Ion Trafficking through T-type Ca2+ Channels

Voltage-gated Ca 2 channels play a key role in controlling Ca 2 entry during cell depolarization. At least 10 genes encode the main ( 1 ) subunits of voltage-gated Ca 2 channels, which have been grouped into two main classes: the high voltage–activated (HVA) and the low voltage–activated (LVA) channels. HVA channels are primarily involved in muscle contraction, synaptic transmission, and hormone secretion, while LVA channels are associated with action potential generation and repetitive electrical activity. Structurally speaking, the Ca 2 channel 1 subunits forming the pore share strong similarities with other voltage-gated ion channels, in particular with Na -conducting pores (Hille, 2001). Each 1 subunit has four domains (I–IV) linked together in a single polypeptide chain and each domain contains six putative transmembrane segments (S1–S6), plus a loop (P) that dips partially into the pore to, presumably, form the pore lining. Functionally speaking, the most striking similarity between Ca 2 and Na channels is represented by the LVA (T-type) channels, which have comparably low threshold for activation ( 50 to 40 mV in 5 mM Ca 2 ) and inactivate fully and rapidly, if at a 20to 40-fold lower rate than Na channels. Like Na channels, fast inactivation of T-type channels is strictly voltage rather than Ca 2 dependent, as in the case of channel types of the HVA family (L, N, P/Q, and R). T-type channels, however, possess other properties that are unique in comparison to other Ca 2 channels: (a) they deactivate more slowly ( deact 2.5 ms at 110 mV in 5 mM Ca 2 ; Carbone and Lux, 1984a); (b) they inactivate at relatively negative holding potentials; (c) they are equally permeable to Ca 2 and Ba 2 ; (d) they have small single channel conductance; and (e) they outlast membrane-patch excision since they do not require specific metabolic factors to preserve their activity (Carbone and Lux, 1984b, 1987). T-type channels were identified 20 years ago by several groups. Since then their biophysical, pharmacological, and functional properties have been widely investigated (for reviews see Huguenard, 1996; Perez-Reyes, 2003). A main drawback that significantly limited the analysis of ion permeability and gating properties of T-type channels was the lack of selective toxins or drugs that allowed for their pharmacological isolation. New impetus for approaching these issues was provided by the molecular cloning of three different pore-forming 1 subunits ( 1G , 1H , 1I also denoted as Ca V 3.1, Ca V 3.2, Ca V 3.3) with biophysical properties that clearly identify them as T-type channels (Perez-Reyes et al., 1998). This opened for a new era of biophysical studies of LVA channels, which led important new insights into the features of ion selectivity and gating that distinguish LVA from HVA channels. Among the new findings on ion permeation through cloned T-type channels, those concerning the blocking action of divalent and trivalent cations deserve particular attention. They show clear evidence for the following: (a) a voltage-dependent blocking action of Ni 2 (Lee et al., 1999), which is more effective on inward Ca 2 currents through 1H channels compared with other T-type channel subunits; (b) a more effective blocking capability of Mg 2 on inward Ba 2 as compared with Ca 2 currents in 1G channels (Serrano et al., 2000), uncovering a Ca 2 /Ba 2 selectivity that is absent in Mg 2 -free media; and (c) the existence of potent T-type channel blockers among trivalent cations, with yttrium (Y 3 ) being the most potent blocker of 1G currents (Beedle et al., 2002). The common aspect of these studies is that T-type channels can be blocked by multivalent ions larger or more hydrated than Ca 2 and that blocking ions can be effectively removed from their blocking position in a voltageand current-dependent manner by strong depolarization. This is particularly evident in the case of Ni 2 , in which effective unblocking occurs while outward ion currents clear the channel. The unblock persists in the absence of permeating ions, proving that “ion–ion repulsion” (or single file diffusion) in a multi-ion pore favors but does not fully