Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na(+) channel alpha subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na(+) channels involve mechanical destabilization. Our observation that the beta1 subunit causes similar changes in function of the Na(+) channel alpha subunit suggests that beta1 may act through interaction with cytoskeleton.