Two types of modified cardiac Na+ channels after cytosolic interventions at the alpha-subunit capable of removing Na+ inactivation.
Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the alpha-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodatemodified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with alpha-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. Invitro cleavage of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the alpha-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.
Benz
I
I
Physiological Institute University of Freiburg, Germany.
Beck
W
W
Kraas
W
W
Stoll
D
D
Jung
G
G
Kohlhardt
M
M
D000081:Acetamides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002154:Calpain / Q000494:pharmacology; D003600:Cytosol / Q000502:physiology; D006321:Heart / Q000502:physiology; D007452:Iodates / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D012964:Sodium / Q000502:physiology*; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D013329:Structure-Activity Relationship
D000081:Acetamides; D000906:Antibodies; D007452:Iodates; D046911:Macromolecular Substances; D015222:Sodium Channels; C016272:N-bromoacetamide; D012964:Sodium; D010447:Peptide Hydrolases; D002154:Calpain