Action of internal pronase on the f-channel kinetics in the rabbit SA node

1. The hyperpolarization-activated I-f current was recorded in inside-out m acropatches from sino-atrial (SA) node myocytes during exposure of their in tracellular side to pronase, in an attempt to verify if cytoplasmic f-chann el domains are involved in both voltage- and cAMP-dependent gating. 2. Superfusion with pronase caused a quick, dramatic acceleration of channe l opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cle avage of channel regions which contribute to gating. 3. I-f recorded from patches normally responding to cAMP became totally ins ensitive to cAMP following pronase treatment, indicating partial or total r emoval of channel regions involved in the cAMP-dependent activation. 4. The fully activated I-V relationship was not modified by pronase, indica ting that internal proteolysis did not affect the f-channel conductance. 5. The changes in I-f kinetics induced by pronase were due to a large depol arizing shift of the f-channel open probability curve (56.5 +/- 1.1 mV, n = 7). 6. These results are consistent with the hypothesis that cytoplasmic f-chan nel regions are implicated in dual voltage- and cAMP-dependent gating; also , since pronase does not abolish hyperpolarization-activated opening, an in trinsic voltage-dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thu s includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.