Effect of transjunctional KCl gradients on the spermine inhibition of connexin40 gap junctions.
Spermine inhibits rat connexin40 (Cx40) gap junctions. Glutamate residues at positions 9 and 13 and a basic amino acid (HKH) motif at positions 15-17 on the amino terminal domain are essential for this inhibitory activity. Questions remain as to whether spermine occludes the channel within the ion permeation pathway. To examine this question, cis or trans [KCl] was systematically lowered and the equilibrium dissociation constants (K(d)) and kinetics of unilateral spermine block on wild-type Cx40 gap junctions were determined. Asymmetric reductions in the trans [KCl] produced noticeable asymmetric shifts in the V(1/2) and G(min) values that progressively resembled G(j)-V(j) relationships observed in heterotypic connexin gap junction combinations. As cis or trans [KCl] was reduced by 25%, 50%, or 75% relative to the spermine-containing side, the transjunctional voltage (V(j))-dependent K(d) values increased or decreased, respectively. The spermine on-rates and off-rates, calculated from the junctional current decay and recovery time constants, were similarly affected. Hill coefficients for the spermine dose-response curves were approximately 0.58, indicative of negative cooperativity and possible multiple spermine inhibitory sites. The equivalent "electrical distance" (delta) ranged from 0.61 at 25% cis [KCl] to 1.4 at 25% trans [KCl], with a Hill coefficient of 1.0. Symmetrical reductions in [KCl] resulted in intermediate decreases in the spermine K(d)s, indicative of a minor electrostatic effect and a more significant effect of the transjunctional KCl electrodiffusion potential on the spermine association and dissociation rates. These data are consistent with a single spermine molecule being sufficient to occlude the Cx40 gap junction channel within the KCl permeation pathway.
Lin
Xianming
X
Department of Pharmacology, The State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Veenstra
Richard D
RD
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002460:Cell Line; D017630:Connexins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000187:drug effects* / Q000378:metabolism; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D007700:Kinetics; D011189:Potassium Chloride / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology*; D014162:Transfection; D000097003:Gap Junction alpha-5 Protein
D017630:Connexins; D011189:Potassium Chloride; D011994:Recombinant Proteins; D013096:Spermine; D000097003:Gap Junction alpha-5 Protein