Membrane excitability in different tissues is due, in large part, to the selective expression of distinct genes encoding the voltage-dependent sodium channel. Although the predominant sodium channels in brain, skeletal muscle, and cardiac muscle have been identified, the major sodium channel types responsible for excitability within the peripheral nervous system have remained elusive. We now describe the deduced primary structure of a sodium channel, peripheral nerve type 1 (PN1), which is expressed at high levels throughout the peripheral nervous system and is targeted to nerve terminals of cultured dorsal root ganglion neurons. Studies using cultured PC12 cells indicate that both expression and targeting of PN1 is induced by treatment of the cells with nerve growth factor. The preferential localization suggests that the PN1 sodium channel plays a specific role in nerve excitability.

Functional large-conductance Ca2+- and voltage-activated K+ (BK) channels can be assembled from four α subunits (Slo1) alone, or together with four auxiliary β1 subunits to greatly increase the apparent Ca2+ sensitivity of the channel. We examined the structural features involved in this modulation with two types of experiments. In the first, the tail domain of the α subunit, which includes the RCK2 (regulator of K+ conductance) domain and Ca2+ bowl, was replaced with the tail domain of Slo3, a BK-related channel that lacks both a Ca2+ bowl and high affinity Ca2+ sensitivity. In the second, the Ca2+ bowl was disrupted by mutations that greatly reduce the apparent Ca2+ sensitivity. We found that the β1 subunit increased the apparent Ca2+ sensitivity of Slo1 channels, independently of whether the α subunits were expressed as separate cores (S0-S8) and tails (S9-S10) or full length, and this increase was still observed after the Ca2+ bowl was mutated. In contrast, β1 subunits no longer increased Ca2+ sensitivity when Slo1 tails were replaced by Slo3 tails. The β1 subunits were still functionally coupled to channels with Slo3 tails, as DHS-I and 17 β-estradiol activated these channels in the presence of β1 subunits, but not in their absence. These findings indicate that the increase in apparent Ca2+ sensitivity induced by the β1 subunit does not require either the Ca2+ bowl or the linker between the RCK1 and RCK2 domains, and that Slo3 tails cannot substitute for Slo1 tails. The β1 subunit also induced a decrease in voltage sensitivity that occurred with either Slo1 or Slo3 tails. In contrast, the β1 subunit–induced increase in apparent Ca2+ sensitivity required Slo1 tails. This suggests that the allosteric activation pathways for these two types of actions of the β1 subunit may be different.

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The COOH-terminal S9–S10 tail domain of large conductance Ca2+-activated K+ (BK) channels is a major determinant of Ca2+ sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416–421). To investigate whether the tail domain also modulates Ca2+-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:3509–3516). Compared with mSlo1 channels, mSlo3 channels showed little Ca2+ sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca2+; reduced the Ca2+ sensitivity greatly by allowing only partial activation by Ca2+ and by reducing the Hill coefficient for Ca2+ activation; decreased the voltage dependence ∼28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance ∼14%; decreased the K d for block by TEAi ∼30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca2+ sensitivity.

modulates axo-axonal coupling between neurons critical for learning in the leech. J Neurophysiol 94: [2575][2576][2577][2578][2579][2580][2581][2582][2583][2584][2585][2586][2587][2588][2589] 2005. First published June 29, 2005; doi:10.1152/jn.00322.2005. S cells form a chain of electrically coupled neurons that extends the length of the leech CNS and plays a critical role in sensitization during wholebody shortening. This process requires serotonin, which acts in part by altering the pattern of activity in the S-cell network. Serotonincontaining axons and varicosities were observed in Faivre's nerve where the S-to-S-cell electrical synapses are located. To determine whether serotonin modulates these synapses, S-cell action-potential (AP) propagation was studied in a two-ganglion chain containing one electrical synapse. Suction electrodes were placed on the cut ends of the connectives to stimulate one S cell while recording the other, coupled S cell's APs. A third electrode, placed en passant, recorded the APs near the electrical synapse before they propagated through it. Low concentrations of the gap junction inhibitor octanol increased AP latency across the two-ganglion chain, and this effect was localized to the region of axon containing the electrical synapse. At higher concentrations, APs failed to propagate across the synapse. Serotonin also increased AP latency across the electrical synapse, suggesting that serotonin reduced coupling between S cells. This effect was independent of the direction of propagation and increased with the number of electrical synapses in progressively longer chains. Furthermore, serotonin modulated instantaneous AP frequency when APs were initiated in separate S cells and in a computational model of S-cell activity after mechanosensory input. Thus serotonergic modulation of S-cell electrical synapses may contribute to changes in the pattern of activity in the S-cell network.

The N-methyl-D-aspartate receptor (NMDAR) is involved in a number of physiological and pathophysiological processes in vertebrates, but there have been few studies examining the role of invertebrate NMDA receptors. In the leech, pharmacological evidence suggests that NMDARs contribute to synaptic plasticity, but there has been no molecular identification of NMDA receptors. In this report, a partial cDNA encoding the leech NR1 subunit of the NMDA receptor (HirNR1) is presented. Reverse transcriptase-polymerase chain reaction from single neurons of the leech central nervous system confirms HirNR1 expression in the Retzius (R), Anterior Pagoda (AP), Pressure (P), and Touch (T) neurons. Immunoblotting with an anti-NR1 antibody yielded a approximately 110 kDa protein, similar to the expected weight of the NR1 subunit (approximately 116 kDa). Finally, pairing pre- and postsynaptic activity elicited long-term potentiation in synapses between neurons expressing NR1 mRNA (P-to-AP synapse) and this potentiation was blocked by the NMDAR antagonist AP5.

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.

The Ca2+-dependent gating mechanism of cloned BK channels from Drosophila (dSlo) was studied. Both a natural variant (A1/C2/E1/G3/IO) and a mutant (S942A) were expressed in Xenopus oocytes, and single-channel currents were recorded from excised patches of membrane. Stability plots were used to define stable segments of data. Unlike native BK channels from rat skeletal muscle in which increasing internal Ca2+ concentration (Cai2+) in the range of 5 to 30 microM increases mean open time, increasing Cai2+ in this range for dSlo had little effect on mean open time. However, further increases in Cai2+ to 300 or 3000 microM then typically increased dSlo mean open time. Kinetic schemes for the observed Ca2+-dependent gating kinetics of dSlo were evaluated by fitting two-dimensional dwell-time distributions using maximum likelihood techniques and by comparing observed dependency plots with those predicted by the models. Previously described kinetic schemes that largely account for the Ca2+-dependent kinetics of native BK channels from rat skeletal muscle did not adequately describe the Ca2+ dependence of dSlo. An expanded version of these schemes which, in addition to the Ca2+-activation steps, permitted a Ca2+-facilitated transition from each open state to a closed state, could approximate the Ca2+-dependent kinetics of dSlo, suggesting that Ca2+ may exert dual effects on gating.