Amino acid substitutions in the pore of the Ca(V)1.2 calcium channel reduce barium currents without affecting calcium currents.
Ba(2+) currents through Ca(V)1.2 Ca(2+) channels are typically twice as large as Ca(2+) currents. Replacing Phe-1144 in the pore-loop of domain III with glycine and lysine, and Tyr-1152 with lysine, reduces whole-cell G(Ba)/G(Ca) from 2.2 (wild-type) to 0.95, 1.21, and 0.90, respectively. Whole-cell and single-channel measurements indicate that reductions in G(Ba)/G(Ca) result specifically from a decrease in Ba(2+) conductance and not changes in V(h) or P(O). Half-maximal block of I(Li) is increased by 3.2-, 3.8-, and 1.6-fold in Ca(2+), and 3.8-, 4.2-, and 1.8-fold in Ba(2+) for F1144G, Y1152K, and F1144K, respectively. High affinity interactions of individual divalent cations to the pore are not important for determining G(Ba)/G(Ca), because the fold increases in IC(50) values for Ba(2+) and Ca(2+) are similar. On the contrary, conductance-concentration curves indicate that G(Ba)/G(Ca) is reduced because the interactions of multiple Ba(2+) ions in the mutant pores are altered. The complexity of these interactions is exemplified by the anomalous mole fraction effect, which is flattened for F1144G and FY/GK but accentuated for F1144K. In summary, the physicochemical properties of the amino acid residues at positions 1144 and 1152 are crucial to the pore's ability to distinguish between multiple Ba(2+) ions and Ca(2+) ions.
Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Xianming
X
Wang
Tudor A
TA
Ponoran
Randall L
RL
Rasmusson
David S
DS
Ragsdale
Blaise Z
BZ
Peterson
D000595:Amino Acid Sequence; D001464:Barium / Q000737:chemistry*; D001692:Biological Transport; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000502:physiology*; D002412:Cations; D002413:Cations, Divalent; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D015640:Ion Channel Gating; D007477:Ions; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000737:chemistry
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002412:Cations; D002413:Cations, Divalent; D018076:DNA, Complementary; D007477:Ions; C419385:L-type calcium channel alpha(1C); D001464:Barium; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D008239:Lysine; D002118:Calcium; D005998:Glycine