2 TYPES OF MODIFIED CARDIAC NA-SUBUNIT CAPABLE OF REMOVING NA+ INACTIVATION( CHANNELS AFTER CYTOSOLIC INTERVENTIONS AT THE ALPHA)

Failure of inactivation is the typical response of voltage-gated Na+ c hannels to the cytosolic presence of proteolytic enzymes, protein reag ents such as N-bromo-acetamide (NBA) or iodate, and antibodies directe d 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 occurren ce of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1 496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inac tivation to induce burst activity which was accompanied by the occurre nce of two open states. A deactivation process terminated channel acti vity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodate-modified Na+ chann els were kinetically indistinguishable from the anti-SLP2-modified typ e since they likewise deactivate and, thus, attain an only moderate P- o of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis wi th alpha-chymotrypsin, trypsin or pronase, mean P-o during membrane de polarization amounted to approximately 40% because deactivation operat ed extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. In-vitro cleava ge of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or rea ctants for NBA but can-not be chemically modified by iodate. This ioda te resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involve d in Na+ inactivation, besides the linker between domains III and IV o f the alpha-subunit. Endogenous peptidases such as calpain did not aff ect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactiva ting mode.