The recently cloned rat vanilloid receptor, VR1, can be activated by capsaicin, acid, and heat. To determine the molecular mechanisms facilitating channel opening in response to these stimuli, VR1 and six channels containing charge neutralization point mutations surrounding the putative channel pore domain were expressed and characterized in Xenopus laevis oocytes. Steady-state doseresponse relationships, current-voltage relationships, ionic selectivities, and single-channel properties were recorded using voltage-clamp techniques. Three of the mutant channels are significantly more sensitive to capsaicin than is wild-type VR1, whereas none differed in their activation by acidic pH or temperature. Furthermore, one of the mutants has lost all positive cooperativity for capsaicin activation (Hill coefficient Х 1, VR1 Х 2), is much more selective for Ca 2؉ , and exhibits a lower efficacy for acid than for capsaicin activation. Single-channel recordings show that capsaicin-and acid-activated channels have the same conductance, that the three mutants with increased capsaicin sensitivity exhibit higher open probabilities at submaximal capsaicin concentrations, and that the gating properties of capsaicin activation differ from those of acid activation. These data indicate that VR1 undergoes conformational changes upon capsaicin binding that it does not undergo in response to activation by protons or thermal stimuli. Furthermore, these structural rearrangements include the putative pore domain and reveal the location of an intracellular domain that contributes to the positive cooperativity seen for capsaicin activation.C apsaicin, the primary pungent compound in ''hot'' chili peppers, produces pain and inflammation when placed on skin or mucus membranes. These responses are a consequence of capsaicin activating nonselective cation channel receptors within C and A␦ nociceptors and inducing the release of peptides and other transmitters from their peripheral and central terminals (1). Although a number of reports have demonstrated the existence of capsaicin receptors in sensory neurons (2, 3), the cloning of rat vanilloid receptor 1 (VR1) (4) was a breakthrough in understanding their properties and physiological functions (3, 5). VR1 subunits have now been identified in peripheral and central terminals of neurons in sensory ganglia (1,3,4,6,7). Electrophysiological studies of expressed VR1 receptors show that they are activated independently by capsaicin (EC 50 ϭ 0.7 M), acid (pH 50 ϭ 5.4), and elevated temperatures Ն 43°C (3, 6). These conditions are similar to those found in some physiological states (2,3,8). Further findings were that two or more of these activation pathways can act in concert to increase the sensitivity of VR1 receptors to any one stimulus (6,8). To determine protein domains important for the activation of VR1 by capsaicin, protons, and heat, we constructed a number of VR1 point mutants and measured their responsiveness to these three activators. We found that mutations near the pore of VR1 channels ca...

1. Cloned large-conductance Ca2+-activated K+ channels from Drosophila (dslo) and human (hslo) were expressed in Xenopus oocytes. The effects of Ca2+ and voltage on these channels were compared by analysing both macroscopic currents and single-channel transitions.2. The activation kinetics of dslo Ca2+-activated K+ channels are strongly influenced by the intracellular Ca2+ concentration, but are only minimally affected by membrane voltage. Current activation kinetics increase more than 60-fold in response to Ca2+ concentration increases in the range 0f56-405 /M, but increase less than 2-fold by voltage changes from -60 to +80 mV. 3. The activation kinetics of hslo channels are similarly influenced by increases in Ca2+ concentration; however, these kinetics are also increased 5-to 10-fold by voltage changes from -60 to +80 mV.4. The deactivation kinetics of both dslo and hslo channels are also more Ca2+ sensitive than voltage sensitive. Increasing concentrations of Ca2+ slow deactivation kinetics more than 40-fold, while changes in the membrane voltage cause less than 2-fold changes.

Type-2 calcium-dependent potassium (KCa) channels from mammalian brain, reconstituted into planar phospholipid bilayers, are modulated by ATP or ATP analogs via an endogenous protein kinase activity intimately associated with the channel (Chung et al., 1991). We show here that the endogenous protein kinase activity is protein kinase C (PKC)-like because (1) modulation by ATP can be mimicked by exogenous PKC, and (2) the effects of ATP can be blocked by PKC(19-36), a specific peptide inhibitor of PKC. Furthermore, adding the PKC inhibitor peptide after the addition of ATP reverses the modulation produced by ATP, suggesting that there is a phosphoprotein phosphatase activity closely associated with type-2 KCa channels. Consistent with this idea is the finding that microcystin, a non-specific phosphatase inhibitor, enhances the modulation of KCa channel activity by ATP. Inhibitor-1, a specific protein inhibitor of phosphoprotein phosphatase-1, also enhances the effect of ATP, suggesting that the endogenous phosphatase activity is phosphatase-1-like. The results imply that type-2 KCa channels exist as part of a regulatory complex that includes a PKC-like protein kinase and a phosphatase-1-like phosphoprotein phosphatase, both of which participate in the modulation of channel function.