HYPOOSMOTIC CHALLENGE STIMULATES TRANSEPITHELIAL K-SK CHANNEL IN VESTIBULAR DARK CELLS( SECRETION AND ACTIVATES APICAL I)

Volume regulation of vestibular dark cells from the gerbilline inner e ar in response to a hypoosmotic challenge depends on the presence of c ytosolic K+ and Cl-. The present study addresses the questions: (i) wh ether and by what mechanism K+ is released during volume regulation, ( ii) whether the osmolarity of the basolateral medium has an effect on the steady-state rate of transepithelial K+ transport and (iii) whethe r there is cross-talk between the basolateral membrane responsible for K+ uptake and the apical membrane responsible for K+ release. K+ secr etion (J(K+,probe)) and current density (I-sc,I-probe) were measured w ith vibrating probes in the vicinity of the apical membrane and the tr ansepithelial potential (V-t) and resistance (R(t)) were measured in a micro-Ussing chamber. The equivalent short-circuit current (I-sc) was calculated. The current (I-IsK), conductance (g(IsK)) and inactivatio n time constant (tau(IsK)) Of the I-sK channel and the apparent revers al potential of the apical membrane (V-r) were obtained with the cell- attached macropatch technique. V-r was corrected (V-rc) for the membra ne voltage (V-m) measured separately with microelectrodes. A hypo-osmo tic challenge (294 to 154 MOSM by removal of 150 mM mannitol) on the b asolateral side of the epithelium increased J(K+,probe) and I-sc,I-pro be by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was ap plied to both sides of the epithelium V-t and I-sc increased from 5 to 14 mV and from 189 to 824 mu A/cm(2) whereas R(t) decreased from 27 t o 19 Omega-cm(2). With 3.6 mM K+ in the pipette I-IsK was outwardly di rected, tau(IsK) was 267 msec and the hypo-osmotic challenge caused I- IsK and g(IsK) to increase from 14 to 37 pA and from 292 to 732 pS. V- rc hyperpolarized from -44 to -76 mV. With 150 mM K+ in the pipette I- IsK was inwardly directed, tau(IsK) was 208 msec and the hypo-osmotic challenge caused I-IsK and g(IsK) to increase in magnitude from 0 to - 21 pA and from 107 to 1101 pS. V-rc remained unchanged (-2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transe pithelial K+ secretion and activates the apical I-sK channel. The hypo osmotically-induced increase in K+ secretion exceeded the estimated am ount of K+ release necessary for the maintenance of constant cell volu me, suggesting that the rate of basolateral K+ uptake was upregulated in the presence of the hypo-osmotic challenge and that cross-talk exis ts between the apical membrane and the basolateral membrane.