- cle 9, metacerebral giant cells 13, marine eggs 6, tunicate embryos 19 and different vertebrate neurones 3'14'22'26. More recently, another type of inward rectification car- ded by Na + and K + was found in cardiac muscle (called I h (Ref. 27) or If (Ref. 1)) and hippocampal neurones (termed Iq (Ref. 10)). It has no instantanous voltage-de- pendent component, shows slower activation kinetics than the former and its activation curve is independent of [K÷]o . Finally, an inward rectifier mediated by Cl- has been reported in Aplysia neurones 2, amphibian oocytes 21 and mammalian hippocampal neurones 17. Also, a de- layed decrease in input resistance elicited by hyperpo- larizing pulses was described in crayfish muscle 5, and in- terpreted to be due to Cl- activation 2°. In the present study, inward rectification in crayfish (Procambarus clarkii) muscle fibres was examined with two electrode voltage clamp techniques. Opener muscles from the first walking leg were isolated and transferred to a 2 ml chamber. Small crayfish (<5 cm) with short muscle fibres (<400 /tm) were used for better space clamp characteristics. Composition of the modified Van Harreveld solution 25 was (in mM): NaC1 210, KC1 5.4, CaC12 13.5, MgCI 2 2.6, and Tris buffer 10, adjusted with HC1 to pH 7.2. Different concentrations of chloride salts of monovalent (Cs +) and divalent (Ba 2+, Mg 2+ and Cd 2+) cations were added in equimolar exchange with NaCl and CaCl 2, respectively. In experiments with in- creased [K+]o, NaC1 was equimolarly replaced by KCI; experiments in low [K+]o did not provide accurate re- sults, probably because the muscle membrane is too complex to allow a good control of low [K÷]o, and were excluded. Solutions were continuously superfused at room temperature (20-22 °C). Twenty-seven fibres were impaled with two 1 M KC1 pipettes (1-5 Mf~) and volt- age clamped with an Axoclamp-2A amplifier and a x 10 current probe. The time required to reach the command pulse potential was ~<0.5 ms and there were no voltage variations during the pulse; recordings which did not meet these criteria were discarded. In several experi- ments, the current electrode (filled with 1 M KC1) was substituted, after control recording, by a new electrode filled with 700 mM EGTA (ethyleneglycol-bis-(fl-amino- ethyl ether)N,N,N',N'-tetraacetic acid) neutralized with KOH. EGTA was ionophoretically injected with 100 nA negative current pulses during 15 min. The mean potentials of the 27 fibres was -65.5 mV (S.D. -+ 5.8) and they were clamped at -60 mV. This holding potential (Vh) was selected to minimize steady state currents and to avoid possible interference of the depolarizing-activated currents which could not be to- tally blocked without modifying the inward rectification under analysis. Hyperpolarizing pulses evoked an instan- taneous inward current followed by a gradually develop- ing inward current. Both components of the total cur- Correspondence: W. Bufio, Neurofisiologia, Instituto Cajal, Avenida Doctor Arce 37, E-28002 Madrid, Spain. Fax: (34) (1) 585 41 54.