Slack and Slick KNa channels are required for the depolarizing afterpotential of acutely isolated, medium diameter rat dorsal root ganglion neurons.

AIM: Na+-activated K+ (K(Na)) channels set and stabilize resting membrane potential in rat small dorsal root ganglion (DRG) neurons. However, whether K(Na) channels play the same role in other size DRG neurons is still elusive. The aim of this study is to identify the existence and potential physiological functions of K(Na) channels in medium diameter (25-35 microm) DRG neurons. METHODS: Inside-out and whole-cell patch-clamp were used to study the electrophysiological characterizations of native K(Na) channels. RT-PCR was used to identify the existence of Slack and Slick genes. RESULTS: We report that K(Na) channels are required for depolarizing afterpotential (DAP) in medium sized rat DRG neurons. In inside-out patches, K(Na) channels represented 201 pS unitary chord conductance and were activated by cytoplasmic Na+ [the half maximal effective concentration (EC50): 35 mmol/L] in 160 mmol/L symmetrical K+o/K+i solution. Additionally, these K(Na) channels also represented cytoplasmic Cl(-)-dependent activation. RT-PCR confirmed the existence of Slack and Slick genes in DRG neurons. Tetrodotoxin (TTX, 100 nmol/L) completely blocked the DRG inward Na+ currents, and the following outward currents which were thought to be K(Na) currents. The DAP was increased when extracellular Na+ was replaced by Li+. CONCLUSION: We conclude that Slack and Slick K(Na) channels are required for DAP of medium diameter rat DRG neurons that regulate DRG action potential repolarization.

Cerebrosides of baifuzi, a novel potential blocker of calcium-activated chloride channels in rat pulmonary artery smooth muscle cells.

Calcium-activated chloride channels (CaCCs) are crucial regulators of vascular tone by promoting a depolarizing influence on the resting membrane potential of vascular smooth muscle cells. However, the lack of a special blocker of CaCCs has limited the investigation of its functions for long time. Here, we report that CB is a novel potential blocker of I(Cl(Ca)) in rat pulmonary artery smooth muscle cells (PASMC). Cerebrosides (CB) were isolated from Baifuzi which is dried root tuber of the herb Typhonium giganteum Engl used for treatment of stroke in traditional medicine. Using the voltage-clamp technique, sustained Ca(2+)-activated Cl(-) current (I(Cl(Ca))) was evoked by a K(+)-free pipette solution containing 500nM Ca(2+) which exhibited typical outwardly rectifying and voltage-/time-dependence characterization. Data showed that CB played a distinct inhibitory role in modulating the CaCCs. Moreover, we investigated the kinetic effect of CB on I(Cl(Ca)) and found that it could slow the activation dynamics of the outward current, accelerate the decay of the inward tail current and change the time-dependence characterization. We conclude that CB is a novel potent blocker of CaCCs. The interaction between CB and CaCCs is discussed.

Characterization of voltage-and Ca2+-activated K+ channels in rat dorsal root ganglion neurons.

Auxiliary beta-subunits associated with pore-forming Slo1 alpha-subunits play an essential role in regulating functional properties of large-conductance, voltage- and Ca(2+)-activated K(+) channels commonly termed BK channels. Even though both noninactivating and inactivating BK channels are thought to be regulated by beta-subunits (beta1, beta2, beta3, or beta4), the molecular determinants underlying inactivating BK channels in native cells have not been extensively demonstrated. In this study, rbeta2 (but not rbeta3-subunit) was identified as a molecular component in rat lumbar L4-6 dorsal root ganglia (DRG) by RT-PCR responsible for inactivating large-conductance Ca(2+)-dependent K(+) currents (BK(i) currents) in small sensory neurons. The properties of native BK(i) currents obtained from both whole-cell and inside-out patches are very similar to inactivating BK channels produced by co-expressing mSlo1 alpha- and hbeta2-subunits in Xenopus oocytes. Intracellular application of 0.5 mg/ml trypsin removes inactivation of BK(i) channels, and the specific blockers of BK channels, charybdotoxin (ChTX) and iberiotoxin (IbTX), inhibit these BK(i) currents. Single BK(i) channel currents derived from inside-out patches revealed that one BK(i) channel contained three rbeta2-subunits (on average), with a single-channel conductance about 217 pS under 160 K(+) symmetrical recording conditions. Blockade of BK(i) channels by 100 nM IbTX augmented firing frequency, broadened action potential waveform and reduced after-hyperpolarization. We propose that the BK(i) channels in small diameter DRG sensory neurons might play an important role in regulating nociceptive input to the central nervous system (CNS).