Todorovic, Slobodan M. and Christopher J. Lingle. Pharmacological properties of T-type Ca²⁺current in adult rat sensory neurons: effects of anticonvulsant and anesthetic agents. J. Neurophysiol. 79: 240–252, 1998. We have used the whole cell patch-clamp method to study pharmacological properties of low-voltage–activated (LVA) Ca²⁺current in freshly dissociated neurons from dorsal root ganglia of adult rats. Inward barium current [in the presence of internal fluoride to reduce L-type high-voltage–activated (HVA) and external 1 μM ω-conotoxin GVIA to blockN-type HVA current] was evoked from negative holding potentials of −90 mV to test potentials of −25 mV and showed complete inactivation during 200-ms test pulses. Amiloride blocked ∼90% of current with half-maximal block (EC₅₀) of 75 μM and a Hill coefficient ( n) of 0.99. LVA current was blocked completely by inorganic Ca²⁺channel blockers: lanthanum (EC₅₀= 0.53 μM) > zinc (EC₅₀= 11.3 μM) > cadmium (EC₅₀= 20 μM)> nickel (EC₅₀= 51 μM). The antiepileptics, ethosuximide (EC₅₀= 23.7 mM, n = 1.4), phenytoin (EC₅₀= 7.3 μM, n = 1.3), α-methyl-α-phenylsuccinimide (EC₅₀= 170 μM, n = 2.1), and valproic acid (EC₅₀= 330 μM, n = 1.9) maximally blocked ∼100, 60, 26, and 17% of T current, respectively. Another antiepileptic, carbamazepine (≤100 μM), and convulsants such as pentylenetetrazole (1 mM) and tert-butyl-bicyclo [2.2.2] phosphorothionate (50 μM) had no effect on T current. Barbiturates completely blocked T current: thiopental (EC₅₀= 153 μM, n =1.2) > pentobarbital (EC₅₀= 334 μM, n = 1.2) > methohexital (EC₅₀= 502 μM, n = 1.3) > phenobarbital (EC₅₀= 1.7 mM, n = 1.2). Blockade by thiopental and pentobarbital did not show voltage or use dependence. General anesthetics blocked T current completely and reversibly: propofol (EC₅₀= 12.9 μM, n = 1.3) > octanol(EC₅₀= 122 μM, n = 1.2) > etomidate (EC₅₀= 205 μM, n =1.3) > isoflurane (EC₅₀= 303 μM, n = 2.3) > halothane (EC₅₀= 655 μM, n = 2.0) > ketamine (EC₅₀= 2.5 mM, n = 1.1). Mibefradil, a novel Ca²⁺channel blocker, blocked dorsal root ganglion T current in a voltage- and use-dependent fashion with an EC₅₀of ∼3 μM( n = 1.3). When compared with results on other T currents, these data indicate that significant differences exist among different T currents in terms of pharmacological sensitivities. Furthermore, differences in pharmacological sensitivity of T currents among peripheral neurons, CNS, and neuroendocrine cells may contribute to the spectrum of effects of particular analgesic, anticonvulsant, and anesthetic drugs.

Large-conductance Ca²⁺- and voltage-dependent potassium (BK) channels exhibit functional diversity not explained by known splice variants of the singleSloα-subunit. Here we describe an accessory subunit (β3) with homology to other β-subunits of BK channels that confers inactivation when it is coexpressed withSlo. Message encoding the β3 subunit is found in rat insulinoma tumor (RINm5f) cells and adrenal chromaffin cells, both of which express inactivating BK channels. Channels resulting from coexpression ofSloα and β3 subunits exhibit properties characteristic of native inactivating BK channels. Inactivation involves multiple cytosolic, trypsin-sensitive domains. The time constant of inactivation reaches a limiting value ∼25–30 msec at Ca²⁺of 10 μmand positive activation potentials. UnlikeShakerN-terminal inactivation, but like native inactivating BK channels, a cytosolic channel blocker does not compete with the native inactivation process. Finally, the β3 subunit confers a reduced sensitivity to charybdotoxin, as seen with native inactivating BK channels. Inactivation arises from the N terminal of the β3 subunit. Removal of the β3 N terminal (33 amino acids) abolishes inactivation, whereas the addition of the β3 N terminal onto the β1 subunit confers inactivation. The β3 subunit shares with the β1 subunit an ability to shift the range of voltages over which channels are activated at a given Ca²⁺. Thus, the β-subunit family of BK channels regulates a number of critical aspects of BK channel phenotype, including inactivation and apparent Ca²⁺sensitivity.

Mouse spermatozoa express a pH-dependent K⁺current (KSper) thought to be composed of subunits encoded by theSlo3gene. However, the equivalence of KSper andSlo3-dependent current remains uncertain, because heterologous expression ofSlo3results in currents that are less effectively activated by alkalization than are native KSper currents. Here, we show that genetic deletion ofSlo3abolishes all pH-dependent K⁺current at physiological membrane potentials in corpus epididymal sperm. A residual pH-dependent outward current (I_Kres) is observed inSlo3^−/−sperm at potentials of >0 mV. Differential inhibition of KSper/Slo3 andI_Kresby clofilium reveals that the amplitude ofI_Kresis similar in both wild-type (wt) andSlo3^−/−sperm. The properties ofI_Kressuggest that it likely represents outward monovalent cation flux through CatSper channels. Thus, KSper/Slo3 may account for essentially all mouse sperm K⁺current and is the sole pH-dependent K⁺conductance in these sperm. With physiological ionic gradients, alkalization depolarizesSlo3^−/−spermatozoa, presumably from CatSper activation, in contrast to Slo3/KSper-mediated hyperpolarization in wt sperm.Slo3^−/−male mice are infertile, butSlo3^−/−sperm exhibit some fertility within in vitro fertilization assays.Slo3^−/−sperm exhibit a higher incidence of morphological abnormalities accentuated by hypotonic challenge and also exhibit deficits in motility in the absence of bicarbonate, revealing a role of KSper under unstimulated conditions. Together, these results show that KSper/Slo3 is the primary spermatozoan K⁺current, that KSper may play a critical role in acquisition of normal morphology and sperm motility when faced with hyperosmotic challenges, and that Slo3 is critical for fertility.

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10–300 μM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.